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Patent Analysis of

Lipid comprising polyunsaturated fatty acids

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US9999607

Application Number

US15/678002

Application Date

15 August 2017

Publication Date

19 June 2018

Current Assignee

COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION,NUSEED PTY. LTD.,GRAINS RESEARCH AND DEVELOPMENT CORPORATION

Original Assignee (Applicant)

PETRIE, JAMES ROBERTSON,SINGH, SURINDER PAL,DE FEYTER, ROBERT CHARLES

International Classification

A23D9/00,C11B1/10,A61K31/231,A23L33/12,A23K20/158

Cooperative Classification

A61K31/232,A23K20/158,A23L33/12,A61K36/31,C11B1/10

Inventor

PETRIE, JAMES ROBERTSON,SINGH, SURINDER PAL,DE FEYTER, ROBERT CHARLES

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US9999607 Lipid comprising polyunsaturated fatty acids 1 US9999607 Lipid comprising polyunsaturated fatty acids 2 US9999607 Lipid comprising polyunsaturated fatty acids 3
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Abstract

The present invention relates to extracted plant lipid, comprising fatty acids in an esterified form.

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Claims

1. Oil extracted from seeds which are Brassica napes or Arabidopsis thaliana seeds, wherein the oil comprises a total fatty acid content which comprises a) a total monounsaturated fatty acid content which comprises oleic acid, b) a total saturated fatty acid content which comprises palmitic acid, or palmitic acid and myristic acid (C14:0), c) a total ω6 fatty acid content which comprises linoleic acid (LA) and γ-linolenic acid (GLA), d) a total ω3 fatty acid content which comprises α-linolenic acid (ALA), docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein i) DHA is present in the extracted oil at a level of about 3% of the total fatty acid content, wherein at least 70% of the DHA which is esterified in the form of triacylglycerols (TAG) is esterified at the sn-1or sn-3 position of the TAG, ii) palmitic acid is present in the extracted oil at a level of between 2% and 16% of the total fatty acid content, iii) oleic acid is present in the extracted oil at a level of between 1% and 30% of the total fatty acid content, iv) LA is present in the extracted oil at a level of between 4% and 35% of the total fatty acid content, v) GLA is present in the extracted oil at a level of less than 4% of the total fatty acid content, vi) ALA is present in the extracted oil at a level of between 4% and 40% of the total fatty acid content, vii) the total saturated fatty acid content of the extracted oil is between 4% and 25% of the total fatty acid content, viii) the ratio of the total ω6 fatty acid content to the total ω3 fatty acid content of the extracted oil is between 0.1 and 3.0, ix) myristic acid, if present, is present at a level of less than 1% of the total fatty acid content, and x) eicosatrienoic acid (ETrA), if present, is present at a level of less than 4% of the total fatty acid content.

2. The oil of claim 1, wherein γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.

3. The oil of claim 1, wherein oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.

4. The oil of claim 1, wherein the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.

5. The oil of claim 1, comprising new ω3 fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.

6. The oil of claim 1, comprising tri-DHA TAG (TAG 66:18).

7. The oil of claim 1, wherein at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.

8. Oil extracted from seeds which are Brassica napus or Arabidopsis thaliana seeds, wherein the oil comprises a total fatty acid content which comprises a) a total monounsaturated fatty acid content which comprises oleic acid, b) a total saturated fatty acid content which comprises palmitic acid, or palmitic acid and myristic acid (C14:0), c) a total ω6 fatty acid content which comprises linoleic acid (LA) and γ-linolenic acid (GLA), d) a total ω3 fatty acid content which comprises α-linolenic acid (ALA), docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein i) DHA is present in the extracted oil at a level of about 4% of the total fatty acid content, wherein at least 70% of the DHA which is esterified in the form of triacylglycerols (TAG) is esterified at the sn-1 or sn-3 position of the TAG, ii) palmitic acid is present in the extracted oil at a level of between 2% and 16% of the total fatty acid content, iii) oleic acid is present in the extracted oil at a level of between 1% and 30% of the total fatty acid content, iv) LA is present in the extracted oil at a level of between 4% and 35% of the total fatty acid content, v) GLA is present in the extracted oil at a level of less than 4% of the total fatty acid content, vi) ALA is present in the extracted oil at a level of between 4% and 40% of the total fatty acid content, vii) the total saturated fatty acid content of the extracted oil is between 4% and 25% of the total fatty acid content, viii) the ratio of the total ω6 fatty acid content to the total ω3 fatty acid content of the extracted oil is between 0.1 and 3.0, ix) myristic acid, if present, is present at a level of less than 1% of the total fatty acid content, and x) eicosatrienoic acid (ETrA), if present, is present at a level of less than 4% of the total fatty acid content.

9. The oil of claim 8, wherein γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.

10. The oil of claim 8, wherein oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.

11. The oil of claim 8, wherein the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.

12. The oil of claim 8, comprising new ω3fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.

13. The oil of claim 8, comprising tri-DHA TAG (TAG 66:18).

14. The oil of claim 8, wherein at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.

15. Oil extracted from seeds which are Brassica napus or Arabidopsis thaliana seeds, wherein the oil comprises a total fatty acid content which comprises a) a total monounsaturated fatty acid content which comprises oleic acid, b) a total saturated fatty acid content which comprises palmitic acid, or palmitic acid and myristic acid (C14:0), c) a total ω6 fatty acid content which comprises linoleic acid (LA) and γ-linolenic acid (GLA), d) a total ω3 fatty acid content which comprises α-linolenic acid (ALA), docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein i) DHA is present in the extracted oil at a level of about 5% of the total fatty acid content, wherein at least 70% of the DHA which is esterified in the form of triacylglycerols (TAG) is esterified at the sn-1 or sn-3 position of the TAG, ii) palmitic acid is present in the extracted oil at a level of between 2% and 16% of the total fatty acid content, iii) oleic acid is present in the extracted oil at a level of between 1% and 30% of the total fatty acid content, iv) LA is present in the extracted oil at a level of between 4% and 35% of the total fatty acid content, v) GLA is present in the extracted oil at a level of less than 4% of the total fatty acid content, vi) ALA is present in the extracted oil at a level of between 4% and 40% of the total fatty acid content, vii) the total saturated fatty acid content of the extracted oil is between 4% and 25% of the total fatty acid content, viii) the ratio of the total ω6 fatty acid content to the total ω3 fatty acid content of the extracted oil is between 0.1 and 3.0, ix) myristic acid, if present, is present at a level of less than 1% of the total fatty acid content, and x) eicosatrienoic acid (ETrA), if present, is present at a level of less than 4% of the total fatty acid content.

16. The oil of claim 15, wherein γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.

17. The oil of claim 15, wherein oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.

18. The oil of claim 15, wherein the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.

19. The oil of claim 15, comprising new ω3 fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.

20. The oil of claim 15, comprising tri-DHA TAG (TAG 66:18).

21. The oil of claim 15, wherein at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.

22. Oil extracted from seeds which are Brassica napus or Arabidopsis thaliana seeds, wherein the oil comprises a total fatty acid content which comprises a) a total monounsaturated fatty acid content which comprises oleic acid, b) a total saturated fatty acid content which comprises palmitic acid, or palmitic acid and myristic acid (C14:0), c) a total ω6 fatty acid content which comprises linoleic acid (LA) and γ-linolenic acid (GLA), d) a total ω3 fatty acid content which comprises α-linolenic acid (ALA), docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein i) DHA is present in the extracted oil at a level of about 6% of the total fatty acid content, wherein at least 70% of the DHA which is esterified in the form of triacylglycerols (TAG) is esterified at the sn-1 or sn-3 position of the TAG, ii) palmitic acid is present in the extracted oil at a level of between 2% and 16% of the total fatty acid content, iii) oleic acid is present in the extracted oil at a level of between 1% and 30% of the total fatty acid content, iv) LA is present in the extracted oil at a level of between 4% and 35% of the total fatty acid content, v) GLA is present in the extracted oil at a level of less than 4% of the total fatty acid content, vi) ALA is present in the extracted oil at a level of between 4% and 40% of the total fatty acid content, vii) the total saturated fatty acid content of the extracted oil is between 4% and 25% of the total fatty acid content, viii) the ratio of the total ω6 fatty acid content to the total ω3 fatty acid content of the extracted oil is between 0.1 and 3.0, ix) myristic acid, if present, is present at a level of less than 1% of the total fatty acid content, and x) eicosatrienoic acid (ETrA), if present, is present at a level of less than 4% of the total fatty acid content.

23. The oil of claim 22, wherein γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.

24. The oil of claim 22, wherein oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.

25. The oil of claim 22, wherein the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.

26. The oil of claim 22, comprising new ω3 fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.

27. The oil of claim 22, comprising tri-DHA TAG (TAG 66:18).

28. The oil of claim 22, wherein at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.

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Claim Tree

  • 1
    1. Oil extracted from seeds which are Brassica napes or Arabidopsis thaliana seeds, wherein
    • the oil comprises a total fatty acid content which comprises
    • 2. The oil of claim 1, wherein
      • γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.
    • 3. The oil of claim 1, wherein
      • oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.
    • 4. The oil of claim 1, wherein
      • the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.
    • 5. The oil of claim 1, comprising
      • new ω3 fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.
    • 6. The oil of claim 1, comprising
      • tri-DHA TAG (TAG 66:18).
    • 7. The oil of claim 1, wherein
      • at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.
  • 8
    8. Oil extracted from seeds which are Brassica napus or Arabidopsis thaliana seeds, wherein
    • the oil comprises a total fatty acid content which comprises
    • 9. The oil of claim 8, wherein
      • γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.
    • 10. The oil of claim 8, wherein
      • oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.
    • 11. The oil of claim 8, wherein
      • the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.
    • 12. The oil of claim 8, comprising
      • new ω3fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.
    • 13. The oil of claim 8, comprising
      • tri-DHA TAG (TAG 66:18).
    • 14. The oil of claim 8, wherein
      • at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.
  • 15
    15. Oil extracted from seeds which are Brassica napus or Arabidopsis thaliana seeds, wherein
    • the oil comprises a total fatty acid content which comprises
    • 16. The oil of claim 15, wherein
      • γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.
    • 17. The oil of claim 15, wherein
      • oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.
    • 18. The oil of claim 15, wherein
      • the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.
    • 19. The oil of claim 15, comprising
      • new ω3 fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.
    • 20. The oil of claim 15, comprising
      • tri-DHA TAG (TAG 66:18).
    • 21. The oil of claim 15, wherein
      • at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.
  • 22
    22. Oil extracted from seeds which are Brassica napus or Arabidopsis thaliana seeds, wherein
    • the oil comprises a total fatty acid content which comprises
    • 23. The oil of claim 22, wherein
      • γ-linolenic acid (GLA) is present in the extracted oil at a level of less than 3% of the total fatty acid content.
    • 24. The oil of claim 22, wherein
      • oleic acid is present in the extracted oil at a level of between 6% and 30% of the total fatty acid content.
    • 25. The oil of claim 22, wherein
      • the total saturated fatty acid content of the extracted oil is between 6% and 20% of the total fatty acid content.
    • 26. The oil of claim 22, comprising
      • new ω3 fatty acids in the total ω3 fatty acid content and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the extracted canola oil is between 0.1 and 1.
    • 27. The oil of claim 22, comprising
      • tri-DHA TAG (TAG 66:18).
    • 28. The oil of claim 22, wherein
      • at least 80% of the DHA esterified in the form of TAG is at the sn-1 or sn-3 position of the TAG.
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Description

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “170815_84199-AAAA Substitute_Sequence_Listing_DH,” which is 369 kilobytes in size, and which was created Aug. 15, 2017 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Aug. 15, 2017 as part of this application.

FIELD OF THE INVENTION

The present invention relates to extracted plant lipid, comprising fatty acids in an esterified form.

BACKGROUND OF THE INVENTION

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) are now widely recognized as important compounds for human and animal health. These fatty acids may be obtained from dietary sources or by conversion of linoleic (LA, 18:2ω6) or α-linolenic (ALA, 18:3ω3) fatty acids, both of which are regarded as essential fatty acids in the human diet. While humans and many other vertebrate animals are able to convert LA or ALA, obtained from plant sources to C22 they carry out this conversion at a very low rate. Moreover, most modern societies have imbalanced diets in which at least 90% of polyunsaturated fatty acids (PUFA) are of the ω6 fatty acids, instead of the 4:1 ratio or less for ω6:ω3 fatty acids that is regarded as ideal (Trautwein, 2001). The immediate dietary source of LC-PUFAs such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) for humans is mostly from fish or fish oil. Health professionals have therefore recommended the regular inclusion of fish containing significant levels of LC-PUFA into the human diet. Increasingly, fish-derived LC-PUFA oils are being incorporated into food products and in infant formula, for example. However, due to a decline in global and national fisheries, alternative sources of these beneficial health-enhancing oils are needed.

Flowering plants, in contrast to animals, lack the capacity to synthesise polyunsaturated fatty acids with chain lengths longer than 18 carbons. In particular, crop and horticultural plants along with other angiosperms do not have the enzymes needed to synthesize the longer chain ω3 fatty acids such as EPA, docosapentaenoic acid (DPA, 22:5ω3) and DHA that are derived from ALA. An important goal in plant biotechnology is therefore the engineering of crop plants which produce substantial quantities of LC-PUFA, thus providing an alternative source of these compounds.

LC-PUFA Biosynthesis Pathways

Biosynthesis of LC-PUFAs in organisms such as microalgae, mosses and fungi usually occurs as a series of oxygen-dependent desaturation and elongation reactions (FIG. 1). The most common pathway that produces EPA in these organisms includes a Δ6-desaturation, Δ6-elongation and Δ5-desaturation (termed the Δ6-desaturation pathway) whilst a less common pathway uses a Δ9-elongation, Δ8-desaturation and Δ5-desaturation (termed the Δ9-desaturation pathway). These consecutive desaturation and elongation reactions can begin with either the ω6 fatty acid substrate LA, shown schematically as the upper left part of FIG. 1 (ω6) or the ω3 substrate ALA through to EPA, shown as the lower right part of FIG. 1 (ω3). If the initial Δ6-desaturation is performed on the ω6 substrate LA, the LC-PUFA product of the series of three enzymes will be the ω6 fatty acid ARA. LC-PUFA synthesising organisms may convert ω6 fatty acids to ω3 fatty acids using an ω3-desaturase, shown as the Δ17-desaturase step in FIG. 1 for conversion of arachidonic acid (ARA, 20:4ω6) to EPA. Some members of the ω3-desaturase family can act on a variety of substrates ranging from LA to ARA. Plant ω3-desaturases often specifically catalyse the Δ15-desaturation of LA to ALA, while fungal and yeast ω3-desaturases may be specific for the Δ17-desaturation of ARA to EPA (Pereira et al., 2004a; Zank et al., 2005). Some reports suggest that non-specific ω3-desaturases may exist which can convert a wide variety of ω6 substrates to their corresponding ω3 products (Zhang et al., 2008).

The conversion of EPA to DHA in these organisms occurs by a Δ5-elongation of EPA to produce DPA, followed by a Δ4-desaturation to produce DHA (FIG. 1). In contrast, mammals use the so-called “Sprecher” pathway which converts DPA to DHA by three separate reactions that are independent of a Δ4-desaturase (Sprecher et al., 1995).

The front-end desaturases generally found in plants, mosses, microalgae, and lower animals such as Caenorhabditis elegans predominantly accept fatty acid substrates esterified to the sn-2 position of a phosphatidylcholine (PC) substrate. These desaturases are therefore known as acyl-PC, lipid-linked, front-end desaturases (Domergue et al., 2003). In contrast, higher animal front-end desaturases generally accept acyl-CoA substrates where the fatty acid substrate is linked to CoA rather than PC (Domergue et al., 2005). Some microalgal desaturases and one plant desaturase are known to use fatty acid substrates esterified to CoA (Table 2).

Each PUFA elongation reaction consists of four steps catalysed by a multi-component protein complex: first, a condensation reaction results in the addition of a 2C unit from malonyl-CoA to the fatty acid, resulting in the formation of a β-ketoacyl intermediate. This is then reduced by NADPH, followed by a dehydration to yield an enoyl intermediate. This intermediate is finally reduced a second time to produce the elongated fatty acid. It is generally thought that the condensation step of these four reactions is substrate specific whilst the other steps are not. In practice, this means that native plant elongation machinery is capable of elongating PUFA providing that the condensation enzyme (typically called an ‘elongase’) specific to the PUFA is introduced, although the efficiency of the native plant elongation machinery in elongating the non-native PUFA substrates may be low. In 2007 the identification and characterisation of the yeast elongation cycle dehydratase was published (Denic and Weissman, 2007).

PUFA desaturation in plants, mosses and microalgae naturally occurs to fatty acid substrates predominantly in the acyl-PC pool whilst elongation occurs to substrates in the acyl-CoA pool. Transfer of fatty acids from acyl-PC molecules to a CoA carrier is performed by phospholipases (PLAs) whilst the transfer of acyl-CoA fatty acids to a PC carrier is performed by lysophosphatidyl-choline acyltransferases (LPCATs) (FIG. 21) (Singh et al., 2005).

Engineered Production of LC-PUFA

Most LC-PUFA metabolic engineering has been performed using the aerobic Δ6-desaturation/elongation pathway. The biosynthesis of γ-linolenic acid (GLA, 18:3ω6) in tobacco was first reported in 1996 using a Δ6-desaturase from the cyanobacterium Synechocystis (Reddy and Thomas, 1996). More recently, GLA has been produced in crop plants such as safflower (73% GLA in seedoil; Knauf et al., 2006) and soybean (28% GLA; Sato et al., 2004). The production of LC-PUFA such as EPA and DHA involves more complicated engineering due to the increased number of desaturation and elongation steps involved. EPA production in a land plant was first reported by Qi et al. (2004) who introduced genes encoding a Δ9-elongase from Isochrysis galbana, a Δ8-desaturase from Euglena gracilis and a Δ5-desaturase from Mortierella alpina into Arabidopsis yielding up to 3% EPA. This work was followed by Abbadi et al. (2004) who reported the production of up to 0.8% EPA in flax seed using genes encoding a Δ6-desaturase and Δ6-elongase from Physcomitrella patens and a Δ5-desaturase from Phaeodactylum tricornutum.

The first report of DHA production, and to date the highest levels of VLC-PUFA production reported, was in WO 04/017467 where the production of 3% DHA in soybean embryos is described, but not seed, by introducing genes encoding the Saprolegnia diclina Δ6-desaturase, Mortierella alpina Δ6-desaturase, Mortierella alpina Δ5-desaturase, Saprolegnia diclina Δ4-desaturase, Saprolegnia diclina Δ17-desaturase, Mortierella alpina Δ6-elongase and Pavlova lutheri Δ5-elongase. The maximal EPA level in embryos also producing DHA was 19.6%, indicating that the efficiency of conversion of EPA to DHA was poor (WO 2004/071467). This finding was similar to that published by Robert et al. (2005), where the flux from EPA to DHA was low, with the production of 3% EPA and 0.5% DHA in Arabidopsis using the Danio rerio Δ5/6-desaturase, the Caenorhabditis elegans Δ6-elongase, and the Pavlova salina Δ5-elongase and Δ4-desaturase. Also in 2005, Wu et al. published the production of 25% ARA, 15% EPA, and 1.5% DHA in Brassica juncea using the Pythium irregulare Δ6-desaturase, a Thraustochytrid Δ5-desaturase, the Physcomitrella patens Δ6-elongase, the Calendula officianalis Δ12-desaturase, a Thraustochytrid Δ5-elongase, the Phytophthora infestans Δ17-desaturase, the Oncorhyncus mykiss LC-PUFA elongase, a Thraustochytrid Δ4-desaturase and a Thraustochytrid LPCAT (Wu et al., 2005). Summaries of efforts to produce oil-seed crops which synthesize ω3 LC-PUFAs is provided in Venegas-Caleron et al. (2010) and Ruiz-Lopez et al. (2012). As indicated by Ruiz-Lopez et al. (2012), results obtained to date for the production of DHA in transgenic plants has been no where near the levels seen in fish oils.

There therefore remains a need for more efficient production of LC-PUFA in recombinant cells, in particular of DHA in seeds of oilseed plants.

SUMMARY OF THE INVENTION

The present inventors have identified methods and plants for producing lipid with high levels of DHA.

In a first aspect, the present invention provides extracted plant lipid, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%.

In an embodiment, the extracted lipid has one or more or all of the following features

    • i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between about 2% and 18%, between about 2% and 16%, or between about 2% and 15%,
    • ii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than about 6%, less than about 3%, less than about 2%, or less than about 1%,
    • iii) the level of oleic acid in the total fatty acid content of the extracted lipid is between about 1% and about 30%, between about 3% and about 30%, between about 6% and about 30%, between 1% and about 20%, between about 30% and about 60%, between about 45% to about 60%, or is about 30%,
    • iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 4% and about 20%, or between about 4% and 17%,
    • v) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between about 4% and about 40%, between about 7% and about 40%, between about 10% and about 35%, between about 20% and about 35%, between about 4% and about 16%, or between about 2% and about 16%,
    • vi) the level of γ-linolenic acid (GLA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, between 0.05% and about 7%, between 0.05% and about 4%, between 0.05% and about 3%, or between 0.05% and about 2%,
    • vii) the level of stearidonic acid (SDA) in the total fatty acid content of the extracted lipid is less than about 7%, less than about 6%, less than about 4%, less than about 3%, between about 0.05% and about 7%, between about 0.05% and about 6%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between 0.05% and about 2%,
    • viii) the level of eicosatetraenoic acid (ETA) in the total fatty acid content of the extracted lipid is less than about 6%, less than about 5%, less than about 4%, less than about 1%, less than about 0.5%, between about 0.05% and about 6%, between about 0.05% and about 5%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between about 0.05% and about 2%,
    • ix) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 2%, less than about 1%, between about 0.05% and about 4%, between about 0.05% and about 3%, between about 0.05% and about 2%, or between about 0.05% and about 1%,
    • x) the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, less than about 2%, between about 0.05% and about 10%, between about 0.05% and about 5%, between about 0.05% and about 3%, or between about 0.05% and about 2%,
    • xi) the level of docosapentaenoic acid (DPA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, less than about 2%, between about 0.05% and about 8%, between about 0.05% and about 5%, between about 0.05% and about 3%, or between about 0.05% and about 2%,
    • xii) the level of DHA in the total fatty acid content of the extracted lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, between about 8% and 20%, between about 10% and 20%, between about 11% and 20%, between about 10% and about 16%, or between about 14% and 20%,
    • xiii) the lipid comprises ω6-docosapentaenoic acid (22:5Δ4,7,10,13,16) in its fatty acid content,
    • xiv) the lipid is essentially free of ω6-docosapentaenoic acid (22:5Δ4,7,10,13,16) in its fatty acid content,
    • xv) the lipid is essentially free of SDA, EPA and ETA in its fatty acid content,
    • xvi) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, between about 4% and about 20%, between about 6% and about 20%, between about 4% and about 60%, between about 30% and about 60%, or between about 45% and about 60%,
    • xvii) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 8% and about 25%, or between 8% and about 22%,
    • xviii) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, between about 50% and about 75%, or between about 60% and about 75%,
    • xix) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 35% and about 50%, between about 20% and about 35%, between about 6% and 20%, less than about 20%, less than about 16%, less than about 10%, between about 1% and about 16%, between about 2% and about 10%, or between about 4% and about 10%,
    • xx) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10%, less than about 8%, less than about 6%, less than 4%, between about 1% and about 20%, between about 1% and about 10%, between about 0.5% and about 8%, or between about 0.5% and 4%,
    • xxi) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 65%, between about 40% and about 60%, between about 20% and about 35%, between about 10% and about 20%, about 25%, about 30%, about 35% or about 40%,
    • xxii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 9% and about 33%, between about 10% and about 20%, between about 20% and about 30%, between about 12% and about 25%, about 13%, about 15%, about 17% or about 20%,
    • xxiii) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1 or about 0.2,
    • xxiv) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.1, about 0.2 or about 1.0,
    • xxv) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to LA by Δ12-desaturase of at least about 60%, at least about 70%, at least about 80%, between about 60% and about 98%, between about 70% and about 95%, or between about 75% and about 90%,
    • xxvi) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to SDA by Δ6-desaturase of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, between about 30% and about 70%, between about 35% and about 60%, or between about 50% and about 70%,
    • xxvii) the fatty acid composition of the lipid is based on an efficiency of conversion of SDA to ETA acid by Δ6-elongase of at least about 60%, at least about 70%, at least about 75%, between about 60% and about 95%, between about 70% and about 88%, or between about 75% and about 85%,
    • xxviii) the fatty acid composition of the lipid is based on an efficiency of conversion of ETA to EPA by Δ5-desaturase of at least about 60%, at least about 70%, at least about 75%, between about 60% and about 99%, between about 70% and about 99%, or between about 75% and about 98%,
    • xxix) the fatty acid composition of the lipid is based on an efficiency of conversion of EPA to DPA by Δ5-elongase of at least about 80%, at least about 85%, at least about 90%, between about 50% and about 95%, or between about 85% and about 95%,
    • xxx) the fatty acid composition of the lipid is based on an efficiency of conversion of DPA to DHA by Δ4-desaturase of at least about 80%, at least about 90%, at least about 93%, between about 50% and about 95%, between about 80% and about 95%, or between about 85% and about 95%,
    • xxxi) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to DHA of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,
    • xxxii) the fatty acid composition of the lipid is based on an efficiency of conversion of LA to DHA of at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,
    • xxxiii) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to DHA of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,
    • xxxiv) the total fatty acid in the extracted lipid has less than 1% C20:1,
    • xxxv) the triacylglycerol (TAG) content of the lipid is at least about 70%, at least about 80%, at least about 90%, at least 95%, between about 70% and about 99%, or between about 90% and about 99%,
    • xxxvi) the lipid comprises diacylglycerol (DAG),
    • xxxvii) the lipid comprises less than about 10%, less than about 5%, less than about 1%, or between about 0.001% and about 5%, free (non-esterified) fatty acids and/or phospholipid, or is essentially free thereof,
    • xxxviii) at least 70%, or at least 80%, of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG,
    • xxxix) the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12), and
    • xl) the lipid comprises tri-DHA TAG (TAG 66:18).

In another embodiment, the extracted lipid is in the form of an oil, wherein at least about 90%, or least about 95%, at least about 98%, or between about 95% and about 98%, by weight of the oil is the lipid.

In a preferred embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid or oil, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 6%, the level of oleic acid is between about 1% and about 30%, the level of LA is between about 4% and about 35%, ALA is present, GLA is present, the level of SDA is between about 0.05% and about 7%, the level of ETA is less than about 4%, the level of EPA is between about 0.05% and about 10%, the level of DPA is between about 0.05% and about 8%, the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0, preferably less than about 0.50, the fatty acid composition of the lipid is based on: an efficiency of conversion of oleic acid to LA by Δ12-desaturase of at least about 60%, an efficiency of conversion of SDA to ETA acid by Δ6-elongase of at least about 60%, an efficiency of conversion of EPA to DPA by Δ5-elongase of between about 50% and about 95%, an efficiency of conversion of DPA to DHA by Δ4-desaturase of between about 50% and about 95%, an efficiency of conversion of oleic acid to DHA of at least about 10%, and the triacylglycerol (TAG) content of the lipid is at least about 70%, and optionally the lipid is essentially free of cholesterol and/or the lipid comprises tri-DHA TAG (TAG 66:18).

In a more preferred embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 2%, the level of oleic acid is between about 1% and about 30%, the level of LA is between about 4% and about 35%, the level of ALA is between about 7% and about 40%, the level of GLA is less than about 4%, the level of SDA is between about 0.05% and about 7%, the level of ETA is less than about 4%, the level of ETrA is between about 0.05% and about 4%, the level of EPA is between about 0.05% and about 10%, the level of DPA is between about 0.05% and about 8%, the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%, the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 75%, the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 9% and about 33%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0, the fatty acid composition of the lipid is based on: an efficiency of conversion of oleic acid to LA by Δ12-desaturase of at least about 60%, an efficiency of conversion of SDA to ETA acid by Δ6-elongase of at least about 60%, an efficiency of conversion of ETA to EPA by Δ5-desaturase of at least about 60%, an efficiency of conversion of EPA to DPA by Δ5-elongase of between about 50% and about 95%, an efficiency of conversion of DPA to DHA by Δ4-desaturase of between about 50% and about 95%, an efficiency of conversion of oleic acid to DHA of at least about 10%, an efficiency of conversion of LA to DHA of at least about 15%, an efficiency of conversion of ALA to DHA of at least about 17%, and the total fatty acid content in the extracted lipid has less than 1% C20:1, the triacylglycerol (TAG) content of the lipid is at least about 70%, the lipid is essentially free of cholesterol, and the lipid comprises tri-DHA TAG (TAG 66:18). Preferably, the lipid or oil is canola oil and/or has not been treated with a transesterification process after it was extracted from the plant or plant part. In a particular embodiment, the lipid or canola oil may subsequently be treated to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters. Further treatment may be applied to enrich the lipid or oil for the DHA.

In an embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 2%, the level of oleic acid is between about 30% and about 60%, preferably between about 45% and about 60%, the level of LA is between about 4% and about 20%, the level of ALA is between about 2% and about 16%, the level of GLA is less than about 3%, the level of SDA is less than about 3%, the level of ETA is less than about 4%, the level of ETrA less than about 2%, the level of EPA is less than about 4%, the level of DPA is less than about 4%, the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 30% and about 60%, or between about 40% and about 60%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%, the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 10% and about 20%, the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 9% and about 20%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, preferably less than about 0.50, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0, the triacylglycerol (TAG) content of the lipid is at least about 70%, the lipid is essentially free of cholesterol, and the lipid comprises tri-DHA TAG (TAG 66:18). Preferably, the lipid or oil is essentially free of SDA, EPA and ETA and/or is canola oil and/or has not been treated with a transesterification process after it was extracted from the plant or plant part. In a particular embodiment, the lipid or canola oil may subsequently be treated to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters. Further treatment may be applied to enrich the lipid or oil for the DHA.

In a further preferred embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid or oil, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 6%, the level of oleic acid is between about 1% and about 30%, the level of LA is between about 4% and about 35%, ALA is present, GLA is present, the level of SDA is between about 0.05% and about 7%, the level of ETA is less than about 6%, the level of EPA is between about 0.05% and about 10%, the level of DPA is between about 0.05% and about 8%.

In a further embodiment, the extracted lipid further comprises one or more sterols, preferably plant sterols.

In another embodiment, the extracted lipid is in the form of an oil, and comprises less than about 10 mg of sterols/g of oil, less than about 7 mg of sterols/g of oil, between about 1.5 mg and about 10 mg of sterols/g of oil, or between about 1.5 mg and about 7 mg of sterols/g of oil.

Examples of sterols which can be in the extracted lipid include, but are not necessarily limited to, one or more or all of campesterol/24-methylcholesterol, Δ5-stigmasterol, eburicol, β-sitosterol/24-ethylcholesterol, Δ5-avenasterol/isofucosterol, Δ7-stigmasterol/stigmast-7-en-3β-ol, and Δ7-avenasterol.

In an embodiment, the plant species is one listed in Table 26, such as canola, and the level of sterols are about the same as that listed in Table 26 for that particular plant species.

In an embodiment, the extracted lipid comprises less than about 0.5 mg of cholesterol/g of oil, less than about 0.25 mg of cholesterol/g of oil, between about 0 mg and about 0.5 mg of cholesterol/g of oil, or between about 0 mg and about 0.25 mg of cholesterol/g of oil, or which is essentially free of cholesterol.

In a further embodiment, the lipid is an oil, preferably oil from an oilseed. Examples of such oils include, but are not limited to, Brassica sp. oil such as canola oil, Gossypium hirsutum oil, Linum usitatissimum oil, Helianthus sp. oil, Carthamus tinctorius oil, Glycine max oil, Zea mays oil, Arabidopsis thaliana oil, Sorghum bicolor oil, Sorghum vulgare oil, Avena sativa oil, Trifolium sp. oil, Elaesis guineenis oil, Nicotiana benthamiana oil, Hordeum vulgare oil, Lupinus angustifolius oil, Oryza sativa oil, Oryza glaberrima oil, Camelina sativa oil, Crambe abyssinica oil, Miscanthus×giganteus oil, or Miscanthus sinensis oil.

Also provided is extracted plant lipid, preferably extracted canola seedoil, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein lipid has the following features in the total fatty acid content of the lipid;

i) the level of DHA is about 3%, about 4%, about 5%, about 6% or about 7%,

ii) the level of palmitic acid is between about 2% and about 16%,

iii) the level of myristic acid is less than about 2%,

iv) the level of oleic acid is between about 30% and about 60%, preferably between about 45% and about 60%,

v) the level of LA is between about 4% and about 20%,

vi) the level of ALA is between about 2% and about 16%,

vii) the level of GLA is less than about 4%,

viii) the level of SDA is less than about 6%, or less than about 4%,

ix) the level of ETA is less than about 6%, or less than about 4%,

x) the level of ETrA less than about 1%,

xi) the level of EPA is less than about 10% and/or the level of EPA is 0.5-2.0 fold the level of DHA,

xii) the level of DPA is less than about 4%,

xiii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%,

xiv) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 30% and about 70%,

xv) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 15% and about 75%, preferably between about 15% and about 30%,

xvi) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%,

xvii) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 10% and about 20%,

xviii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 3% and about 20%,

xix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, preferably less than about 0.50,

xx) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0,

xxi) the triacylglycerol (TAG) content of the lipid is at least about 70%, and

xxii) the lipid is essentially free of cholesterol. In an embodiment, the lipid comprises tri-DHA TAG (TAG 66:18). More preferably, the lipid is essentially free of SDA and ETA, and/or has not been treated with a transesterification process after it was extracted from the plant or plant part.

In another aspect, provided is extracted plant lipid, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoic acid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHA in the total fatty acid content of the extracted lipid is between 7% and 20%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (iii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 6%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is between 1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between 4% and 40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than 4%, (viii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between 1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG. In an embodiment, one or more or all of the following features

    • i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 15%,
    • ii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 1%,
    • iii) the level of oleic acid in the total fatty acid content of the extracted lipid is between about 3% and about 30%, between about 6% and about 30%, between 1% and about 20%, between about 45% and about 60%, or is about 30%,
    • iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 4% and 17%,
    • v) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between about 7% and about 40%, between about 10% and about 35%, between about 20% and about 35%, or between about 4% and 16%,
    • vi) the level of γ-linolenic acid (GLA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, between 0.05% and 7%, between 0.05% and 4%, or between 0.05% and about 3%, or between 0.05% and about 2%,
    • vii) the level of stearidonic acid (SDA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, between about 0.05% and about 7%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between 0.05% and about 2%,
    • viii) the level of eicosatetraenoic acid (ETA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 1%, less than about 0.5%, between about 0.05% and about 5%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between about 0.05% and about 2%,
    • ix) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than about 2%, less than about 1%, between 0.05% and 4%, between 0.05% and 3%, or between 0.05% and about 2%, or between 0.05% and about 1%,
    • x) the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 10%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,
    • xi) the level of docosapentaenoic acid (DPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 8%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,
    • xii) the level of DHA in the total fatty acid content of the extracted lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, between about 8% and 20%, between about 10% and 20%, between about 11% and 20%, between about 10% and about 16%, or between about 14% and 20%,
    • xiii) the lipid comprises ω6-docosapentaenoic acid (22:5Δ4,7,10,13,16) in its fatty acid content,
    • xiv) the lipid is essentially free of ω6-docosapentaenoic acid (22:5Δ4,7,10,13,16) in its fatty acid content,
    • xv) the lipid is essentially free of SDA, EPA and ETA in its fatty acid content,
    • xvi) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 6% and about 20%,
    • xvii) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 8% and about 25%, or between 8% and about 22%,
    • xviii) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, between about 50% and about 75%, or between about 60% and about 75%,
    • xix) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 35% and about 50%, between about 20% and about 35%, between about 6% and 20%, less than 20%, less than about 16%, less than about 10%, between about 1% and about 16%, between about 2% and about 10%, or between about 4% and about 10%,
    • xx) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10%, less than about 8%, less than about 6%, less than 4%, between about 1% and about 20%, between about 1% and about 10%, between about 0.5% and about 8%, or between about 0.5% and 4%,
    • xxi) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 65%, between 40% and about 60%, between about 20% and about 35%, between about 10% and about 20%, about 25%, about 30%, about 35% or about 40%,
    • xxii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between 9% and about 33%, between about 10% and about 20%, between about 20% and about 30%, between about 12% and about 25%, about 13%, about 15%, about 17% or about 20%,
    • xxiii) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1 or about 0.2,
    • xxiv) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.1, about 0.2 or about 1.0,
    • xxv) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to DHA of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,
    • xxvi) the fatty acid composition of the lipid is based on an efficiency of conversion of LA to DHA of at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,
    • xxvii) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to DHA of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,
    • xxviii) the total fatty acid in the extracted lipid has less than 1% C20:1,
    • xxix) the triacylglycerol (TAG) content of the lipid is at least about 80%, at least about 90%, at least 95%, between about 70% and about 99%, or between about 90% and about 99%,
    • xxx) the lipid comprises diacylglycerol (DAG),
    • xxxi) the lipid comprises less than about 10%, less than about 5%, less than about 1%, or between about 0.001% and about 5%, free (non-esterified) fatty acids and/or phospholipid, or is essentially free thereof,
    • xxxii) at least 80%, of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG,
    • xxxiii) the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12), and
    • xxxiv) the lipid comprises tri-DHA TAG (TAG 66:18).

With specific regard to the above aspect, in an embodiment

i) the lipid is in the form of an oil, wherein the oil comprises one or more sterols such as one or more or all of campesterol, Δ5-stigmasterol, eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol and Δ7-avenasterol, and optionally the oil comprises less than 10 mg of sterols/g of oil and/or the oil is essentially free of cholesterol, and/or

ii) the lipid is in the form of an oil from an oilseed such as oilseed is a Brassica sp oilseed or canola seed.

In another aspect, the present invention provides a process for producing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of eicosapentaenoic acid (EPA), stearidonic acid (SDA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of extractable lipid in the plant part is about 7% to 20%, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%.

In a preferred embodiment, the extracted lipid has one or more of the features defined above.

In an embodiment, wherein the plant part is a seed, preferably an oilseed. Examples of such seeds include, but are not limited to, Brassica sp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, or Crambe abyssinica, preferably a Brassica napus, B. juncea or C. sativa seed.

In another embodiment, the seed comprises at least about 18 mg, at least about 22 mg, at least about 26 mg, between about 18 mg and about 100 mg, between about 22 mg and about 70 mg, or between about 24 mg and about 50 mg, of DHA per gram of seed.

In a further embodiment, the plant part comprises exogenous polynucleotides encoding one of the following sets of enzymes;

i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and a Δ5-elongase,

vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase, or

viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

and wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in a cell of the plant part.

In yet a further embodiment, the plant part has one or more or all of the following features

i) the Δ12-desaturase converts oleic acid to linoleic acid in one or more cells of the plant with an efficiency of at least about 60%, at least about 70%, at least about 80%, between about 60% and about 98%, between about 70% and about 95%, or between about 75% and about 90%,

ii) the ω3-desaturase converts ω6 fatty acids to ω3 fatty acids in one or more cells of the plant with an efficiency of at least about 65%, at least about 75%, at least about 85%, between about 65% and about 95%, between about 75% and about 95%, or between about 80% and about 95%,

iii) the Δ6-desaturase converts ALA to SDA in one or more cells of the plant with an efficiency of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, between about 30% and about 70%, between about 35% and about 60%, or between about 50% and about 70%,

iv) the Δ6-desaturase converts linoleic acid to γ-linolenic acid in one or more cells of the plant with an efficiency of less than about 5%, less than about 2.5%, less than about 1%, between about 0.1% and about 5%, between about 0.5% and about 2.5%, or between about 0.5% and about 1%,

v) the Δ6-elongase converts SDA to ETA in one or more cells of the plant with an efficiency of at least about 60%, at least about 70%, at least about 75%, between about 60% and about 95%, between about 70% and about 88%, or between about 75% and about 85%,

vi) the Δ5-desaturase converts ETA to EPA in one or more cells of the plant with an efficiency of at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, between about 60% and about 99%, between about 70% and about 99%, or between about 75% and about 98%,

vii) the Δ5-elongase converts EPA to DPA in one or more cells of the plant with an efficiency of at least about 80%, at least about 85%, at least about 90%, between about 50% and about 95%, or between about 85% and about 95%,

viii) the Δ4-desaturase converts DPA to DHA in one or more cells of the plant with an efficiency of at least about 80%, at least about 90%, at least about 93%, between about 50% and about 95%, between about 80% and about 95%, or between about 85% and about 95%,

ix) the efficiency of conversion of oleic acid to DHA in one or more cells of the plant part is at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,

x) the efficiency of conversion of LA to DHA in one or more cells of the plant part is at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,

xi) the efficiency of conversion of ALA to DHA in one or more cells of the plant part is at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,

xii) one or more cells of the plant part comprise at least about 15%, at least about 20%, between about 15% and about 30%, or between about 22.5% and about 27.5%, more ω3 fatty acids than corresponding cells lacking the exogenous polynucleotides,

xiii) the Δ6-desaturase preferentially desaturates α-linolenic acid (ALA) relative to linoleic acid (LA),

xiv) the Δ6-elongase also has Δ9-elongase activity,

xv) the Δ12-desaturase also has Δ15-desaturase activity,

xvi) the Δ6-desaturase also has Δ8-desaturase activity,

xvii) the Δ8-desaturase also has Δ6-desaturase activity or does not have Δ6-desaturase activity,

xviii) the Δ15-desaturase also has ω3-desaturase activity on GLA,

xix) the ω3-desaturase also has Δ15-desaturase activity on LA,

xx) the ω3-desaturase desaturates both LA and/or GLA,

xxi) the ω3-desaturase preferentially desaturates GLA relative to LA,

xxii) the level of DHA in the plant part is based on an efficiency of conversion of oleic acid to DHA in the plant part of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 15% and about 30%, or between about 20% and about 25%,

xxiii) the level of DHA in the plant part is based on an efficiency of conversion of LA to DHA in the plant part of at least about 15%, at least about 20%, at least about 22%, between about 15% and about 60%, between about 20% and about 40%, or between about 22% and about 30%,

xxiv) the level of DHA in the plant part is based on an efficiency of conversion of ALA to DHA in the plant part of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 65%, between about 22% and about 35%, or between about 24% and about 35%

xxx) one or more or all of the desaturases have greater activity on an acyl-CoA substrate than a corresponding acyl-PC substrate,

xxxi) the Δ6-desaturase has greater Δ6-desaturase activity on ALA than LA as fatty acid substrate,

xxxii) the Δ6-desaturase has greater Δ6-desaturase activity on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate,

xxxiii) the Δ6-desaturase has at least about a 2-fold greater Δ6-desaturase activity, at least 3-fold greater activity, at least 4-fold greater activity, or at least 5-fold greater activity, on ALA as a substrate compared to LA,

xxxiv) the Δ6-desaturase has greater activity on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate,

xxxv) the Δ6-desaturase has at least about a 5-fold greater Δ6-desaturase activity or at least 10-fold greater activity, on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate,

xxxvi) the desaturase is a front-end desaturase,

xxxvii) the Δ6-desaturase has no detectable Δ5-desaturase activity on ETA.

In yet a further embodiment, the plant part has one or more or all of the following features

i) the Δ12-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:10, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:10,

ii) the ω3-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:12, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:12,

iii) the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:16, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:16,

iv) the Δ6-elongase comprises amino acids having a sequence as provided in SEQ ID NO:25, a biologically active fragment thereof such as SEQ ID NO:26, or an amino acid sequence which is at least 50% identical to SEQ ID NO:25 and/or SEQ ID NO:26,

v) the Δ5-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:30, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:30,

vi) the Δ5-elongase comprises amino acids having a sequence as provided in SEQ ID NO:37, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:37,

vii) the Δ4-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:41, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:41.

In an embodiment, the plant part further comprises an exogenous polynucleotide encoding a diacylglycerol acyltransferase (DGAT), monoacylglycerol acyltransferase (MGAT), glycerol-3-phosphate acyltransferase (GPAT), 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) preferably an LPAAT which can use a C22 polyunsaturated fatty acyl-CoA substrate, acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT), phospholipase A2 (PLA2), phospholipase C (PLC), phospholipase D (PLD), CDP-choline diacylglycerol choline phosphotransferase (CPT), phoshatidylcholine diacylglycerol acyltransferase (PDAT), phosphatidylcholine:diacylglycerol choline phosphotransferase (PDCT), acyl-CoA synthase (ACS), or a combination of two or more thereof.

In another embodiment, the plant part further comprises an introduced mutation or an exogenous polynucleotide which down regulates the production and/or activity of an endogenous enzyme in a cell of the plant part selected from FAE1, DGAT, MGAT, GPAT, LPAAT, LPCAT, PLA2, PLC, PLD, CPT, PDAT, a thioesterase such as FATB, or a Δ12-desaturase, or a combination of two or more thereof.

In a further embodiment, at least one, or all, of the promoters are seed specific promoters. In an embodiment, at least one, or all, of the promoters have been obtained from oil biosynthesis or accumulation genes such as oleosin, or from seed storage protein genes such as conlinin.

In another embodiment, the promoter(s) directing expression of the exogenous polynucleotides encoding the Δ4-desaturase and the Δ5-elongase initiate expression of the polynucleotides in developing seed of the plant part before, or reach peak expression before, the promoter(s) directing expression of the exogenous polynucleotides encoding the Δ12-desaturase and the ω3-desaturase.

In a further embodiment, the exogenous polynucleotides are covalently linked in a DNA molecule, preferably a T-DNA molecule, integrated into the genome of cells of the plant part and preferably where the number of such DNA molecules integrated into the genome of the cells of the plant part is not more than one, two or three, or is two or three.

In yet another embodiment, the plant comprises at least two different, exogenous polynucleotides each encoding a Δ6-desaturase which have the same or different amino acid sequences.

In a further embodiment, the total oil content of the plant part comprising the exogenous polynucleotides is at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or between about 50% and about 80% of the total oil content of a corresponding plant part lacking the exogenous polynucleotides. In these embodiments, the maximum oil content may be about 100% of the oil content of a corresponding wild-type plant part.

In another embodiment, the lipid is in the form of an oil, preferably a seedoil from an oilseed, and wherein at least about 90%, or about least 95%, at least about 98%, or between about 95% and about 98%, by weight of the lipid is triacylglycerols.

In a further embodiment, the process further comprises treating the lipid to increase the level of DHA as a percentage of the total fatty acid content. For example, the treatment is transesterification. For example, the lipid such as canola oil may be treated to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters, which may then be fractionated to enrich the lipid or oil for the DHA.

Further, provided is a process for producing extracted plant lipid, comprising the steps of

i) obtaining a plant part, preferably canola seed, comprising lipid, the lipid comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of eicosapentaenoic acid (EPA), stearidonic acid (SDA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of extractable lipid in the plant part is about 3%, about 4%, about 5%, about 6% or about 7%, and

ii) extracting lipid from the plant part,

wherein the extracted lipid has the following features in the total fatty acid content of the lipid;

i) the level of DHA is about 3%, about 4%, about 5%, about 6% or about 7%,

ii) the level of palmitic acid is between about 2% and about 16%,

iii) the level of myristic acid is less than about 2%,

iv) the level of oleic acid is between about 30% and about 60%, preferably between about 45% and about 60%,

v) the level of LA is between about 4% and about 20%,

vi) the level of ALA is between about 2% and about 16%,

vii) the level of GLA is less than about 4%,

viii) the level of SDA is less than about 6%, or less than about 4%,

ix) the level of ETA is less than about 6%, or less than about 4%,

x) the level of ETrA less than about 1%,

xi) the level of EPA is less than about 10% and/or the level of EPA is 0.5-2.0 fold the level of DHA,

xii) the level of DPA is less than about 4%,

xiii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%,

xiv) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 30% and about 70%,

xv) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 15% and about 75%, preferably between about 15% and about 30%,

xvi) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%,

xvii) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 10% and about 20%,

xviii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 3% and about 20%,

xix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, preferably less than about 0.50,

xx) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0,

xxi) the triacylglycerol (TAG) content of the lipid is at least about 70%, and

xxii) the lipid is essentially free of cholesterol. In an embodiment, the lipid comprises tri-DHA TAG (TAG 66:18). More preferably, the lipid is essentially free of SDA and ETA, and/or has not been treated with a transesterification process after it was extracted from the plant or plant part.

Also provided is a process for producing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoic acid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHA in the total fatty acid content of the extracted lipid is between 7% and 20%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (iii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 6%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is between 1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between 4% and 40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than 4%, (viii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between 1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG.

%, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%.

Also provided is lipid, or oil comprising the lipid, produced using a process of the invention.

In another aspect, the present invention provides a process for producing ethyl esters of polyunsaturated fatty acids, the process comprising transesterifying triacylglycerols in extracted plant lipid, wherein the extracted plant lipid comprises fatty acids esterified in the form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenicacid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%, thereby producing the ethyl esters.

In a preferred embodiment, the extracted lipid has one or more of the features defined above.

In another aspect, the present invention provides a process for producing ethyl esters of polyunsaturated fatty acids, the process comprising transesterifying triacylglycerols in extracted plant lipid, wherein the extracted plant lipid comprises fatty acids esterified in the form of the triacylglycerols, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoic acid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHA in the total fatty acid content of the extracted lipid is about 3%, about 4%, about 5%, about 6% or between 7% and 20%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (iii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 6%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is between 1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between 4% and 40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than 4%, (viii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between 1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG, thereby producing the ethyl esters. In an embodiment, the extracted plant lipid has one or more or all of the following features

    • i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 15%,
    • ii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 1%,
    • xxxv) the level of oleic acid in the total fatty acid content of the extracted lipid is between about 3% and about 30%, between about 6% and about 30%, between 1% and about 20%, between about 45% and about 60%, or is about 30%,
    • xxxvi) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 4% and 17%,
    • xxxvii) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between about 7% and about 40%, between about 10% and about 35%, between about 20% and about 35%, or between about 4% and 16%,
    • xxxviii) the level of γ-linolenic acid (GLA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, between 0.05% and 7%, between 0.05% and 4%, or between 0.05% and about 3%, or between 0.05% and about 2%,
    • xxxix) the level of stearidonic acid (SDA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, between about 0.05% and about 7%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between 0.05% and about 2%,
    • xl) the level of eicosatetraenoic acid (ETA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 1%, less than about 0.5%, between about 0.05% and about 5%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between about 0.05% and about 2%,
    • xli) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than about 2%, less than about 1%, between 0.05% and 4%, between 0.05% and 3%, or between 0.05% and about 2%, or between 0.05% and about 1%,
    • xlii) the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 10%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,
    • xliii) the level of docosapentaenoic acid (DPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 8%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,
    • xliv) the level of DHA in the total fatty acid content of the extracted lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, between about 8% and 20%, between about 10% and 20%, between about 11% and 20%, between about 10% and about 16%, or between about 14% and 20%,
    • xlv) the lipid comprises ω6-docosapentaenoic acid (22:5Δ4,7,10,13,16) in its fatty acid content,
    • xlvi) the lipid is essentially free of ω6-docosapentaenoic acid (22:5Δ4,7,10,13,16) in its fatty acid content,
    • xlvii) the lipid is essentially free of SDA, EPA and ETA in its fatty acid content,
    • xlviii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 6% and about 20%,
    • xlix) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 8% and about 25%, or between 8% and about 22%,
    • l) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, between about 50% and about 75%, or between about 60% and about 75%,
    • li) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 35% and about 50%, between about 20% and about 35%, between about 6% and 20%, less than 20%, less than about 16%, less than about 10%, between about 1% and about 16%, between about 2% and about 10%, or between about 4% and about 10%,
    • lii) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10%, less than about 8%, less than about 6%, less than 4%, between about 1% and about 20%, between about 1% and about 10%, between about 0.5% and about 8%, or between about 0.5% and 4%,
    • liii) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 65%, between 40% and about 60%, between about 20% and about 35%, between about 10% and about 20%, about 25%, about 30%, about 35% or about 40%,
    • liv) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between 9% and about 33%, between about 10% and about 20%, between about 20% and about 30%, between about 12% and about 25%, about 13%, about 15%, about 17% or about 20%,
    • lv) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1 or about 0.2,
    • lvi) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.1, about 0.2 or about 1.0,
    • lvii) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to DHA of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,
    • lviii) the fatty acid composition of the lipid is based on an efficiency of conversion of LA to DHA of at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,
    • lix) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to DHA of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,
    • lx) the total fatty acid in the extracted lipid has less than 1% C20:1,
    • lxi) the triacylglycerol (TAG) content of the lipid is at least about 80%, at least about 90%, at least 95%, between about 70% and about 99%, or between about 90% and about 99%,
    • lxii) the lipid comprises diacylglycerol (DAG),
    • lxiii) the lipid comprises less than about 10%, less than about 5%, less than about 1%, or between about 0.001% and about 5%, free (non-esterified) fatty acids and/or phospholipid, or is essentially free thereof,
    • lxiv) at least 80%, of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG,
    • lxv) the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12), and
    • lxvi) the lipid comprises tri-DHA TAG (TAG 66:18).

With specific regard to the above aspect, in an embodiment one or more or all of the following apply

i) the lipid is in the form of an oil, wherein the oil comprises one or more sterols such as one or more or all of campesterol, Δ5-stigmasterol, eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol and Δ7-avenasterol, and optionally the oil comprises less than 10 mg of sterols/g of oil and/or the oil is essentially free of cholesterol,

ii) the lipid is in the form of an oil from an oilseed such as oilseed is a Brassica sp oilseed or canola seed,

iii) the level of DHA in the total fatty acid content of the extracted plant lipid is about 3%, about 4%, about 5%, about 6%, or is between 7% and 20%.

In a further aspect, the present invention provides a chimeric genetic construct comprising in order a first gene, a second gene, a third gene, a fourth gene, a fifth gene and a sixth gene which are all covalently linked on a single DNA molecule,

  • wherein the first, second and third genes are joined together as a first gene cluster and the fourth, fifth and sixth genes are joined together as a second gene cluster,
  • wherein each gene comprises a promoter, a coding region and a transcription terminator and/or polyadenylation region such that each promoter is operably linked to the coding region and transcription terminator and/or polyadenylation region,
  • wherein each promoter is independently identical or different to the other promoters such that the DNA molecule comprises three, four, five or six different promoters,
  • wherein one or more or all of the promoters are heterologous with respect to the coding region to which it is operably linked,
  • wherein the direction of transcription of the first gene is away from the third gene and opposite to the direction of transcription of the third gene,
  • wherein the direction of transcription of the fourth gene is away from the sixth gene and opposite to the direction of transcription of the sixth gene,
  • wherein the direction of transcription of the second gene is the same as for the first gene or the third gene,
  • wherein the direction of transcription of the fifth gene is the same as for the fourth gene or the sixth gene,
  • wherein the transcription terminator and/or polyadenylation region of the second gene is spaced apart from the promoter of the first or third genes, whichever is closer, by a first spacer region of between about 0.2 and about 3.0 kilobases,
  • wherein the first gene cluster is spaced apart from the second gene cluster by a second spacer region of between about 1.0 and about 10.0 kilobases, and
  • wherein the transcription terminator and/or polyadenylation region of the fifth gene is spaced apart from the promoter of the fourth or sixth genes, whichever is closer, by a third spacer region of between about 0.2 and about 3.0 kilobases.

In an embodiment, the DNA molecule comprises a seventh gene which is spaced apart from the first gene cluster or the second gene cluster, whichever is closer, by a spacer region of between about 1.0 and about 10.0 kilobases.

In another embodiment, the DNA molecule comprises two or more different transcription terminator and/or polyadenylation regions.

In yet a further embodiment, at least one of the spacer regions comprises a matrix attachment region (MAR).

In a further embodiment, the DNA molecule comprises right and left border regions flanking the genes and is a T-DNA molecule.

In another embodiment, the genetic construct is in an Agrobacterium cell or is integrated into the genome of a plant cell.

In a preferred embodiment, at least one of the genes encodes a fatty acid desaturase or a fatty acid elongase.

In another embodiment, the genetic construct comprises genes encoding a set of enzymes as defined herein, and/or wherein one or more of the genes encode an enzyme as defined herein.

In a further aspect, the present invention provides an isolated and/or exogenous polynucleotide comprising:

i) a sequence of nucleotides selected from any one of SEQ ID NOs: 1 to 9, 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, and/or

ii) a sequence of nucleotides which are at least 95% identical or 99% identical to one or more of the sequences set forth in SEQ ID NOs: 1 to 9, 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45.

In a particularly preferred embodiment, the isolated and/or exogenous polynucleotide comprises:

i) a sequence of nucleotides of SEQ ID NO: 2, and/or

ii) a sequence of nucleotides which are at least 95% identical or 99% identical to the sequence set forth in SEQ ID NO: 2.

In another aspect, the present invention provides a vector or genetic construct comprising the polynucleotide of the invention and/or the genetic construct of the invention.

In an embodiment, the sequence of nucleotides selected from any one of SEQ ID NOs: 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, or the sequence of nucleotides which is at least 95% identical or 99% identical to one or more of the sequences set forth in SEQ ID NOs: 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, is operably linked to a promoter.

In a further aspect, the present invention provides a host cell comprising exogenous polynucleotides encoding one of the following sets of enzymes;

i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and a Δ5-elongase,

vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase, or

viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

and wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in the cell.

In an embodiment, the cell comprises lipid as defined above, or wherein one or more or all of the desaturases or elongases have one or more of the features as defined above.

In another aspect, the present invention provides a host cell comprising

i) a first exogenous polynucleotide encoding a Δ12-desaturase which comprises amino acids having a sequence as provided in SEQ ID NO:10, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:10, and

ii) a second exogenous polynucleotide encoding a ω3-desaturase which comprises amino acids having a sequence as provided in SEQ ID NO:12, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:12,

wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in the cell.

In a further aspect, the present invention provides a host cell comprising one or more of the polynucleotide of the invention, the genetic construct of the invention, or the vector or genetic construct of the invention.

In an embodiment, the cell is in a plant, in a plant part and/or is a mature plant seed cell.

In an embodiment, the plant or plant seed is an oilseed plant or an oilseed, respectively.

Also provided is a transgenic non-human organism comprising a cell of the invention. Preferably, the transgenic non-human organism is a transgenic plant, preferably an oilseed plant or Arabidopsis thaliana. In an embodiment, the plant is a Brassica plant, preferably B. napus or B. juncea, or a plant other than Arabidopsis thaliana.

In another aspect, the present invention provides an oilseed plant comprising

a) lipid in its seed, the lipid comprising fatty acids in an esterified form, and

b) exogenous polynucleotides encoding one of the following sets of enzymes;

    • i) a Δ12-desaturase, a fungal ω3-desaturase and/or fungal Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase, or
    • ii) a Δ12-desaturase, a fungal ω3-desaturase and/or fungal Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

wherein each polynucleotide is operably linked to one or more seed-specific promoters that are capable of directing expression of said polynucleotides in developing seed of the plant, wherein the fatty acids comprise oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA) and γ-linolenic acid (GLA), ω3 fatty acids which comprise α-linolenic acid (ALA), stearidonic acid (SDA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), and optionally eicosapentaenoic acid (EPA) and/or eicosatetraenoic acid (ETA), and wherein the level of DHA in the total fatty acid content of the lipid is about 7% to 20%.

Examples of oilseed plants include, but are not limited to, Brassica sp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, or Crambe abyssinica. In an embodiment, the oilseed plant is a canola, Glycine max, Camelina sativa or Arabidopsis thaliana plant. In an alternate embodiment, the oilseed plant is other than A. thaliana.

In an embodiment, one or more of the desaturases is capable of using an acyl-CoA substrate. In a preferred embodiment, one or more of the Δ6-desaturase, Δ5-desaturase, Δ4-desaturase and Δ8-desaturase, if present, is capable of using an acyl-CoA substrate, preferably each of the i) Δ6-desaturase, Δ5-desaturase and Δ4-desaturase or ii) Δ5-desaturase, Δ4-desaturase and Δ8-desaturase is capable of using an acyl-CoA substrate. In an embodiment, a Δ12-desaturase and/or an ω3-desaturase is capable of using an acyl-CoA substrate. The acyl-CoA substrate is preferably an ALA-CoA, ETA-CoA, DPA-CoA, ETrA-CoA, LA-CoA, GLA-CoA, or ARA-CoA.

In an embodiment, mature, harvested seed of the plant has a DHA content of at least about 28 mg per gram seed, preferably at least about 32 mg per gram seed, at least about 36 mg per gram seed, at least about 40 mg per gram seed, more preferably at least about 44 mg per gram seed or at least about 48 mg per gram seed. The maximum DHA content may be about 80 to about 100 mg per gram seed, or about 80 mg or about 100 mg per gram seed.

In a further aspect, the present invention provides a Brassica napus, B. juncea or Camelina sativa plant which is capable of producing seed comprising DHA, wherein mature, harvested seed of the plant has a DHA content of at least about 28 mg per gram seed, preferably at least about 32 mg per gram seed, at least about 36 mg per gram seed, at least about 40 mg per gram seed, more preferably at least about 44 mg per gram seed or at least about 48 mg per gram seed. The maximum DHA content may be about 80 to about 100 mg per gram seed, or about 80 mg or about 100 mg per gram seed.

In another aspect, the present invention provides plant cell of a plant of the invention comprising the exogenous polynucleotides.

Also provided is a plant part, preferably a seed, which has one or more of the following features

i) is from a plant of the invention,

ii) comprises lipid as defined herein,

iii) can be used in a process of the invention,

iv) comprises a genetic construct of the invention, or

v) comprises a set of exogenous polynucleotides as defined herein.

In yet another aspect, the present invention provides mature, harvested Brassica napus, B. juncea or Camelina sativa seed comprising DHA and a moisture content of between about 4% and about 15% by weight, wherein the DHA content of the seed at least about 28 mg per gram seed, preferably at least about 32 mg per gram seed, at least about 36 mg per gram seed, at least about 40 mg per gram seed, more preferably at least about 44 mg per gram seed or at least about 48 mg per gram seed. The maximum DHA content may be about 80 to about 100 mg per gram seed, or about 80 mg or about 100 mg per gram seed.

In an embodiment, the cell of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, or the seed of the invention, which can be used to produce extracted lipid comprising one or more or all of the features defined herein.

In yet a further aspect, the present invention provides a method of producing a cell of the invention, the method comprising

a) introducing into the cell, preferably a cell which is not capable of synthesising a LC-PUFA, the gene construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, one or more of the combinations of exogenous polynucleotides defined herein,

b) optionally, expressing the genes or polynucleotide(s) in the cell;

c) optionally, analysing the fatty acid composition of the cell, and

d) optionally, selecting a cell which express the genes or polynucleotide(s).

In an embodiment, the lipid in the cell has one or more of the features defined herein.

In another embodiment, the gene construct, the isolated and/or exogenous polynucleotide, the vector, the genetic construct or combinations of exogenous polynucleotides, become stably integrated into the genome of the cell.

In a further embodiment, the cell is a plant cell, and the method further comprises the step of regenerating a transformed plant from the cell of step a).

In another embodiment, the genes and/or exogenous polynucleotide(s) are expressed transiently in the cell.

Also provided is a cell produced using a method of the invention.

In another aspect, the present invention provides a method of producing seed, the method comprising,

a) growing a plant of the invention, or a plant which produces a part as defined herein, preferably in a field as part of a population of at least 1000 such plants or in an area of at least 1 hectare planted at a standard planting density,

b) harvesting seed from the plant or plants, and

c) optionally, extracting lipid from the seed, preferably to produce oil with a total DHA yield of at least 60 kg DHA/hectare.

In an embodiment, the plant, plant cell, plant part or seed of the invention has one or more of the following features

    • i) the oil is as defined herein,
    • ii) the plant part or seed is capable of being used in a process of the invention,
    • iii) the exogenous polynucleotides are comprised in a genetic construct of the invention,
    • iv) the exogenous polynucleotides comprise an exogenous polynucleotide of the invention,
    • v) the plant cell is a cell of the invention, and
    • vi) the seed was produced according to the method of the invention.

In another aspect, the present invention provides a method of producing one or more fatty acid desaturases and/or fatty acid elongases, or one or more fatty acid desaturases and one or more fatty acid elongases, the method comprising expressing in a cell or cell free expression system the gene construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, one or more of the combinations of exogenous polynucleotides defined herein, preferably in a developing oilseed in an oilseed plant in the field.

In a further aspect, the present invention provides lipid, or oil, produced by, or obtained from, using the process of the invention, the cell of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, or the plant, plant cell, plant part or seed of the invention.

In an embodiment, the lipid or oil is obtained by extraction of oil from an oilseed. Examples of oil from oilseeds include, but are not limited to, canola oil (Brassica napus, Brassica rapa ssp.), mustard oil (Brassica juncea), other Brassica oil, sunflower oil (Helianthus annus), linseed oil (Linum usitatissimum), soybean oil (Glycine max), safflower oil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotiana tabacum), peanut oil (Arachis hypogaea), palm oil, cottonseed oil (Gossypium hirsutum), coconut oil (Cocos nucifera), avocado oil (Persea americana), olive oil (Olea europaea), cashew oil (Anacardium occidentale), macadamia oil (Macadamia intergrifolia), almond oil (Prunus amygdalus) or Arabidopsis seed oil (Arabidopsis thaliana).

In a further aspect, the present invention provides fatty acid produced by, or obtained from, using the process of the invention, the cell of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, or the plant, plant cell, plant part or seed of the invention. Preferably the fatty acid is DHA. The fatty acid may be in a mixture of fatty acids having a fatty acid composition as described herein. In an embodiment, the fatty acid is non-esterified.

Also provided is seedmeal obtained from seed of the invention. Preferred seedmeal includes, but not necessarily limited to, Brassica napus, B. juncea, Camelina sativa or Glycine max seedmeal. In an embodiment, the seedmeal comprises an exogenous polynucleotide(s) and/or genentic constructs as defined herein.

In another aspect, the present invention provides a composition comprising one or more of a lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, or the seedmeal of the invention. In embodiments, the composition comprises a carrier suitable for pharmaceutical, food or agricultural use, a seed treatment compound, a fertiliser, another food or feed ingredient, or added protein or vitamins.

Also provided is feedstuffs, cosmetics or chemicals comprising one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, or the composition of the invention.

In another aspect, the present invention provides a method of producing a feedstuff, the method comprising mixing one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, or the composition of the invention, with at least one other food ingredient.

In another aspect, the present invention provides a method of treating or preventing a condition which would benefit from a PUFA, the method comprising administering to a subject one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, the composition of the invention, or the feedstuff of the invention.

Examples of conditions which would benefit from a PUFA include, but are not limited to, cardiac arrhythmia's, angioplasty, inflammation, asthma, psoriasis, osteoporosis, kidney stones, AIDS, multiple sclerosis, rheumatoid arthritis, Crohn's disease, schizophrenia, cancer, foetal alcohol syndrome, attention deficient hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, adrenoleukodystophy, coronary heart disease, hypertension, diabetes, obesity, Alzheimer's disease, chronic obstructive pulmonary disease, ulcerative colitis, restenosis after angioplasty, eczema, high blood pressure, platelet aggregation, gastrointestinal bleeding, endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronic fatigue after viral infections or an ocular disease.

Also provided is the use of one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, the composition of the invention, or the feedstuff of the invention for the manufacture of a medicament for treating or preventing a condition which would benefit from a PUFA. The production of the medicament may comprise mixing the oil of the invention with a pharmaceutically acceptable carrier, for treatment of a condition as described herein. The method may comprise firstly purifying the oil and/or transesterification, and/or fractionation of the oil to increase the level of DHA. In a particular embodiment, the method comprises treating the lipid or oil such as canola oil to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters. Further treatment such as fractionation or distillation may be applied to enrich the lipid or oil for the DHA. In a preferred embodiment, the medicament comprises ethyl esters of DHA. In an even more preferred embodiment, the level of ethyl esters of DHA in the medicament is between 30% and 50%. The medicament may further comprise ethyl esters of EPA, such as between 30% and 50% of the total fatty acid content in the medicament. Such medicaments are suitable for administration to human or animal subjects for treatment of medical conditions as described herein.

In another aspect, the present invention provides a method of trading seed, comprising obtaining seed of the invention, and trading the obtained seed for pecuniary gain.

In an embodiment, obtaining the seed comprises cultivating plants of the invention and/or harvesting the seed from the plants.

In another embodiment, obtaining the seed further comprises placing the seed in a container and/or storing the seed.

In a further embodiment, obtaining the seed further comprises transporting the seed to a different location.

In yet another embodiment, the method further comprises transporting the seed to a different location after the seed is traded.

In a further embodiment, the trading is conducted using electronic means such as a computer.

In yet a further aspect, the present invention provides a process of producing bins of seed comprising:

a) swathing, windrowing and/or or reaping above-ground parts of plants comprising seed of the invention,

b) threshing and/or winnowing the parts of the plants to separate the seed from the remainder of the plant parts, and

c) sifting and/or sorting the seed separated in step b), and loading the sifted and/or sorted seed into bins, thereby producing bins of seed.

In an embodiment, where relevant, the lipid or oil, preferably seedoil, of, or useful for, the invention has fatty levels about those provided in a Table in the Examples section, such as seed 14 of Table 16.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Aerobic DHA biosynthesis pathways.

FIG. 2. Map of the T-DNA insertion region between the left and right borders of pJP3416-GA7. RB denotes right border; LB, left border; TER, transcription terminator/polyadenylation region; PRO, promoter; Coding regions are indicated above the arrows, promoters and terminators below the arrows. Micpu-Δ6D, Micromonas pusilla Δ6-desaturase; Pyrco-Δ6E, Pyramimonas cordata Δ6-elongase; Paysa-Δ5D, Pavlova salina Δ5-desaturase; Picpa-ω3D, Pichia pastoris ω3-desaturase; Paysa-Δ4D, P. salina Δ4-desaturase; Lackl-Δ12D, Lachancea kluyveri Δ12-desaturase; Pyrco-Δ5E, Pyramimonas cordata Δ5-elongase. NOS denotes the Agrobacterium tumefaciens nopaline synthase transcription terminator/polyadenylation region; FP1, Brassica napus truncated napin promoter; FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine max lectin transcription terminator/polyadenylation region; Cnl1 and Cnl2 denotes the Linum usitatissimum conlinin1 or conlinin2 promoter or terminator. MAR denotes the Rb7 matrix attachment region from Nicotiana tabacum.

FIG. 3. Map of the T-DNA insertion region between the left and right borders of pJP3404. Labels are as in FIG. 2.

FIG. 4. Map of the insertion region between the left and right borders of pJP3367. Labels are as in FIG. 2.

FIG. 5. DHA levels as a percentage of total fatty acids in seed lipid from multiple independent transgenic Arabidopsis thaliana seeds in both the T2 and T3 generations. The bracketed T2 events were taken to T3. Events from both the Columbia and fad2 mutant A. thaliana backgrounds are shown.

FIG. 6. Oil content (w/w) vs. DHA content, as a percentage of total fatty acid content of lipid from transgenic Arabidopsis thaliana seeds.

FIG. 7. Representative RT-PCR gel showing the low expression of the Δ6-desaturase gene relative to the other transgenes in the T-DNA of B. napus embryos transformed using pJP3416-GA7. Lanes from the left show RT-PCR products: 1, DNA size markers; lane 2, Δ12 desaturase; lane 3, ω3-desaturase; lane 4, Δ6-desaturase (low expression); lane 5, Δ6-elongase; lane 6, Δ5-desaturase; lane 7, Δ5-elongase; lane 8, Δ4-desaturase.

FIG. 8. Percentage of ALA plotted against percentage of oleic acid, each as a percentage of total fatty acids in lipid obtained from transgenic 35S:LEC2 Brassica napus somatic embryos.

FIG. 9. Positional distribution analysis by NMR on A) Tuna oil and, B) transgenic DHA Arabidopsis seed oil. The peaks labelled ‘DHA-alpha’ represent the amount of DHA present at the sn-1 and sn-3 positions of TAG (with no positional preference this would equal 66% of total DHA) whilst the peaks labelled ‘DHA-beta’ represent the amount of DHA present at the sn-2 position of TAG (with no preference this would equal 33% of DHA).

FIG. 10. LC-MS analysis of major DHA-containing triacylglycerol species in transgenic A. thaliana developing (grey) and mature (black) seeds. The number following the DHA denotes the total number of carbon atoms and total number of double bonds in the other two fatty acids. Therefore DHA/34:1 can also be designated TAG 56:7, etc.

FIG. 11. Map of the T-DNA insertion region between the left and right borders of pORE04+11ABGBEC_Cowpea_EPA_insert. Labels are as in FIG. 2; SSU, Arabidopsis thaliana rubisco small subunit promoter.

FIG. 12. Map of the binary vector pJP3364 showing the NotI restriction site into which the candidate Δ12-desaturases were cloned.

FIG. 13. BoxPlot generated using SigmaPlot showing the percentage of fatty acid 20:4ω6 (ARA) in seed lipid of Arabidopsis T2 seed populations transformed with pFN045-pFN050. The boundary of each box closest to zero indicates the 25th percentile, a line within each box marks the median, and the boundary of each box farthest from zero indicates the 75th percentile. Error bars shown above and below each box indicate the 90th and 10th percentiles.

FIG. 14. Average level of ARA as a percentage of the total fatty acid content in seed lipid of Arabidopsis T2 seed transformed with pFN045-pFN050.

FIG. 15. BoxPlot showing the percentage of fatty acid 20:2ω6 (EDA) in seed lipid of Arabidopsis T2 seed populations transformed with pFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

FIG. 16. BoxPlot showing the percentage of ARA in seed lipid of Arabidopsis T4 seed populations transformed with pFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

FIG. 17. Average level of ARA as a percentage of the total fatty acid content in seed lipid of Arabidopsis T4 seed populations transformed with pFN045-pFN050.

FIG. 18. BoxPlot showing the percentage of EDA in seed lipid of Arabidopsis T4 seed populations transformed with pFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

FIG. 19. (A) Basic phytosterol structure with ring and side chain numbering. (B) Chemical structures of some of the phytosterols.

FIG. 20. Phylogenetic tree of known LPAATs.

FIG. 21. The various acyl exchange enzymes which transfer fatty acids between PC, CoA pools, and TAG pools. Adapted from Singh et al. (2005).

KEY TO THE SEQUENCE LISTING

  • SEQ ID NO:1—pJP3416-GA7 nucleotide sequence.
  • SEQ ID NO:2—pGA7-mod_B nucleotide sequence.
  • SEQ ID NO:3—pGA7-mod_C nucleotide sequence.
  • SEQ ID NO:4—pGA7-mod_D nucleotide sequence.
  • SEQ ID NO:5—pGA7-mod_E nucleotide sequence.
  • SEQ ID NO:6—pGA7-mod_F nucleotide sequence.
  • SEQ ID NO:7—pGA7-mod_G nucleotide sequence.
  • SEQ ID NO:8—pORE04+11ABGBEC_Cowpea_EPA_insert nucleotide sequence.
  • SEQ ID NO:9—Codon-optimized open reading frame for expression of Lachancea kluyveri Δ12 desaturase in plants.
  • SEQ ID NO:10—Lachancea kluyveri Δ12-desaturase.
  • SEQ ID NO:11—Codon-optimized open reading frame for expression of Pichia pastoris ω3 desaturase in plants.
  • SEQ ID NO:12—Pichia pastoris ω3 desaturase.
  • SEQ ID NO:13—Open reading frame encoding Micromonas pusilla Δ6-desaturase.
  • SEQ ID NO:14—Codon-optimized open reading frame for expression of Micromonas pusilla Δ6-desaturase in plants (version 1).
  • SEQ ID NO:15—Codon-optimized open reading frame for expression of Micromonas pusilla Δ6-desaturase in plants (version 2).
  • SEQ ID NO:16—Micromonas pusilla Δ6-desaturase.

SEQ ID NO:17—Open reading frame encoding Ostreococcus lucimarinus Δ6-desaturase.

  • SEQ ID NO:18—Codon-optimized open reading frame for expression of Ostreococcus lucimarinus Δ6-desaturase in plants.
  • SEQ ID NO:19—Ostreococcus lucimarinus Δ6-desaturase.
  • SEQ ID NO:20—Ostreococcus tauri Δ6-desaturase.
  • SEQ ID NO:21—Open reading frame encoding Pyramimonas cordata Δ6-elongase.
  • SEQ ID NO:22—Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at 3′ end and encoding functional elongase) (version 1).
  • SEQ ID NO:23—Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at 3′ end and encoding functional elongase) (version 2).
  • SEQ ID NO:24—Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at 3′ end and encoding functional elongase) (version 3).
  • SEQ ID NO:25—Pyramimonas cordata Δ6-elongase.
  • SEQ ID NO:26—Truncated Pyramimonas cordata Δ6-elongase.
  • SEQ ID NO:27—Open reading frame encoding Pavlova salina Δ5-desaturase.
  • SEQ ID NO:28—Codon-optimized open reading frame for expression of Pavlova salina Δ5-desaturase in plants (version 1).
  • SEQ ID NO:29—Codon-optimized open reading frame for expression of Pavlova salina Δ5-desaturase in plants (version 2).
  • SEQ ID NO:30—Pavlova salina Δ5-desaturase.
  • SEQ ID NO:31—Open reading frame encoding Pyramimonas cordata Δ5-desaturase.
  • SEQ ID NO:32—Pyramimonas cordata Δ5-desaturase.
  • SEQ ID NO:33—Open reading frame encoding Pyramimonas cordata Δ5-elongase.
  • SEQ ID NO:34—Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants (version 1).
  • SEQ ID NO:35—Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants (version 2).
  • SEQ ID NO:36—Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants (version 3).
  • SEQ ID NO:37—Pyramimonas cordata Δ5-elongase.
  • SEQ ID NO:38—Open reading frame encoding Pavlova salina Δ4-desaturase.
  • SEQ ID NO:39—Codon-optimized open reading frame for expression of Pavlova salina Δ4-desaturase in plants (version 1).
  • SEQ ID NO:40—Codon-optimized open reading frame for expression of Pavlova salina Δ4-desaturase in plants (version 2).
  • SEQ ID NO:41—Pavlova salina Δ4-desaturase.
  • SEQ ID NO:42—Open reading frame encoding Isochrysis galbana Δ9-elongase.
  • SEQ ID NO:43—Isochrysis galbana Δ9-elongase.
  • SEQ ID NO:44—Open reading frame encoding Emiliania huxleyi CCMP1516 Δ9-elongase.
  • SEQ ID NO:45—Codon-optimized open reading frame for expression of Emiliania huxleyi Δ9-elongase in plants.
  • SEQ ID NO:46—Emiliania huxleyi CCMP1516 Δ9-elongase.
  • SEQ ID NO:47—Open reading frame encoding Pavlova pinguis Δ9-elongase.
  • SEQ ID NO:48—Pavlova pinguis Δ9-elongase.
  • SEQ ID NO:49—Open reading frame encoding Pavlova salina Δ9-elongase.
  • SEQ ID NO:50—Pavlova salina Δ9-elongase.
  • SEQ ID NO:51—Open reading frame encoding Pavlova salina Δ8-desaturase.
  • SEQ ID NO:52—Pavlova salina Δ8-desaturase.
  • SEQ ID NO:53—P19 viral suppressor.
  • SEQ ID NO:54—V2 viral suppressor.
  • SEQ ID NO:55—P38 viral suppressor.
  • SEQ ID NO:56—Pe-P0 viral suppressor.
  • SEQ ID NO:57—RPV-P0 viral suppressor.
  • SEQ ID NO:58—Open reading frame encoding P19 viral suppressor.
  • SEQ ID NO:59—Open reading frame encoding V2 viral suppressor.
  • SEQ ID NO:60—Open reading frame encoding P38 viral suppressor.
  • SEQ ID NO:61—Open reading frame encoding Pe-P0 viral suppressor.
  • SEQ ID NO:62—Open reading frame encoding RPV-P0 viral suppressor.
  • SEQ ID NO: 63—Arabidopsis thaliana LPAAT2.
  • SEQ ID NO: 64—Limnanthes alba LPAAT.
  • SEQ ID NO: 65—Saccharomyces cerevisiae LPAAT.
  • SEQ ID NO: 66—Micromonas pusilla LPAAT.
  • SEQ ID NO: 67—Mortierella alpina LPAAT.
  • SEQ ID NO: 68—Braccisa napus LPAAT.
  • SEQ ID NO: 69—Brassica napus LPAAT.
  • SEQ ID NO: 70—Phytophthora infestans ω3 desaturase.
  • SEQ ID NO: 71—Thalassiosira pseudonana ω3 desaturase.
  • SEQ ID NO: 72—Pythium irregulare ω3 desaturase.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, fatty acid synthesis, transgenic plants, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term “about”, unless stated to the contrary, refers to +/−10%, more preferably +/−5%, more preferably +/−1% of the designated value.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Selected Definitions

As used herein, the terms “extracted plant lipid” and “isolated plant lipid” refer to a lipid composition which has been extracted from, for example by crushing, a plant or part thereof such as seed. The extracted lipid can be a relatively crude composition obtained by, for example, crushing a plant seed, or a more purified composition where most, if not all, of one or more or each of the water, nucleic acids, proteins and carbohydrates derived from the plant material have been removed. Examples of purification methods are described below. In an embodiment, the extracted or isolated plant lipid comprises at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w/w) lipid by weight of the composition. The lipid may be solid or liquid at room temperature, when liquid it is considered to be an oil. In an embodiment, extracted lipid of the invention has not been blended with another lipid such as DHA not produced by another source (for example, DHA from fish oil). In an embodiment, following extraction the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids, has not been significantly altered (for example, no greater than a 10% or 5% alteration) when compared to the ratio in the intact seed or cell. In an another embodiment, the extracted plant lipid has not been exposed to a procedure, such as hydrogenation or fractionation, which may alter the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids, when compared to the ratio in the intact seed or cell. When the extracted plant lipid of the invention is comprised in an oil, the oil may further comprise non-fatty acid molecules such as sterols.

As used herein, the terms “extracted plant oil” and “isolated plant oil” refer to a substance or composition comprising extracted plant lipid or isolated plant lipid and which is a liquid at room temperature. The oil is obtained from a plant or part thereof such as seed. The extracted or isolated oil can be a relatively crude composition obtained by, for example, crushing a plant seed, or a more purified composition where most, if not all, of one or more or each of the water, nucleic acids, proteins and carbohydrates derived from the plant material have been removed. The composition may comprise other components which may be lipid or non-lipid. In an embodiment, the oil composition comprises at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w/w) extracted plant lipid. In an embodiment, extracted oil of the invention has not been blended with another oil such as DHA not produced by another source (for example, DHA from fish oil). In an embodiment, following extraction, the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids, has not been significantly altered (for example, no greater than a 10% or 5% alteration) when compared to the ratio in the intact seed or cell. In an another embodiment, the extracted plant oil has not been exposed to a procedure, such as hydrogenation or fractionation, which may alter the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids, when compared to the ratio in the intact seed or cell. Extracted plant oil of the invention may comprise non-fatty acid molecules such as sterols.

As used herein, an “oil” is a composition comprising predominantly lipid and which is a liquid at room temperature. For instance, oil of the invention preferably comprises at least 75%, at least 80%, at least 85% or at least 90% lipid by weight. Typically, a purified oil comprises at least 90% triacylglycerols (TAG) by weight of the lipid in the oil. Minor components of an oil such as diacylglycerols (DAG), free fatty acids (FFA), phospholipid and sterols may be present as described herein.

As used herein, the term “fatty acid” refers to a carboxylic acid (or organic acid), often with a long aliphatic tail, either saturated or unsaturated. Typically fatty acids have a carbon-carbon bonded chain of at least 8 carbon atoms in length, more preferably at least 12 carbons in length. Most naturally occurring fatty acids have an even number of carbon atoms because their biosynthesis involves acetate which has two carbon atoms. The fatty acids may be in a free state (non-esterified) or in an esterified form such as part of a triglyceride, diacylglyceride, monoacylglyceride, acyl-CoA (thio-ester) bound or other bound form. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms.

“Saturated fatty acids” do not contain any double bonds or other functional groups along the chain. The term “saturated” refers to hydrogen, in that all carbons (apart from the carboxylic acid [—COOH] group) contain as many hydrogens as possible. In other words, the omega (ω) end contains 3 hydrogens (CH3-) and each carbon within the chain contains 2 hydrogens (—CH2-).

“Unsaturated fatty acids” are of similar form to saturated fatty acids, except that one or more alkene functional groups exist along the chain, with each alkene substituting a singly-bonded “—CH2-CH2-” part of the chain with a doubly-bonded “—CH═CH—” portion (that is, a carbon double bonded to another carbon). The two next carbon atoms in the chain that are bound to either side of the double bond can occur in a cis or trans configuration.

As used herein, the term “monounsaturated fatty acid” refers to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and only one alkene group (carbon-carbon double bond) in the chain. As used herein, the terms “polyunsaturated fatty acid” or “PUFA” refer to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and at least two alkene groups (carbon-carbon double bonds).

As used herein, the terms “long-chain polyunsaturated fatty acid” and “LC-PUFA” refer to a fatty acid which comprises at least 20 carbon atoms in its carbon chain and at least two carbon-carbon double bonds, and hence include VLC-PUFAs. As used herein, the terms “very long-chain polyunsaturated fatty acid” and “VLC-PUFA” refer to a fatty acid which comprises at least 22 carbon atoms in its carbon chain and at least three carbon-carbon double bonds. Ordinarily, the number of carbon atoms in the carbon chain of the fatty acids refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes those in sidegroups. In one embodiment, the long-chain polyunsaturated fatty acid is an ω3 fatty acid, that is, having a desaturation (carbon-carbon double bond) in the third carbon-carbon bond from the methyl end of the fatty acid. In another embodiment, the long-chain polyunsaturated fatty acid is an ω6 fatty acid, that is, having a desaturation (carbon-carbon double bond) in the sixth carbon-carbon bond from the methyl end of the fatty acid. In a further embodiment, the long-chain polyunsaturated fatty acid is selected from the group consisting of; arachidonic acid (ARA, 20:4Δ5,8,11,14; ω6), eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3), eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA, 22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19, ω3). The LC-PUFA may also be dihomo-γ-linoleic acid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ11,14,17, ω3). It would readily be apparent that the LC-PUFA that is produced according to the invention may be a mixture of any or all of the above and may include other LC-PUFA or derivatives of any of these LC-PUFA. In a preferred embodiment, the ω3 fatty acids are at least DHA, preferably, DPA and DHA, or EPA, DPA and DHA.

Furthermore, as used herein the terms “long-chain polyunsaturated fatty acid” and “very long-chain polyunsaturated fatty acid” refer to the fatty acid being in a free state (non-esterified) or in an esterified form such as part of a triglyceride, diacylglyceride, monoacylglyceride, acyl-CoA bound or other bound form. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms. Thus, the LC-PUFA may be present as a mixture of forms in the lipid of a cell or a purified oil or lipid extracted from cells, tissues or organisms. In preferred embodiments, the invention provides oil comprising at least 75% or at least 85% triacylglycerols, with the remainder present as other forms of lipid such as those mentioned, with at least said triacylglycerols comprising the LC-PUFA. The oil may subsequently be further purified or treated, for example by hydrolysis with a strong base to release the free fatty acids, or by distillation or the like.

As used herein, “total ω6 fatty acids” or “total ω6 fatty acid content” or the like refers to the sum of all the ω6 fatty acids, esterified and non-esterified, in the extracted lipid, oil, recombinanat cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These ω6 fatty acids include (if present) LA, GLA, DGLA, ARA, EDA and ω6-DPA, and exclude any ω3 fatty acids and monounsaturated fatty acids.

As used herein, “new ω6 fatty acids” or “new ω6 fatty acid content” or the like refers to the sum of all the ω6 fatty acids excluding LA, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These new ω6 fatty acids are the fatty acids that are produced in the cells, plants, plant parts and seeds of the invention by the expression of the genetic constructs (exogenous polynucleotides) introduced into the cells, and include (if present) GLA, DGLA, ARA, EDA and ω6-DPA, but exclude LA and any ω3 fatty acids and monounsaturated fatty acids. Exemplary total ω6 fatty acid contents and new ω6 fatty acid contents are determined by conversion of fatty acids in a sample to FAME and analysis by GC, as described in Example 1.

As used herein, “total ω3 fatty acids” or “total ω3 fatty acid content” or the like refers to the sum of all the ω3 fatty acids, esterified and non-esterified, in the extracted lipid, oil, recombinanat cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These ω3 fatty acids include (if present) ALA, SDA, ETrA, ETA, EPA, DPA and DHA, and exclude any ω6 fatty acids and monounsaturated fatty acids.

As used herein, “new ω3 fatty acids” or “new ω3 fatty acid content” or the like refers to the sum of all the ω3 fatty acids excluding ALA, esterified and non-esterified, in the extracted lipid, oil, recombinanat cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These new ω3 fatty acids are the fatty acids that are produced in the cells, plants, plant parts and seeds of the invention by the expression of the genetic constructs (exogenous polynucleotides) introduced into the cells, and include (if present) SDA, ETrA, ETA, EPA, DPA and DHA, but exclude ALA and any ω6 fatty acids and monounsaturated fatty acids. Exemplary total ω3 fatty acid contents and new ω3 fatty acid contents are determined by conversion of fatty acids in a sample to FAME and analysis by GC, as described in Example 1.

The desaturase, elongase and acyl transferase proteins and genes encoding them that may be used in the invention are any of those known in the art or homologues or derivatives thereof. Examples of such genes and encoded protein sizes are listed in Table 1. The desaturase enzymes that have been shown to participate in LC-PUFA biosynthesis all belong to the group of so-called “front-end” desaturases.

As used herein, the term “front-end desaturase” refers to a member of a class of enzymes that introduce a double bond between the carboxyl group and a pre-existing unsaturated part of the acyl chain of lipids, which are characterized structurally by the presence of an N-terminal cytochrome b5 domain, along with a typical fatty acid desaturase domain that includes three highly conserved histidine boxes (Napier et al., 1997).

Activity of any of the elongases or desaturases for use in the invention may be tested by expressing a gene encoding the enzyme in a cell such as, for example, a yeast cell, a plant cell or preferably in somatic embryos or transgenic plants, and determining whether the cell, embryo or plant has an increased capacity to produce LC-PUFA compared to a comparable cell, embryo or plant in which the enzyme is not expressed.

In one embodiment one or more of the desaturases and/or elongases for use in the invention can purified from a microalga, i.e. is identical in amino acid sequence to a polypeptide which can be purified from a microalga.

Whilst certain enzymes are specifically described herein as “bifunctional”, the absence of such a term does not necessarily imply that a particular enzyme does not possess an activity other than that specifically defined.

Desaturases

As used herein, the term “desaturase” refers to an enzyme which is capable of introducing a carbon-carbon double bond into the acyl group of a fatty acid substrate which is typically in an esterified form such as, for example, acyl-CoA esters. The acyl group may be esterified to a phospholipid such as phosphatidylcholine (PC), or to acyl carrier protein (ACP), or in a preferred embodiment to CoA. Desaturases generally may be categorized into three groups accordingly. In one embodiment, the desaturase is a front-end desaturase.

As used herein, a “Δ4-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 4th carbon-carbon bond from the carboxyl end of a fatty acid substrate. The “Δ4-desaturase” is at least capable of converting DPA to DHA. The desaturation step to produce DHA from DPA is catalysed by a Δ4-desaturase in organisms other than mammals, and a gene encoding this enzyme has been isolated from the freshwater protist species Euglena gracilis and the marine species Thraustochytrium sp. (Qiu et al., 2001; Meyer et al., 2003). In one embodiment, the Δ4-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:41, or a Thraustochytrium sp. Δ4-desaturase, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:41.


TABLE 1
Cloned genes involved in LC-PUFA biosynthesis
Protein
Type of
Accession
size
Enzyme
organism
Species
nos.
(aa's)
References
Δ4-
Protist
Euglena gracilis
AY278558
541
Meyer et al., 2003
desaturase
Algae
Pavlova lutherii
AY332747
445
Tonon et al., 2003
Isochrysis galbana
AAV33631
433
Pereira et al., 2004b
Pavlova salina
AAY15136
447
Zhou et al., 2007
Thraustochytrid
Thraustochytrium aureum
AAN75707
515
N/A
AAN75708
AAN75709
AAN75710
Thraustochytrium sp.
AAM09688
519
Qiu et al. 2001
ATCC21685
Δ5-
Mammals
Homo sapiens
AF199596
444
Cho et al., 1999b
desaturase
Leonard et al., 2000b
Nematode
Caenorhabditis elegans
AF11440,
447
Michaelson et al., 1998b;
NM_069350
Watts and Browse, 1999b
Fungi
Mortierella alpina
AF067654
446
Michaelson et al., 1998a;
Knutzon et al., 1998
Pythium irregulare
AF419297
456
Hong et al., 2002a
Dictyostelium discoideum
AB022097
467
Saito et al., 2000
Saprolegnia diclina
470
WO02081668
Diatom
Phaeodactylum tricornutum
AY082392
469
Domergue et al., 2002
Algae
Thraustochytrium sp
AF489588
439
Qiu et al., 2001
Thraustochytrium aureum
439
WO02081668
Isochrysis galbana
442
WO02081668
Moss
Marchantia polymorpha
AY583465
484
Kajikawa et al., 2004
Δ6-
Mammals
Homo sapiens
NM_013402
444
Cho et al., 1999a;
desaturase
Leonard et al., 2000
Mus musculus
NM_019699
444
Cho et al., 1999a
Nematode
Caenorhabditis elegans
Z70271
443
Napier et al., 1998
Plants
Borago officinales
U79010
448
Sayanova et al, 1997
Echium
AY055117
Garcia-Maroto et al., 2002
AY055118
Primula vialii
AY234127
453
Sayanova et al., 2003
Anemone leveillei
AF536525
446
Whitney et al., 2003
Mosses
Ceratodon purpureus
AJ250735
520
Sperling et al., 2000
Marchantia polymorpha
AY583463
481
Kajikawa et al., 2004
Physcomitrella patens
CAA11033
525
Girke et al., 1998
Fungi
Mortierella alpina
AF110510
457
Huang et al., 1999;
AB020032
Sakuradani et al., 1999
Pythium irregulare
AF419296
459
Hong et al., 2002a
Mucor circinelloides
AB052086
467
NCBI*
Rhizopus sp.
AY320288
458
Zhang et al., 2004
Saprolegnia diclina
453
WO02081668
Diatom
Phaeodactylum tricornutum
AY082393
477
Domergue et al., 2002
Bacteria
Synechocystis
L11421
359
Reddy et al., 1993
Algae
Thraustochytrium aureum
456
WO02081668
Bifunctional
Fish
Danio rerio
AF309556
444
Hastings et al., 2001
Δ5/Δ6-
desaturase
C20 Δ8-
Algae
Euglena gracilis
AF139720
419
Wallis and Browse, 1999
desaturase
Plants
Borago officinales
AAG43277
446
Sperling et al., 2001
Δ6-elongase
Nematode
Caenorhabditis elegans
NM_069288
288
Beaudoin et al., 2000
Mosses
Physcomitrella patens
AF428243
290
Zank et al., 2002
Marchantia polymorpha
AY583464
290
Kajikawa et al., 2004
Fungi
Mortierella alpina
AF206662
318
Parker-Barnes et al., 2000
Algae
Pavlova lutheri**
501
WO 03078639
Thraustochytrium
AX951565
271
WO 03093482
Thraustochytrium sp**
AX214454
271
WO 0159128
PUFA-elongase
Mammals
Homo sapiens
AF231981
299
Leonard et al., 2000b;
Leonard et al., 2002
Rattus norvegicus
AB071985
299
Inagaki et al., 2002
Rattus norvegicus**
AB071986
267
Inagaki et al., 2002
Mus musculus
AF170907
279
Tvrdik et al., 2000
Mus musculus
AF170908
292
Tvrdik et al., 2000
Fish
Danio rerio
AF532782
291 (282)
Agaba et al., 2004
Danio rerio**
NM_199532
266
Lo et al., 2003
Worm
Caenorhabditis elegans
Z68749
309
Abbott et al., 1998
Beaudoin et al., 2000
Algae
Thraustochytrium aureum**
AX464802
272
WO 0208401-A2
Pavlova lutheri**
320
WO 03078639
Δ9-elongase
Algae
Isochrysis galbana
AF390174
263
Qi et al., 2002
Euglena gracilis
258
WO 08/128241
Δ5-elongase
Algae
Ostreococcus tauri
AAV67798
300
Meyer et al., 2004
Pyramimonas cordata
268
WO 2010/057246
Pavlova sp. CCMP459
AAV33630
277
Pereira et al., 2004b
Pavlova salina
AAY15135
302
Robert et al., 2009
Diatom
Thalassiosira pseudonana
AAV67800
358
Meyer et al., 2004
Fish
Oncorhynchus mykiss
CAM55862
295
WO 06/008099
Moss
Marchantia polymorpha
BAE71129
348
Kajikawa et al., 2006
*http://www.ncbi.nlm.nih.gov/
**Function not proven/not demonstrated

As used herein, a “Δ5-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 5th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Examples of Δ5-desaturases are listed in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a) and in Table 1 herein. In one embodiment, the Δ5-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:30, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:30. In another embodiment, the Δ5-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:32, a biologically active fragment thereof, or an amino acid sequence which is at least 53% identical to SEQ ID NO:32. In another embodiment, the Δ5-desaturase is from Thraustochytrium sp or Emiliania huxleyi.

As used herein, a “Δ6-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 6th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Examples of Δ6-desaturases are listed in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a) and in Table 1 herein. Preferred Δ6-desaturases are from Micromonas pusilla, Pythium irregulare or Ostreococcus taurii.

In an embodiment, the Δ6-desaturase is further characterised by having at least two, preferably all three and preferably in a plant cell, of the following: i) greater Δ6-desaturase activity on α-linolenic acid (ALA, 18:3Δ9,12,15, ω3) than linoleic acid (LA, 18:2Δ9,12, ω6) as fatty acid substrate; ii) greater Δ6-desaturase activity on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate; and iii) Δ8-desaturase activity on ETrA. Examples of such Δ6-desaturases are provided in Table 2.

In an embodiment the Δ6-desaturase has greater activity on an ω3 substrate than the corresponding ω6 substrate and has activity on ALA to produce octadecatetraenoic acid (stearidonic acid, SDA, 18:4Δ6,9,12,15, ω3) with an efficiency of at least 30%, more preferably at least 40%, or most preferably at least 50% when expressed from an exogenous polynucleotide in a recombinant cell such as a plant cell, or at least 35% when expressed in a yeast cell. In one embodiment, the Δ6-desaturase has greater activity, for example, at least about a 2-fold greater Δ6-desaturase activity, on ALA than LA as fatty acid substrate. In another embodiment, the Δ6-desaturase has greater activity, for example, at least about 5 fold greater Δ6-desaturase activity or at least 10-fold greater activity, on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate. In a further embodiment, the Δ6-desaturase has activity on both fatty acid substrates ALA-CoA and on ALA joined to the sn-2 position of PC.


TABLE 2
Desaturases demonstrated to have activity on an acyl-CoA substrate
Protein
Type of
Accession
size
Enzyme
organism
Species
nos.
(aa's)
References
Δ6-desaturase
Algae
Mantoniella squamata
CAQ30479
449
Hoffmann et al., 2008
Ostreococcus tauri
AAW70159
456
Domergue et al., 2005
Micromonas pusilla
EEH58637
Petrie et al., 2010a
(SEQ ID NO: 13)
Δ5-desaturase
Algae
Mantoniella squamata
CAQ30478
482
Hoffmann et al., 2008
Plant
Anemone leveillei
N/A
Sayanova et al., 2007
ω3-desaturase
Fungi
Pythium aphanidermatum
FW362186.1
359
Xue et al., 2012;
WO2008/054565
Fungi
Phytophthora sojae
FW362214.1
363
Xue et al., 2012;
(oomycete)
WO2008/054565
Fungi
Phytophthora ramorum
FW362213.1
361
Xue et al., 2012;
(oomycete)
WO2008/054565

In one embodiment, the Δ6-desaturase has no detectable Δ5-desaturase activity on ETA. In another embodiment, the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:16, SEQ ID NO:19 or SEQ ID NO:20, a biologically active fragment thereof, or an amino acid sequence which is at least 77% identical to SEQ ID NO:16, SEQ ID NO:19 or SEQ ID NO:20. In another embodiment, the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:19 or SEQ ID NO:20, a biologically active fragment thereof, or an amino acid sequence which is at least 67% identical to one or both of SEQ ID NO:19 or SEQ ID NO:20. The Δ6-desaturase may also have Δ8-desaturase activity.

As used herein, a “Δ8-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 8th carbon-carbon bond from the carboxyl end of a fatty acid substrate. The Δ8-desaturase is at least capable of converting ETrA to ETA. Examples of Δ8-desaturases are listed in Table 1. In one embodiment, the Δ8-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:52, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:52.

As used herein, an “ω3-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 3rd carbon-carbon bond from the methyl end of a fatty acid substrate. A ω3-desaturase therefore may convert LA to ALA and GLA to SDA (all C18 fatty acids), or DGLA to ETA and/or ARA to EPA (C20 fatty acids). Some ω3-desaturases (group I) have activity only on C18 substrates, such as plant and cyanobacterial ω3-desaturases. Such ω3-desaturases are also Δ15-desaturases. Other ω3-desaturases have activity on C20 substrates with no activity (group II) or some activity (group III) on C18 substrates. Such ω3-desaturases are also Δ17-desaturases. Preferred ω3-desaturases are group III type which convert LA to ALA, GLA to SDA, DGLA to ETA and ARA to EPA, such as the Pichia pastoris ω3-desaturase (SEQ ID NO: 12). Examples of ω3-desaturases include those described by Pereira et al. (2004a) (Saprolegnia diclina ω3-desaturase, group II), Horiguchi et al. (1998), Berberich et al. (1998) and Spychalla et al. (1997) (C. elegans ω3-desaturase, group III). In a preferred embodiment, the ω3-desaturase is a fungal ω3-desaturase. As used herein, a “fungal ω3-desaturase” refers to an ω3-desaturase which is from a fungal source, including an oomycete source, or a variant thereof whose amino acid sequence is at least 95% identical thereto. Genes encoding numerous ω3-desaturases have been isolated from fungal sources such as, for example, from Phytophthora infestans (Accession No. CAJ30870, WO2005083053; SEQ ID NO: 70), Saprolegnia diclina (Accession No. AAR20444, Pereira et al., 2004a & U.S. Pat. No. 7,211,656), Pythium irregulare (WO2008022963, Group II; SEQ ID NO: 72), Mortierella alpina (Sakuradani et al., 2005; Accession No. BAD91495; WO2006019192), Thalassiosira pseudonana (Armbrust et al., 2004; Accession No. XP_002291057; WO2005012316, SEQ ID NO: 71), Lachancea kluyveri (also known as Saccharomyces kluyveri; Oura et al., 2004; Accession No. AB118663). Xue et al. (2012) describes ω3-desaturases from the oomycetes Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum which were able to efficiently convert ω6 fatty acid substrates to the corresponding ω3 fatty acids, with a preference for C20 substrates, i.e. they had stronger Δ17-desaturase activity than Δ15-desaturase activity. These enzymes lacked Δ12-desaturase activity, but could use fatty acids in both acyl-CoA and phospholipid fraction as substrates.

In a more preferred embodiment, the fungal ω3-desaturase is the Pichia pastoris (also known as Komagataella pastoris) ω3-desaturase/Δ15-desaturase (Zhang et al., 2008; Accession No. EF116884; SEQ ID NO: 12), or a polypeptide which is at least 95% identical thereto.

In an embodiment, the ω3-desaturase is at least capable of converting one of ARA to EPA, DGLA to ETA, GLA to SDA, both ARA to EPA and DGLA to ETA, both ARA to EPA and GLA to SDA, or all three of these.

In one embodiment, the ω3-desaturase has Δ17-desaturase activity on a C20 fatty acid which has at least three carbon-carbon double bonds, preferably ARA. In another embodiment, the ω3-desaturase has Δ15-desaturase activity on a C18 fatty acid which has three carbon-carbon double bonds, preferably GLA. Preferably, both activities are present.

As used herein, a “Δ12-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 12th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Δ12-desaturases typically convert either oleoyl-phosphatidylcholine or oleoyl-CoA to linoleoyl-phosphatidylcholine (18:1-PC) or linoleoyl-CoA (18:1-CoA), respectively. The subclass using the PC linked substrate are referred to as phospholipid-dependent Δ12-desaturases, the latter subclass as acyl-CoA dependent Δ12-desaturases. Plant and fungal Δ12-desaturases are generally of the former sub-class, whereas animal Δ12-desaturases are of the latter subclass, for example the Δ12-desaturases encoded by genes cloned from insects by Zhou et al. (2008). Many other Δ12-desaturase sequences can be easily identified by searching sequence databases.

As used herein, a “Δ15-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 15th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Numerous genes encoding Δ15-desaturases have been cloned from plant and fungal species. For example, U.S. Pat. No. 5,952,544 describes nucleic acids encoding plant Δ15-desaturases (FAD3). These enzymes comprise amino acid motifs that were characteristic of plant Δ15-desaturases. WO200114538 describes a gene encoding soybean FAD3. Many other Δ15-desaturase sequences can be easily identified by searching sequence databases.

As used herein, a “Δ17-desaturase” refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 17th carbon-carbon bond from the carboxyl end of a fatty acid substrate. A Δ17-desaturase is also regarded as an ω3-desaturase if it acts on a C20 substrate to introduce a desaturation at the ω3 bond.

In a preferred embodiment, the Δ12-desaturase and/or Δ15-desaturase is a fungal Δ12-desaturase or fungal Δ15-desaturase. As used herein, a “fungal Δ12-desaturase” or “a fungal Δ15-desaturase” refers to a Δ12-desaturase or Δ15-desaturase which is from a fungal source, including an oomycete source, or a variant thereof whose amino acid sequence is at least 95% identical thereto. Genes encoding numerous desaturases have been isolated from fungal sources. U.S. Pat. No. 7,211,656 describes a Δ12 desaturase from Saprolegnia diclina. WO2009016202 describes fungal desaturases from Helobdella robusta, Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosiga brevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola, Naegleria gruben, Nectria haematococca, Nematostella vectensis, Phycomyces blakesleeanus, Trichoderma resii, Physcomitrella patens, Postia placenta, Selaginella moellendorffii and Microdochium nivale. WO2005/012316 describes a Δ12-desaturase from Thalassiosira pseudonana and other fungi. WO2003/099216 describes genes encoding fungal Δ12-desaturases and Δ15-desaturases isolated from Neurospora crassa, Aspergillus nidulans, Botrytis cinerea and Mortierella alpina. WO2007133425 describes fungal Δ15 desaturases isolated from: Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans, Neurospora crassa, Fusarium graminearum, Fusarium moniliforme and Magnaporthe grisea. A preferred Δ12 desaturase is from Phytophthora sojae (Ruiz-Lopez et al., 2012).

A distinct subclass of fungal Δ12-desaturases, and of fungal Δ15-desaturases, are the bifunctional fungal Δ12/Δ15-desaturases. Genes encoding these have been cloned from Fusarium monoliforme (Accession No. DQ272516, Damude et al., 2006), Acanthamoeba castellanii (Accession No. EF017656, Sayanova et al., 2006), Perkinsus marinus (WO2007042510), Claviceps purpurea (Accession No. EF536898, Meesapyodsuk et al., 2007) and Coprinus cinereus (Accession No. AF269266, Zhang et al., 2007).

In another embodiment, the ω3-desaturase has at least some activity on, preferably greater activity on, an acyl-CoA substrate than a corresponding acyl-PC substrate. As used herein, a “corresponding acyl-PC substrate” refers to the fatty acid esterified at the sn-2 position of phosphatidylcholine (PC) where the fatty acid is the same fatty acid as in the acyl-CoA substrate. For example, the acyl-CoA substrate may be ARA-CoA and the corresponding acyl-PC substrate is sn-2 ARA-PC. In an embodiment, the activity is at least two-fold greater. Preferably, the ω3-desaturase has at least some activity on both an acyl-CoA substrate and its corresponding acyl-PC substrate and has activity on both C18 and C20 substrates. Examples of such ω3-desaturases are known amongst the cloned fungal desaturases listed above.

In a further embodiment, the ω3-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:12, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO:12, preferably at least 90% or at least 95% identical to SEQ ID NO:12.

In yet a further embodiment, a desaturase for use in the present invention has greater activity on an acyl-CoA substrate than a corresponding acyl-PC substrate. In another embodiment, a desaturase for use in the present invention has greater activity on an acyl-PC substrate than a corresponding acyl-CoA substrate, but has some activity on both substrates. As outlined above, a “corresponding acyl-PC substrate” refers to the fatty acid esterified at the sn-2 position of phosphatidylcholine (PC) where the fatty acid is the same fatty acid as in the acyl-CoA substrate. In an embodiment, the greater activity is at least two-fold greater. In an embodiment, the desaturase is a Δ5 or Δ6-desaturase, or an ω3-desaturase, examples of which are provided, but not limited to, those listed in Table 2. To test which substrate a desaturase acts on, namely an acyl-CoA or an acyl-PC substrate, assays can be carried out in yeast cells as described in Domergue et al. (2003) and (2005). Acyl-CoA substrate capability for a desaturase can also be inferred when an elongase, when expressed together with the desturase, has an enzymatic conversion efficiency in plant cells of at least about 90% where the elongase catalyses the elongation of the product of the desaturase. On this basis, the Δ5-desaturase and Δ4-desaturases expressed from the GA7 construct (Examples 2 and 3) and variants thereof (Example 5) are capable of desaturating their respective acyl-CoA substrates, ETA-CoA and DPA-CoA.

Elongases

Biochemical evidence suggests that the fatty acid elongation consists of 4 steps: condensation, reduction, dehydration and a second reduction. In the context of this invention, an “elongase” refers to the polypeptide that catalyses the condensing step in the presence of the other members of the elongation complex, under suitable physiological conditions. It has been shown that heterologous or homologous expression in a cell of only the condensing component (“elongase”) of the elongation protein complex is required for the elongation of the respective acyl chain. Thus, the introduced elongase is able to successfully recruit the reduction and dehydration activities from the transgenic host to carry out successful acyl elongations. The specificity of the elongation reaction with respect to chain length and the degree of desaturation of fatty acid substrates is thought to reside in the condensing component. This component is also thought to be rate limiting in the elongation reaction.

As used herein, a “Δ5-elongase” is at least capable of converting EPA to DPA. Examples of Δ5-elongases include those disclosed in WO2005/103253. In one embodiment, the Δ5-elongase has activity on EPA to produce DPA with an efficiency of at least 60%, more preferably at least 65%, more preferably at least 70% or most preferably at least 80% or 90%. In a further embodiment, the Δ5-elongase comprises an amino acid sequence as provided in SEQ ID NO:37, a biologically active fragment thereof, or an amino acid sequence which is at least 47% identical to SEQ ID NO:37. In a further embodiment, the Δ6-elongase is from Ostreococcus taurii or Ostreococcus lucimarinus (US2010/088776).

As used herein, a “Δ6-elongase” is at least capable of converting SDA to ETA. Examples of Δ6-elongases include those listed in Table 1. In one embodiment, the elongase comprises amino acids having a sequence as provided in SEQ ID NO:25, a biologically active fragment thereof (such as the fragment provided as SEQ ID NO:26), or an amino acid sequence which is at least 55% identical to one or both of SEQ ID NO:25 or SEQ ID NO:26. In an embodiment, the Δ6-elongase is from Physcomitrella patens (Zank et al., 2002; Accession No. AF428243) or Thalassiosira pseudonana (Ruiz-Lopez et al., 2012).

As used herein, a “Δ9-elongase” is at least capable of converting ALA to ETrA. Examples of Δ9-elongases include those listed in Table 1. In one embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:43, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:43. In another embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:46, a biologically active fragment thereof, or an amino acid sequence which is at least 81% identical to SEQ ID NO:46. In another embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:48, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:48. In another embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:50, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:50. In a further embodiment, the Δ9-elongase has greater activity on an ω6 substrate than the corresponding ω3 substrate, or the converse.

As used herein, the term “has greater activity on an ω6 substrate than the corresponding ω3 substrate” refers to the relative activity of the enzyme on substrates that differ by the action of an ω3 desaturase. Preferably, the ω6 substrate is LA and the ω3 substrate is ALA.

An elongase with Δ6-elongase and Δ9-elongase activity is at least capable of (i) converting SDA to ETA and (ii) converting ALA to ETrA and has greater Δ6-elongase activity than Δ9-elongase activity. In one embodiment, the elongase has an efficiency of conversion on SDA to produce ETA which is at least 50%, more preferably at least 60%, and/or an efficiency of conversion on ALA to produce ETrA which is at least 6% or more preferably at least 9%. In another embodiment, the elongase has at least about 6.5 fold greater Δ6-elongase activity than Δ9-elongase activity. In a further embodiment, the elongase has no detectable Δ5-elongase activity

Other Enzymes

As used herein, the term “1-acyl-glycerol-3-phosphate acyltransferase” (LPAAT), also termed lysophosphatidic acid-acyltransferase or acylCoA-lysophosphatidate-acyltransferase, refers to a protein which acylates sn-1-acyl-glycerol-3-phosphate (sn-1 G-3-P) at the sn-2 position to form phosphatidic acid (PA). Thus, the term “1-acyl-glycerol-3-phosphate acyltransferase activity” refers to the acylation of (sn-1 G-3-P) at the sn-2 position to produce PA (EC 2.3.1.51). Preferred LPAATs are those that can use a polyunsaturated C22 acyl-CoA as substrate to transfer the polyunsaturated C22 acyl group to the sn-2 position of LPA, forming PA. Such LPAATs are exemplified in Example 13 and can be tested as described therein. In an embodiment, an LPAAT useful for the invention comprises amino acids having a sequence as provided in any one of SEQ ID NOs: 63 to 69, a biologically active fragment thereof, or an amino acid sequence which is at least 40% identical to any one or more of SEQ ID NOs: 63 to 69. In a preferred embodiment, an LPAAT useful for the invention comprises amino acids having a sequence as provided in any one of SEQ ID NOs: 64, 65 and 67, a biologically active fragment thereof, or an amino acid sequence which is at least 40% identical to any one or more of SEQ ID NOs: 64, 65 and 67.

As used herein, the term “diacylglycerol acyltransferase” (EC 2.3.1.20; DGAT) refers to a protein which transfers a fatty acyl group from acyl-CoA to a diacylglycerol substrate to produce a triacylglycerol. Thus, the term “diacylglycerol acyltransferase activity” refers to the transfer of acyl-CoA to diacylglycerol to produce triacylglycerol. There are three known types of DGAT referred to as DGAT1, DGAT2 and DGAT3 respectively. DGAT1 polypeptides typically have 10 transmembrane domains, DGAT2 typically have 2 transmembrane domains, whilst DGAT3 is typically soluble. Examples of DGAT1 polypeptides include polypeptides encoded by DGAT1 genes from Aspergillus fumigatus (Accession No. XP_755172), Arabidopsis thaliana (CAB44774), Ricinus communis (AAR11479), Vernicia fordii (ABC94472), Vernonia galamensis (ABV21945, ABV21946), Euonymus alatus (AAV31083), Caenorhabditis elegans (AAF82410), Rattus norvegicus (NP 445889), Homo sapiens (NP_036211), as well as variants and/or mutants thereof. Examples of DGAT2 polypeptides include polypeptides encoded by DGAT2 genes from Arabidopsis thaliana (Accession No. NP_566952), Ricinus communis (AAY16324), Vernicia fordii (ABC94474), Mortierella ramanniana (AAK84179), Homo sapiens (Q96PD7, Q58HT5), Bos taurus (Q70VD8), Mus musculus (AAK84175), Micromonas CCMP1545, as well as variants and/or mutants thereof. Examples of DGAT3 polypeptides include polypeptides encoded by DGAT3 genes from peanut (Arachis hypogaea, Saha, et al., 2006), as well as variants and/or mutants thereof.

Polypeptides/Peptides

The term “recombinant” in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate, compared to its native state if it is produced naturally. In one embodiment the cell is a cell that does not naturally produce the polypeptide.

However, the cell may be a cell which comprises a non-endogenous gene that causes an altered amount of the polypeptide to be produced. A recombinant polypeptide of the invention includes polypeptides in the cell, tissue, organ or organism, or cell-free expression system, in which it is produced i.e. a polypeptide which has not been purified or separated from other components of the transgenic (recombinant) cell in which it was produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.

The terms “polypeptide” and “protein” are generally used interchangeably.

A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns two sequences over their entire length. The polypeptide or class of polypeptides may have the same enzymatic activity as, or a different activity than, or lack the activity of, the reference polypeptide. Preferably, the polypeptide has an enzymatic activity of at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the reference polypeptide.

As used herein a “biologically active” fragment is a portion of a polypeptide defined herein which maintains a defined activity of a full-length reference polypeptide, for example possessing desaturase and/or elongase activity or other enzyme activity. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size portion as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.

With regard to a defined polypeptide or enzyme, it will be appreciated that % identity figures higher than those provided herein will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide/enzyme comprises an amino acid sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Amino acid sequence variants/mutants of the polypeptides of the defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such variants/mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired enzyme activity.

Mutant (altered) peptides can be prepared using any technique known in the art. For example, a polynucleotide defined herein can be subjected to in vitro mutagenesis or DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess, for example, desaturase or elongase activity.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites which are not conserved amongst naturally occurring desaturases or elongases. These sites are preferably substituted in a relatively conservative manner in order to maintain enzyme activity. Such conservative substitutions are shown in Table 3 under the heading of “exemplary substitutions”.

In a preferred embodiment a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 3. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.

Polypeptides can be produced in a variety of ways, including production and recovery of natural polypeptides or recombinant polypeptides according to methods known in the art. In one embodiment, a recombinant polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, such as a host cell defined herein. A more preferred cell to produce the polypeptide is a cell in a plant, especially in a seed in a plant.

Polynucleotides

The invention also provides and/or uses polynucleotides which may be, for example, a gene, an isolated polynucleotide, a chimeric genetic construct such as a T-DNA molecule, or a chimeric DNA. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein or other materials to perform a particular activity defined herein. The term “polynucleotide” is used interchangeably herein with the term “nucleic acid molecule”. By “isolated polynucleotide” we mean a polynucleotide which, if obtained from a natural source, has been separated from the polynucleotide sequences with which it is associated or linked in its native state, or a non-naturally occurring polynucleotide. Preferably, the isolated polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.


TABLE 3
Exemplary substitutions.
Original
Exemplary
Residue
Substitutions
Ala (A)
val; leu; ile; gly
Arg (R)
lys
Asn (N)
gln; his
Asp (D)
glu
Cys (C)
ser
Gln (Q)
asn; his
Glu (E)
asp
Gly (G)
pro, ala
His (H)
asn; gln
Ile (I)
leu; val; ala
Leu (L)
ile; val; met; ala; phe
Lys (K)
arg
Met (M)
leu; phe
Phe (F)
leu; val; ala
Pro (P)
gly
Ser (S)
thr
Thr (T)
ser
Trp (W)
tyr
Tyr (Y)
trp; phe
Val (V)
ile; leu; met; phe, ala

In an embodiment, a polynucleotide of the invention is non-naturally occurring. Examples of non-naturally occurring polynucleotides include, but are not limited to, those that have been mutated (such as by using methods described herein), and polynucleotides where an open reading frame encoding a protein is operably linked to a promoter to which it is not naturally associated (such as in the constructs described herein).

As used herein, the term “gene” is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the transcribed region and, if translated, the protein coding region, of a structural gene and including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of at least about 2 kb on either end and which are involved in expression of the gene. In this regard, the gene includes control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals in which case the gene is referred to as a “chimeric gene”. The sequences which are located 5′ of the protein coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the protein coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.

As used herein, a “chimeric DNA” or “chimeric genetic construct” refers to any DNA molecule that is not a native DNA molecule in its native location, also referred to herein as a “DNA construct”. Typically, a chimeric DNA or chimeric gene comprises regulatory and transcribed or protein coding sequences that are not found operably linked together in nature i.e. that are heterologous with respect to each other. Accordingly, a chimeric DNA or chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.

The term “endogenous” is used herein to refer to a substance that is normally present or produced in, for example, an unmodified plant at the same developmental stage as the plant under investigation. An “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, “recombinant nucleic acid molecule”, “recombinant polynucleotide” or variations thereof refer to a nucleic acid molecule which has been constructed or modified by recombinant DNA technology. The terms “foreign polynucleotide” or “exogenous polynucleotide” or “heterologous polynucleotide” and the like refer to any nucleic acid which is introduced into the genome of a cell by experimental manipulations. Foreign or exogenous genes may be genes that are inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. The terms “genetically modified”, “transgenic” and variations thereof include introducing genes into cells by transformation or transduction, mutating genes in cells and altering or modulating the regulation of a gene in a cell or organisms to which these acts have been done or their progeny. A “genomic region” as used herein refers to a position within the genome where a transgene, or group of transgenes (also referred to herein as a cluster), have been inserted into a cell, or an ancestor thereof. Such regions only comprise nucleotides that have been incorporated by the intervention of man such as by methods described herein.

The term “exogenous” in the context of a polynucleotide refers to the polynucleotide when present in a cell in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The exogenous polynucleotide (nucleic acid) can be a contiguous stretch of nucleotides existing in nature, or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide. Typically such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest.

As used herein, the term “different exogenous polynucleotides” or variations thereof means that the nucleotide sequence of each polynucleotide are different by at least one, preferably more, nucleotides. The polynucleotides encode RNAs which may or may not be translated to a protein within the cell. In an example, it is preferred that each polynucleotide encodes a protein with a different activity. In another example, each exogenous polynucleotide is less than 95%, less than 90%, or less than 80% identical to the other exogenous polynuclotides. Preferably, the exogenous polynucleotides encode functional proteins/enzymes. Furthermore, it is preferred that the different exogenous polynucleotides are non-overlapping in that each polynucleotide is a distinct region of the, for example, extrachromosomal transfer nucleic acid which does not overlap with another exogenous polynucleotide. At a minimum, each exogenous polnucleotide has a transcription start and stop site, as well as the designated promoter. An individual exogenous polynucloeotide may or may not comprise introns.

With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

A polynucleotide of the present invention may selectively hybridise, under stringent conditions, to a polynucleotide that encodes a polypeptide of the present invention. As used herein, stringent conditions are those that (1) employ during hybridisation a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS and/or (3) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.

Polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above). It is thus apparent that polynucleotides of the invention can be either from a naturally occurring source or recombinant. Preferred polynucleotides are those which have coding regions that are codon-optimised for translation in plant cells, as is known in the art.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector, which comprises at least one polynucleotide molecule defined herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Recombinant vectors include expression vectors. Recombinant vectors contain heterologous polynucleotide sequences, that is, polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules defined herein that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA and typically is a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, or preferably binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (especially plant) cells. The recombinant vector may comprise more than one polynucleotide defined herein, for example three, four, five or six polynucleotides defined herein in combination, preferably a chimeric genetic construct of the invention, each polynucleotide being operably linked to expression control sequences that are operable in the cell of interest. More than one polynucleotide defrined herein, for example 3, 4, 5 or 6 polynucleotides, are preferably covalently joined together in a single recombinant vector, preferably within a single T-DNA molecule, which may then be introduced as a single molecule into a cell to form a recombinant cell according to the invention, and preferably integrated into the genome of the recombinant cell, for example in a transgenic plant. Thereby, the polynucleotides which are so joined will be inherited together as a single genetic locus in progeny of the recombinant cell or plant. The recombinant vector or plant may comprise two or more such recombinant vectors, each containing multiple polynucleotides, for example wherein each recombinant vector comprises 3, 4, 5 or 6 polynucleotides.

“Operably linked” as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter may independently be the same or different.

Recombinant molecules such as the chimeric DNAs or genetic constructs may also contain (a) one or more secretory signals which encode signal peptide sequences, to enable an expressed polypeptide defined herein to be secreted from the cell that produces the polypeptide or which provide for localisation of the expressed polypeptide, for example for retention of the polypeptide in the endoplasmic reticulum (ER) in the cell or transfer into a plastid, and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion or localisation of a polypeptide defined herein. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules defined herein.

To facilitate identification of transformants, the nucleic acid construct desirably comprises a selectable or screenable marker gene as, or in addition to, the foreign or exogenous polynucleotide. By “marker gene” is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can “select” based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, i.e., by “screening” (e.g., β-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells). The marker gene and the nucleotide sequence of interest do not have to be linked. The actual choice of a marker is not crucial as long as it is functional (i.e., selective) in combination with the cells of choice such as a plant cell.

Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin, paromomycin, G418; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as, for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described in WO 87/05327, an acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as, for example, described by Hinchee et al. (1988), a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidA gene encoding a β-glucuronidase (GUS) enzyme for which various chromogenic substrates are known, a green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al., 1986), which allows for bioluminescence detection, and others known in the art. By “reporter molecule” as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that facilitates determination of promoter activity by reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into the genome of the cell, such as the plant cell. Accordingly, the nucleic acid may comprise appropriate elements which allow the molecule to be incorporated into the genome, preferably the right and left border sequences of a T-DNA molecule, or the construct is placed in an appropriate vector which can be incorporated into a chromosome of the cell.

Expression

As used herein, an expression vector is a DNA vector that is capable of transforming a host cell and of effecting expression of one or more specified polynucleotide molecule(s). Preferred expression vectors of the present invention can direct gene expression in yeast and/or plant cells. Expression vectors useful for the invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, polynucleotides or vectors useful for the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter and enhancer sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. The choice of the regulatory sequences used depends on the target organism such as a plant and/or target organ or tissue of interest. Such regulatory sequences may be obtained from any eukaryotic organism such as plants or plant viruses, or may be chemically synthesized. A variety of such transcription control sequences are known to those skilled in the art. Particularly preferred transcription control sequences are promoters active in directing transcription in plants, either constitutively or stage and/or tissue specific, depending on the use of the plant or parts thereof.

A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

A number of constitutive promoters that are active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of the ribulose-1,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate isomerase promoter, the adenine phosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 gene promoter, the mannopine synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll α/β binding protein gene promoter

For the purpose of expression in source tissues of the plant, such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific or -enhanced expression. Examples of such promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose-1,6-biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the serine/threonine kinase promoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana. Also reported to be active in photosynthetically active tissues are ribulose-1,5-bisphosphate carboxylase promoters, and Cab promoters.

A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of genes in plant cells, including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter); (3) hormones, such as abscisic acid, (4) wounding (e.g., WunI); or (5) chemicals, such as methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO97/06269), or it may also be advantageous to employ (6) organ-specific promoters.

As used herein, the term “plant seed specific promoter” or variations thereof refer to a promoter that preferentially, when compared to other plant tissues, directs gene transcription in a developing seed of a plant. In an embodiment, the seed specific promoter is expressed at least 5-fold more strongly in the developing seed of the plant relative to the leaves and/or stems of the plant, and is preferably expressed more strongly in the embryo of the developing seed compared to other plant tissues. Preferably, the promoter only directs expression of a gene of interest in the developing seed, and/or expression of the gene of interest in other parts of the plant such as leaves is not detectable by Northern blot analysis and/or RT-PCR. Typically, the promoter drives expression of genes during growth and development of the seed, in particular during the phase of synthesis and accumulation of storage compounds in the seed. Such promoters may drive gene expression in the entire plant storage organ or only part thereof such as the seedcoat, or cotyledon(s), preferably in the embryos, in seeds of dicotyledonous plants or the endosperm or aleurone layer of a seeds of monocotyledonous plants.

Preferred promoters for seed-specific expression include i) promoters from genes encoding enzymes involved in fatty acid biosynthesis and accumulation in seeds, such as desaturases and elongases, ii) promoters from genes encoding seed storage proteins, and iii) promoters from genes encoding enzymes involved in carbohydrate biosynthesis and accumulation in seeds. Seed specific promoters which are suitable are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO91/13980) or the legumin LeB4 promoter from Vicia faba (Baumlein et al., 1992), and promoters which lead to the seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like. Notable promoters which are suitable are the barley lpt2 or lpt1 gene promoter (WO95/15389 and WO95/23230) or the promoters described in WO99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene). Other promoters include those described by Broun et al. (1998), Potenza et al. (2004), US20070192902 and US20030159173. In an embodiment, the seed specific promoter is preferentially expressed in defined parts of the seed such as the embryo, cotyledon(s) or the endosperm. Examples of such specific promoters include, but are not limited to, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000), the bean phytohemagglutnin promoter (Perrin et al., 2000), the conlinin 1 and conlinin 2 promoters for the genes encoding the flax 2S storage proteins (Cheng et al., 2010), the promoter of the FAE1 gene from Arabidopsis thaliana, the BnGLP promoter of the globulin-like protein gene of Brassica napus, the LPXR promoter of the peroxiredoxin gene from Linum usitatissimum.

The 5′ non-translated leader sequence can be derived from the promoter selected to express the heterologous gene sequence of the polynucleotide of the present invention, or preferably is heterologous with respect to the coding region of the enzyme to be produced, and can be specifically modified if desired so as to increase translation of mRNA. For a review of optimizing expression of transgenes, see Koziel et al. (1996). The 5′ non-translated regions can also be obtained from plant viral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, among others) from suitable eukaryotic genes, plant genes (wheat and maize chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5′ non-translated sequence that accompanies the promoter sequence. The leader sequence could also be derived from an unrelated promoter or coding sequence. Leader sequences useful in context of the present invention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865 and U.S. Pat. No. 5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translated DNA sequence operably linked in the chimeric vector to the polynucleotide of interest. The 3′ non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3′ end of the RNA. The 3′ non-translated region can be obtained from various genes that are expressed in plant cells. The nopaline synthase 3′ untranslated region, the 3′ untranslated region from pea small subunit Rubisco gene, the 3′ untranslated region from soybean 7S seed storage protein gene or a flax conlinin gene are commonly used in this capacity. The 3′ transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules defined herein include, but are not limited to, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of stability sequences to mRNAs, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.

Recombinant Cells

The invention also provides a recombinant cell, preferably a recombinant plant cell, which is a host cell transformed with one or more recombinant molecules, such as the polynucleotides, chimeric genetic constructs or recombinant vectors defined herein. The recombinant cell may comprise any combination thereof, such as two or three recombinant vectors, or a recombinant vector and one or more additional polynucleotides or chimeric DNAs. Suitable cells of the invention include any cell that can be transformed with a polynucleotide, chimeric DNA or recombinant vector of the invention, such as for example, a molecule encoding a polypeptide or enzyme described herein. The cell is preferably a cell which is thereby capable of being used for producing LC-PUFA. The recombinant cell may be a cell in culture, a cell in vitro, or in an organism such as for example a plant, or in an organ such as for example a seed or a leaf. Preferably, the cell is in a plant or plant part, more preferably in the seed of a plant.

Host cells into which the polynucleotide(s) are introduced can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Such nucleic acid molecules may be related to LC-PUFA synthesis, or unrelated. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing proteins defined herein, in which case the recombinant cell derived therefrom has an enhanced capability of producing the polypeptides, or can be capable of producing such proteins only after being transformed with at least one polynucleotide of the invention. In an embodiment, a recombinant cell of the invention has a enhanced capacity to synthesize a long chain polyunsaturated fatty acid. As used herein, the term “cell with an enhanced capacity to synthesize a long chain polyunsaturated fatty acid” is a relative term where the recombinant cell of the invention is compared to the host cell lacking the polynucleotide(s) of the invention, with the recombinant cell producing more long chain polyunsaturated fatty acids, or a greater concentration of LC-PUFA such as DHA (relative to other fatty acids), than the native cell. The cell with an enhanced capacity to synthesize another product, such as for example another fatty acid, a lipid, a carbohydrate such as starch, an RNA molecule, a polypeptide, a pharmaceutical or other product has a corresponding meaning.

Host cells of the present invention can be any cell capable of producing at least one protein described herein, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. The cells may be prokaryotic or eukaryotic. Preferred host cells are yeast and plant cells. In a preferred embodiment, the plant cell is a seed cell, in particular a cell in a cotyledon or endosperm of a seed. In one embodiment, the cell is an animal cell or an algal cell. The animal cell may be of any type of animal such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or cells of aquatic animals such as, fish or crustacea, invertebrates, insects, etc. The cells may be of an organism suitable for a fermentation process. As used herein, the term the “fermentation process” refers to any fermentation process or any process comprising a fermentation step. Examples of fermenting microorganisms include fungal organisms, such as yeast. As used herein, “yeast” includes Saccharomyces spp., Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowia lipolytica. Preferred yeast include strains of the Saccharomyces spp., and in particular, Saccharomyces cerevisiae.

Transgenic Plants

The invention also provides a plant comprising a cell of the invention, such as a transgenic plant comprising one or more polynucleotides of the invention. The term “plant” as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. The term “plant part” refers to all plant parts that comprise the plant DNA, including vegetative structures such as, for example, leaves or stems, roots, floral organs or structures, pollen, seed, seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue such as, for example, vascular tissue, cells and progeny of the same, as long as the plant part synthesizes lipid according to the invention.

A “transgenic plant”, “genetically modified plant” or variations thereof refers to a plant that contains a gene construct (“transgene”) not found in a wild-type plant of the same species, variety or cultivar. Transgenic plants as defined in the context of the present invention include plants and their progeny which have been genetically modified using recombinant techniques to cause production of the lipid or at least one polypeptide defined herein in the desired plant or plant organ. Transgenic plant cells and transgenic plant parts have corresponding meanings. A “transgene” as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into a cell of the invention, preferably a plant cell. The transgene may include genetic sequences derived from a plant cell which may be of the same species, variety or cultivar as the plant cell into which the transgene is introduced or of a different species, variety or cultivar, or from a cell other than a plant cell. Typically, the transgene has been introduced into the cell, such as a plant, by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes.

The terms “seed” and “grain” are used interchangeably herein. “Grain” refers to mature grain such as harvested grain or grain which is still on a plant but ready for harvesting, but can also refer to grain after imbibition or germination, according to the context. Mature grain or seed commonly has a moisture content of less than about 18-20%. “Developing seed” as used herein refers to a seed prior to maturity, typically found in the reproductive structures of the plant after fertilisation or anthesis, but can also refer to such seeds prior to maturity which are isolated from a plant.

As used herein, the term “obtaining a plant part” or “obtaining a seed” refers to any means of obtaining a plant part or seed, respectively, including harvesting of the plant parts or seed from plants in the field or in containment such as a greenhouse or growth chamber, or by purchase or receipt from a supplier of the plant parts or seed.

The seed may be suitable for planting i.e. able to germinate and produce progeny plants, or alternatively has been processed in such a way that it is no longer able to germinate, e.g. cracked, polished or milled seed which is useful for food or feed applications, or for extraction of lipid of the invention.

As used herein, the term “plant storage organ” refers to a part of a plant specialized to storage energy in the form of, for example, proteins, carbohydrates, fatty acids and/or oils. Examples of plant storage organs are seed, fruit, tuberous roots, and tubers. A preferred plant storage organ of the invention is seed.

As used herein, the term “phenotypically normal” refers to a genetically modified plant or plant organ, particularly a storage organ such as a seed, tuber or fruit of the invention not having a significantly reduced ability to grow and reproduce when compared to an unmodified plant or plant organ. In an embodiment, the genetically modified plant or plant organ which is phenotypically normal comprises an exogenous polynucleotide encoding a silencing suppressor operably linked to a plant storage organ specific promoter and has an ability to grow or reproduce which is essentially the same as an isogenic plant or organ not comprising said polynucleotide. Preferably, the biomass, growth rate, germination rate, storage organ size, seed size and/or the number of viable seeds produced is not less than 90% of that of a plant lacking said exogenous polynucleotide when grown under identical conditions. This term does not encompass features of the plant which may be different to the wild-type plant but which do not effect the usefulness of the plant for commercial purposes such as, for example, a ballerina phenotype of seedling leaves.

Plants provided by or contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. In preferred embodiments, the plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or pea), or other legumes. The plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit. The plants may be vegetables or ornamental plants. The plants of the invention may be: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), mustard (Brassica juncea), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, or barley.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an “oilseed plant” is a plant species used for the commercial production of oils from the seeds of the plant. The oilseed plant may be oil-seed rape (such as canola), maize, sunflower, soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseed plant may be other Brassicas, cotton, peanut, poppy, mustard, castor bean, sesame, safflower, or nut producing plants. The plant may produce high levels of oil in its fruit, such as olive, oil palm or coconut. Horticultural plants to which the present invention may be applied are lettuce, endive, or vegetable brassicas including cabbage, broccoli, or cauliflower. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper.

In a further preferred embodiment, the non-transgenic plant used to produce a transgenic plant of the invention produces oil, especially in the seed, which has i) less than 20%, less than 10% or less than 5% 18:2 fatty acids and/or ii) less than 10% or less than 5% 18:3 fatty acids.

In a preferred embodiment, the transgenic plant is homozygous for each and every gene that has been introduced (transgene) so that its progeny do not segregate for the desired phenotype. The transgenic plant may also be heterozygous for the introduced transgene(s), preferably uniformly heterozygous for the transgene, such as for example in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.

Where relevant, the transgenic plants may also comprise additional transgenes encoding enzymes involved in the production of LC-PUFAs such as, but not limited to, a Δ6-desaturase, a Δ9-elongase, a Δ8-desaturase, a Δ6-elongase, a Δ5-desaturase, an ω3-desaturase, a Δ4-desaturase, a Δ5-elongase, diacylglycerol acyltransferase, LPAAT, a Δ17-desaturase, a Δ15-desaturase and/or a Δ12 desaturase. Examples of such enzymes with one of more of these activities are known in the art and include those described herein. In specific examples, the transgenic plant at least comprises exogenous polynucleotides encoding;

a) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase and a Δ6-elongase,

b) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase and a Δ9-elongase,

c) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, a Δ6-elongase, and a Δ15-desaturase,

d) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, a Δ9-elongase, and a Δ15-desaturase,

e) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, a Δ6-elongase, and a Δ17-desaturase, or

f) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, a Δ9-elongase, and a Δ17-desaturase.

In an embodiment, the exogenous polynucleotides encode set of polypeptides which are a Pythium irregulare Δ6-desaturase, a Thraustochytrid Δ5-desaturase or an Emiliana huxleyi Δ5-desaturase, a Physcomitrella patens Δ6-elongase, a Thraustochytrid Δ5-elongase or an Ostreocccus taurii Δ5-elongase, a Phytophthora infestans ω3-desaturase or a Pythium irregulare ω3-desaturase, and a Thraustochytrid Δ4-desaturase.

In an embodiment, plants of the invention are grown in the field, preferably as a population of at least 1,000 or 1,000,000 plants that are essentially the same, or in an area of at least 1 hectare. Planting densities differ according to the plant species, plant variety, climate, soil conditions, fertiliser rates and other factors as known in the art. For example, canola is typically grown at a planting density of 1.2-1.5 million plants per hectare. Plants are harvested as is known in the art, which may comprise swathing, windrowing and/or reaping of plants, followed by threshing and/or winnowing of the plant material to separate the seed from the remainder of the plant parts often in the form of chaff. Alternatively, seed may be harvested from plants in the field in a single process, namely combining.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology—The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed” and variations thereof refer to the integration of the exogenous nucleic acid molecules into the genome of the cell such that they are transferred to progeny cells during cell division without the need for positively selecting for their presence. Stable transformants, or progeny thereof, can be selected by any means known in the art such as Southern blots on chromosomal DNA or in situ hybridization of genomic DNA.

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because DNA can be introduced into cells in whole plant tissues or plant organs or explants in tissue culture, for either transient expression or for stable integration of the DNA in the plant cell genome. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863 or U.S. Pat. No. 5,159,135) including floral dipping methods using Agrobacterium or other bacteria that can transfer DNA into plant cells. The region of DNA to be transferred is defined by the border sequences, and the intervening DNA (T-DNA) is usually inserted into the plant genome. Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. In those plant varieties where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer. Preferred Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203 (1985).

Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required.

In another alternative embodiment, plastids can be stably transformed. Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402, 5,932,479, and WO99/05265).

Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.

To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

A transgenic plant formed using Agrobacterium or other transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene(s). More preferred is a transgenic plant that is homozygous for the added gene(s); i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by self-fertilising a hemizygous transgenic plant, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants that contain two independently segregating exogenous genes or loci can also be crossed (mated) to produce offspring that contain both sets of genes or loci. Selfing of appropriate F1 progeny can produce plants that are homozygous for both exogenous genes or loci. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Enhancing Exogenous RNA Levels and Stabilized Expression

Silencing Suppressors

In an embodiment, a cell, plant or plant part of the invention comprises an exogenous polynucleotide encoding a silencing suppressor protein.

Post-transcriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs for degradation PTGS occurs in plants or fungi stably or transiently transformed with foreign (heterologous) or endogenous DNA and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced nucleic acid.

It has widely been considered that co-expression of a silencing suppressor with a transgene of interest will increase the levels of RNA present in the cell transcribed from the transgene. Whilst this has proven true for cells in vitro, significant side-effects have been observed in many whole plant co-expression studies. More specifically, as described in Mallory et al. (2002), Chapman et al. (2004), Chen et al. (2004), Dunoyer et al. (2004), Zhang et al. (2006), Lewsey et al. (2007) and Meng et al. (2008) plants expressing silencing suppressors, generally under constitutive promoters, are often phenotypically abnormal to the extent that they are not useful for commercial production.

Recently, it has been found that RNA molecule levels can be increased, and/or RNA molecule levels stabilized over numerous generations, by limiting the expression of the silencing suppressor to a seed of a plant or part thereof (WO2010/057246). As used herein, a “silencing suppressor protein” or SSP is any polypeptide that can be expressed in a plant cell that enhances the level of expression product from a different transgene in the plant cell, particularly over repeated generations from the initially transformed plant. In an embodiment, the SSP is a viral silencing suppressor or mutant thereof. A large number of viral silencing suppressors are known in the art and include, but are not limited to P19, V2, P38, Pe-Po and RPV-P0. In an embodiment, the viral silencing suppressor comprises amino acids having a sequence as provided in any one of SEQ ID NOs 53 to 57, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 53 to 57 and which has activity as a silencing suppressor.

As used herein, the terms “stabilising expression”, “stably expressed”, “stabilised expression” and variations thereof refer to level of the RNA molecule being essentially the same or higher in progeny plants over repeated generations, for example at least three, at least five or at least 10 generations, when compared to isogenic plants lacking the exogenous polynucleotide encoding the silencing suppressor. However, this term(s) does not exclude the possibility that over repeated generations there is some loss of levels of the RNA molecule when compared to a previous generation, for example not less than a 10% loss per generation.

The suppressor can be selected from any source e.g. plant, viral, mammal etc. See WO2010/057246 for a list of viruses from which the suppressor can be obtained and the protein (eg B2, P14 etc) or coding region designation for the suppressor from each particular virus. Multiple copies of a suppressor may be used. Different suppressors may be used together (e. g., in tandem).

RNA Molecules

Essentially any RNA molecule which is desirable to be expressed in a plant seed can be co-expressed with the silencing suppressor. The encoded polypeptides may be involved in metabolism of oil, starch, carbohydrates, nutrients, etc., or may be responsible for the synthesis of proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids. hormones, polymers, flavonoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins, glycolipids, etc, preferably the biosynthesis or assembly of TAG.

In a particular example, the plants produced increased levels of enzymes for oil production in plants such as Brassicas, for example canola or sunflower, safflower, flax, cotton, soya bean, Camelina or maize.

Levels of LC-PUFA Produced

The levels of the LC-PUFA or combination of LC-PUFAs that are produced in the recombinant cell or plant part such as seed are of importance. The levels may be expressed as a composition (in percent) of the total fatty acid that is a particular LC-PUFA or group of related LC-PUFA, for example the ω3 LC-PUFA or the ω6 LC-PUFA, or the VLC-PUFA, or other which may be determined by methods known in the art. The level may also be expressed as a LC-PUFA content, such as for example the percentage of LC-PUFA in the dry weight of material comprising the recombinant cells, for example the percentage of the weight of seed that is LC-PUFA. It will be appreciated that the LC-PUFA that is produced in an oilseed may be considerably higher in terms of LC-PUFA content than in a vegetable or a grain that is not grown for oil production, yet both may have similar LC-PUFA compositions, and both may be used as sources of LC-PUFA for human or animal consumption.

The levels of LC-PUFA may be determined by any of the methods known in the art. In a preferred method, total lipid is extracted from the cells, tissues or organisms and the fatty acid converted to methyl esters before analysis by gas chromatography (GC). Such techniques are described in Example 1. The peak position in the chromatogram may be used to identify each particular fatty acid, and the area under each peak integrated to determine the amount. As used herein, unless stated to the contrary, the percentage of particular fatty acid in a sample is determined as the area under the peak for that fatty acid as a percentage of the total area for fatty acids in the chromatogram. This corresponds essentially to a weight percentage (w/w). The identity of fatty acids may be confirmed by GC-MS. Total lipid may be separated by techniques known in the art to purify fractions such as the TAG fraction. For example, thin-layer chromatography (TLC) may be performed at an analytical scale to separate TAG from other lipid fractions such as DAG, acyl-CoAs or phospholipid in order to determine the fatty acid composition specifically of TAG.

In one embodiment, the sum total of ARA, EPA, DPA and DHA in the fatty acids in the extracted lipid is between about 7% and about 25% of the total fatty acids in the cell. In a further embodiment, the total fatty acid in the cell has less than 1% C20:1. In preferred embodiments, the extractable TAG in the cell comprises the fatty acids at the levels referred to herein. Each possible combination of the features defining the lipid as described herein is also encompassed.

The level of production of LC-PUFA in the recombinant cell, plant or plant part such as seed may also be expressed as a conversion percentage of a specific substrate fatty acid to one or more product fatty acids, which is also referred to herein as a “conversion efficiency” or “enzymatic efficiency”. This parameter is based on the fatty acid composition in the lipid extracted from the cell, plant, plant part or seed, i.e., the amount of the LC-PUFA formed (including other LC-PUFA derived therefrom) as a percentage of one or more substrate fatty acids (including all other fatty acids derived therefrom). The general formula for a conversion percentage is: 100×(the sum of percentages of the product LC-PUFA and all products derived therefrom)/(the sum of the percentages of the substrate fatty acid and all products derived therefrom). With regard to DHA, for example, this may be expressed as the ratio of the level of DHA (as a percentage in the total fatty acid content in the lipid) to the level of a substrate fatty acid (e.g. OA, LA, ALA, SDA, ETA or EPA) and all products other than DHA derived from the substrate. The conversion percentage or efficiency of conversion can be expressed for a single enzymatic step in a pathway, or for part or the whole of a pathway.

Specific conversion efficiencies are calculated herein according to the formulae:

  • 1. OA to DHA=100×(% DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 2. LA to DHA=100×(% DHA)/(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 3. ALA to DHA=100×(% DHA)/(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 4. EPA to DHA=100×(% DHA)/(sum % for EPA, DPA and DHA).
  • 5. DPA to DHA (Δ4-desaturase efficiency)=100×(% DHA)/(sum % for DPA and DHA).
  • 6. Δ12-desaturase efficiency=100×(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 7. ω3-desaturase efficiency=100×(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 8. OA to ALA=100×(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 9. Δ6-desaturase efficiency (on ω3 substrate ALA)=100×(sum % for SDA, ETA, EPA, DPA and DHA)/(% ALA, SDA, ETrA, ETA, EPA, DPA and DHA).
  • 10. Δ6-elongase efficiency (on ω3 substrate SDA)=100×(sum % for ETA, EPA, DPA and DHA)/(sum % for SDA, ETA, EPA, DPA and DHA).
  • 11. Δ5-desaturase efficiency (on ω3 substrate ETA)=100×(sum % for EPA, DPA and DHA)/(sum % for ETA, EPA, DPA and DHA).
  • 12. Δ5-elongase efficiency (on ω3 substrate EPA)=100×(sum % for DPA and DHA)/(sum % for EPA, DPA and DHA).

The fatty acid composition of the lipid, preferably seedoil, of the invention, is also characterised by the ratio of ω6 fatty acids:ω3 fatty acids in the total fatty acid content, for either total ω6 fatty acids:total ω3 fatty acids or for new ω6 fatty acids:new ω3 fatty acids. The terms total ω6 fatty acids, total ω3 fatty acids, new ω6 fatty acids and new ω3 fatty acids have the meanings as defined herein. The ratios are calculated from the fatty acid composition in the lipid extracted from the cell, plant, plant part or seed, in the manner as exemplified herein. It is desirable to have a greater level of ω3 than ω6 fatty acids in the lipid, and therefore an ω6:ω3 ratio of less than 1.0 is preferred. A ratio of 0.0 indicates a complete absence of the defined ω6 fatty acids; a ratio of 0.03 was achieved as described in Example 6. Such low ratios can be achieved through the combined use of a Δ6-desaturase which has an ω3 substrate preference together with an ω3-desaturase, particularly a fungal ω3-desaturase such as the Pichia pastoris ω3-desaturase as exemplified herein.

The yield of LC-PUFA per weight of seed may also be calculated based on the total oil content in the seed and the % DHA in the oil. For example, if the oil content of canola seed is about 40% (w/w) and about 12% of the total fatty acid content of the oil is DHA, the DHA content of the seed is about 4.8% or about 48 mg per gram of seed. As described in Example 2, the DHA content of Arabidopsis seed having about 9% DHA, which has a lower oil content than canola, was about 25 mg/g seed. At a DHA content of about 7%, canola seed or Camelina sativa seed has a DHA content of about 28 mg per gram of seed. The present invention therefore provides Brassica napus, B. juncea and Camelina sativa plants, and seed obtained therefrom, comprising at least about 28 mg DHA per gram seed. The seed has a moisture content as is standard for harvested mature seed after drying down (4-15% moisture). The invention also provides a process for obtaining oil, comprising obtaining the seed and extracting the oil from the seed, and uses of the oil and methods of obtaining the seed comprising harvesting the seeds from the plants according to the invention.

The amount of DHA produced per hectare can also be calculated if the seed yield per hectare is known or can be estimated. For example, canola in Australia typically yields about 2.5 tonnes seed per hectare, which at 40% oil content yields about 1000 kg of oil. At 12% DHA in the total oil, this provides about 120 kg of DHA per hectare. If the oil content is reduced by 50%, this still provides about 60 kg DHA/ha.

Evidence to date suggests that some desaturases expressed heterologously in yeast or plants have relatively low activity in combination with some elongases. This may be alleviated by providing a desaturase with the capacity of to use an acyl-CoA form of the fatty acid as a substrate in LC-PUFA synthesis, and this is thought to be advantageous in recombinant cells particularly in plant cells. A particularly advantageous combination for efficient DHA synthesis is a fungal ω3-desaturase, for example such as the Pichia pastoris ω3-desaturase (SEQ ID NO: 12), with a Δ6-desaturase which has a preference for ω3 acyl substrates such as, for example, the Micromonas pusilla Δ6-desaturase (SEQ ID NO: 13), or variants thereof which have at least 95% amino acid sequence identity.

As used herein, the term “essentially free” means that the composition (for example lipid or oil) comprises little (for example, less than about 0.5%, less than about 0.25%, less than about 0.1%, or less than about 0.01%) or none of the defined component. In an embodiment, “essentially free” means that the component is undetectable using a routine analytical technique, for example a specific fatty acid (such as ω6-docosapentaenoic acid) cannot be detected using gas chromatography as outlined in Example 1.

Production of Oils

Techniques that are routinely practiced in the art can be used to extract, process, and analyze the oils produced by cells, plants, seeds, etc of the instant invention. Typically, plant seeds are cooked, pressed, and extracted to produce crude oil, which is then degummed, refined, bleached, and deodorized. Generally, techniques for crushing seed are known in the art. For example, oilseeds can be tempered by spraying them with water to raise the moisture content to, e.g., 8.5%, and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm. Depending on the type of seed, water may not be added prior to crushing. Application of heat deactivates enzymes, facilitates further cell rupturing, coalesces the oil droplets, and agglomerates protein particles, all of which facilitate the extraction process.

In an embodiment, the majority of the seed oil is released by passage through a screw press. Cakes expelled from the screw press are then solvent extracted, e.g., with hexane, using a heat traced column. Alternatively, crude oil produced by the pressing operation can be passed through a settling tank with a slotted wire drainage top to remove the solids that are expressed with the oil during the pressing operation. The clarified oil can be passed through a plate and frame filter to remove any remaining fine solid particles. If desired, the oil recovered from the extraction process can be combined with the clarified oil to produce a blended crude oil.

Once the solvent is stripped from the crude oil, the pressed and extracted portions are combined and subjected to normal oil processing procedures. As used herein, the term “purified” when used in connection with lipid or oil of the invention typically means that that the extracted lipid or oil has been subjected to one or more processing steps of increase the purity of the lipid/oil component. For example, a purification step may comprise one or more or all of the group consisting of: degumming, deodorising, decolourising, drying and/or fractionating the extracted oil. However, as used herein, the term “purified” does not include a transesterification process or other process which alters the fatty acid composition of the lipid or oil of the invention so as to increase the DHA content as a percentage of the total fatty acid content. Expressed in other words, the fatty acid composition of the purified lipid or oil is essentially the same as that of the unpurified lipid or oil.

Degumming

Degumming is an early step in the refining of oils and its primary purpose is the removal of most of the phospholipids from the oil, which may be present as approximately 1-2% of the total extracted lipid. Addition of ˜2% of water, typically containing phosphoric acid, at 70-80° C. to the crude oil results in the separation of most of the phospholipids accompanied by trace metals and pigments. The insoluble material that is removed is mainly a mixture of phospholipids and triacylglycerols and is also known as lecithin. Degumming can be performed by addition of concentrated phosphoric acid to the crude seedoil to convert non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present. Gum is separated from the seedoil by centrifugation.

Alkali Refining

Alkali refining is one of the refining processes for treating crude oil, sometimes also referred to as neutralization. It usually follows degumming and precedes bleaching. Following degumming, the seedoil can treated by the addition of a sufficient amount of an alkali solution to titrate all of the fatty acids and phosphoric acids, and removing the soaps thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate and ammonium hydroxide. This process is typically carried out at room temperature and removes the free fatty acid fraction. Soap is removed by centrifugation or by extraction into a solvent for the soap, and the neutralised oil is washed with water. If required, any excess alkali in the oil may be neutralized with a suitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

Bleaching is a refining process in which oils are heated at 90-120° C. for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and in the absence of oxygen by operating with nitrogen or steam or in a vacuum. This step in oil processing is designed to remove unwanted pigments (carotenoids, chlorophyll, gossypol etc), and the process also removes oxidation products, trace metals, sulphur compounds and traces of soap.

Deodorization

Deodorization is a treatment of oils and fats at a high temperature (200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achieved by introducing steam into the seedoil at a rate of about 0.1 ml/minute/100 ml of seedoil. After about 30 minutes of sparging, the seedoil is allowed to cool under vacuum. The seedoil is typically transferred to a glass container and flushed with argon before being stored under refrigeration. This treatment improves the colour of the seedoil and removes a majority of the volatile substances or odorous compounds including any remaining free fatty acids, monoacylglycerols and oxidation products.

Winterisation

Winterization is a process sometimes used in commercial production of oils for the separation of oils and fats into solid (stearin) and liquid (olein) fractions by crystallization at sub-ambient temperatures. It was applied originally to cottonseed oil to produce a solid-free product. It is typically used to decrease the saturated fatty acid content of oils.

Transesterification

Transesterification is a process that exchanges the fatty acids within and between TAGs or transfers the fatty acids to another alcohol to form an ester, initially by releasing fatty acids from the TAGs either as free fatty acids or as fatty acid esters, usually fatty acid methyl esters or ethyl esters. When combined with a fractionation process, transesterification can be used to modify the fatty acid composition of lipids (Marangoni et al., 1995). Transesterification can use either chemical (e.g. strong acid or base catalysed) or enzymatic means, the latter using lipases which may be position-specific (sn-1/3 or sn-2 specific) for the fatty acid on the TAG, or having a preference for some fatty acids over others (Speranza et al, 2012). The fatty acid fractionation to increase the concentration of LC-PUFA in an oil can be achieved by any of the methods known in the art, such as, for example, freezing crystallization, complex formation using urea, molecular distillation, supercritical fluid extraction and silver ion complexing. Complex formation with urea is a preferred method for its simplicity and efficiency in reducing the level of saturated and monounsaturated fatty acids in the oil (Gamez et al., 2003). Initially, the TAGs of the oil are split into their constituent fatty acids, often in the form of fatty acid esters, by hydrolysis under either acid or base catalysed reaction conditions, whereby one mol of TAG is reacted with at least 3 mol of alcohol (e.g. ethanol for ethyl esters or methanol for methyl esters) with excess alcohol used to enable separation of the formed alkyl esters and the glycerol that is also formed, or by lipases. These free fatty acids or fatty acid esters, which are usually unaltered in fatty acid composition by the treatment, may then be mixed with an ethanolic solution of urea for complex formation. The saturated and monounsaturated fatty acids easily complex with urea and crystallize out on cooling and may subsequently be removed by filtration. The non-urea complexed fraction is thereby enriched with LC-PUFA.

Feedstuffs

The present invention includes compositions which can be used as feedstuffs. For purposes of the present invention, “feedstuffs” include any food or preparation for human or animal consumption which when taken into the body (a) serve to nourish or build up tissues or supply energy; and/or (b) maintain, restore or support adequate nutritional status or metabolic function. Feedstuffs of the invention include nutritional compositions for babies and/or young children such as, for example, infant formula, and seedmeal of the invention.

Feedstuffs of the invention comprise, for example, a cell of the invention, a plant of the invention, the plant part of the invention, the seed of the invention, an extract of the invention, the product of the method of the invention, the product of the fermentation process of the invention, or a composition along with a suitable carrier(s). The term “carrier” is used in its broadest sense to encompass any component which may or may not have nutritional value. As the skilled addressee will appreciate, the carrier must be suitable for use (or used in a sufficiently low concentration) in a feedstuff such that it does not have deleterious effect on an organism which consumes the feedstuff.

The feedstuff of the present invention comprises an oil, fatty acid ester, or fatty acid produced directly or indirectly by use of the methods, cells or plants disclosed herein. The composition may either be in a solid or liquid form. Additionally, the composition may include edible macronutrients, protein, carbohydrate, vitamins, and/or minerals in amounts desired for a particular use. The amounts of these ingredients will vary depending on whether the composition is intended for use with normal individuals or for use with individuals having specialized needs, such as individuals suffering from metabolic disorders and the like.

Examples of suitable carriers with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins. Examples of such edible fats include, but are not limited to, coconut oil, borage oil, fungal oil, black current oil, soy oil, and mono- and diglycerides. Examples of such carbohydrates include (but are not limited to): glucose, edible lactose, and hydrolyzed starch. Additionally, examples of proteins which may be utilized in the nutritional composition of the invention include (but are not limited to) soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to the feedstuff compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the present invention can be of semi-purified or purified origin. By semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added to food even when supplementation of the diet is not required. For example, the composition may be added to food of any type, including (but not limited to): margarine, modified butter, cheeses, milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats, fish and beverages.

The genus Saccharomyces spp is used in both brewing of beer and wine making and also as an agent in baking, particularly bread. Other yeasts such as oleaginous yeast including, for example, Yarrowia spp, are also useful in LC-PUFA production. Yeasts may be used as an additive in animal feed, such as in aquaculture. It will be apparent that genetically engineered yeast strains can be provided which are adapted to synthesise LC-PUFA as described herein. These yeast strains, or LC-PUFA produced therein, can then be used in food stuffs and in wine and beer making to provide products which have enhanced fatty acid content.

Additionally, fatty acids produced in accordance with the present invention or host cells transformed to contain and express the subject genes may also be used as animal food supplements to alter an animal's tissue, egg or milk fatty acid composition to one more desirable for human or animal consumption. Examples of such animals include sheep, cattle, horses, poultry such as chickens and the like.

Furthermore, feedstuffs of the invention can be used in aquaculture to increase the levels of fatty acids in fish or crustaceans such as, for example, prawns for human or animal consumption. Preferred fish are salmon.

Preferred feedstuffs of the invention are the plants, seed and other plant parts such as leaves and stems which may be used directly as food or feed for humans or other animals. For example, animals may graze directly on such plants grown in the field or be fed more measured amounts in controlled feeding. The invention includes the use of such plants and plant parts as feed for increasing the LC-PUFA levels in humans and other animals.

Compositions

The present invention also encompasses compositions, particularly pharmaceutical compositions, comprising one or more of the fatty acids and/or resulting oils produced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the fatty acids and/or oils, in combination with a standard, well-known, non-toxic pharmaceutically-acceptable carrier, adjuvant or vehicle such as phosphate-buffered saline, water, ethanol, polyols, vegetable oils, a wetting agent or an emulsion such as a water/oil emulsion. The composition may be in either a liquid or solid form. For example, the composition may be in the form of a tablet, capsule, ingestible liquid or powder, injectible, or topical ointment or cream. Proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfuming agents.

Suspensions, in addition to the active compounds, may comprise suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth or mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art. For example, fatty acids produced in accordance with the present invention can be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate. Capsules can be prepared by incorporating these excipients into a gelatin capsule along with antioxidants and the relevant fatty acid(s).

For intravenous administration, the fatty acids produced in accordance with the present invention or derivatives thereof may be incorporated into commercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g, taken from one to five times per day (up to 100 g daily) and is preferably in the range of from about 10 mg to about 1, 2, 5, or 10 g daily (taken in one or multiple doses). As known in the art, a minimum of about 300 mg/day of fatty acid, especially LC-PUFA, is desirable. However, it will be appreciated that any amount of fatty acid will be beneficial to the subject.

Possible routes of administration of the pharmaceutical compositions of the present invention include, for example, enteral (e.g., oral and rectal) and parenteral. For example, a liquid preparation may be administered orally or rectally. Additionally, a homogenous mixture can be completely dispersed in water, admixed under sterile conditions with physiologically acceptable diluents, preservatives, buffers or propellants to form a spray or inhalant.

The dosage of the composition to be administered to the patient may be determined by one of ordinary skill in the art and depends upon various factors such as weight of the patient, age of the patient, overall health of the patient, past history of the patient, immune status of the patient, etc.

Additionally, the compositions of the present invention may be utilized for cosmetic purposes. It may be added to pre-existing cosmetic compositions such that a mixture is formed or a fatty acid produced according to the subject invention may be used as the sole “active” ingredient in a cosmetic composition.

EXAMPLES

Example 1

Materials and Methods

Expression of Genes in Plant Cells in a Transient Expression System

Exogenous genetic constructs were expressed in plant cells in a transient expression system essentially as described by Voinnet et al. (2003) and Wood et al. (2009). Plasmids containing a coding region to be expressed from a strong constitutive promoter such as the CaMV 35S promoter were introduced into Agrobacterium tumefaciens strain AGL1. A chimeric gene 35S:p19 for expression of the p19 viral silencing suppressor was separately introduced into AGL1, as described in WO 2010/057246. The recombinant Agrobacterium cells were grown at 28° C. in LB broth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin to stationary phase. The bacteria were then pelleted by centrifugation at 5000 g for 15 min at room temperature before being resuspended to OD600=1.0 in an infiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl2 and 100 μM acetosyringone. The cells were then incubated at 28° C. with shaking for 3 hours before equal volumes of Agrobacterium cultures containing 35S:p19 and the test chimeric construct(s) of interest were mixed prior to infiltration into leaf tissue. The plants were typically grown for a further five days after infiltration before leaf discs were taken and freeze-dried for GC analysis of the fatty acids.

Fatty acid methyl esters (FAME) of total leaf lipids in freeze-dried samples were produced by incubating the samples in methanol/HCl/dichloromethane (10/1/1 v/v) solution for 2 hours at 80° C. together with a known amount of hexadecanoic acid as an internal standard. FAMEs were extracted in hexane/DCM, concentrated to a small volume in hexane and injected into a GC. The amount of individual and total fatty acids present in the lipid fractions were quantified on the basis of the known amount of internal standard.

Gas Chromatography (GC) Analysis of Fatty Acids

FAME were analysed by gas chromatography using an Agilent Technologies 7890A GC (Palo Alto, Calif., USA) equipped with a 30 m SGE-BPX70 column (70% cyanopropyl polysilphenylene-siloxane, 0.25 mm inner diameter, 0.25 mm film thickness), an FID, a split/splitless injector and an Agilent Technologies 7693 Series auto sampler and injector. Helium was used as the carrier gas. Samples were injected in split mode (50:1 ratio) at an oven temperature of 150° C. After injection, the oven temperature was held at 150° C. for 1 min then raised to 210° C. at 3° C. min−1, again raised to 240° C. at 50° C. min−1 and finally holding for 1.4 min at 240° C. Peaks were quantified with Agilent Technologies ChemStation software (Rev B.04.03 (16), Palo Alto, Calif., USA) based on the response of the known amount of the external standard GLC-411 (Nucheck) and C17:0-ME internal standard.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of Lipids

Total lipids were extracted from freeze-dried developing seeds, twelve days after flowering (daf), and mature seeds after adding a known amount of tri-C17:0-TAG as an internal quantitation standard. The extracted lipids were dissolved into 1 mL of 10 mM butylated hydroxytoluene in butanol:methanol (1:1 v/v) per 5 mg dry material and analysed using an Agilent 1200 series LC and 6410b electrospray ionisation triple quadrupole LC-MS. Lipids were chromatographically separated using an Ascentis Express RP-Amide column (50 mm×2.1 mm, 2.7 μm, Supelco) operating a binary gradient with a flow rate of 0.2 mL/min. The mobile phases were: A. 10 mM ammonium formate in H2O:methanol: tetrahydrofuran (50:20:30 v/v/v); B. 10 mM ammonium formate in H2O:methanol: tetrahydrofuran (5:20:75, v/v/v). Multiple reaction monitoring (MRM) lists were based on the following major fatty acids: 16:0, 18:0, 18:1, 18:2, 18:3, 18:4, 20:1, 20:2, 20:3, 20:4, 20:5, 22:4, 22:5, 22:6 using a collision energy of 30 V and fragmentor of 60 V. Individual MRM TAG was identified based on ammoniated precursor ion and product ion from neutral loss of 22:6. TAG was quantified using a 10 μM tristearin external standard.

Determination of Seed Fatty Acid Profile and Oil Content

Where seed oil content was to be determined, seeds were dried in a desiccator for 24 h and approximately 4 mg of seed was transferred to a 2 ml glass vial containing Teflon-lined screw cap. 0.05 mg triheptadecanoin dissolved in 0.1 ml toluene was added to the vial as internal standard.

Seed FAME were prepared by adding 0.7 ml of IN methanolic HCl (Supelco) to the vial containing seed material, vortexed briefly and incubated at 80° C. for 2 h. After cooling to room temperature, 0.3 ml of 0.9% NaCl (w/v) and 0.1 ml hexane was added to the vial and mixed well for 10 min in Heidolph Vibramax 110. The FAME was collected into 0.3 ml glass insert and analysed by GC with a flame ionization detector (FID) as mentioned earlier.

The peak area of individual FAME were first corrected on the basis of the peak area responses of known amount of the same FAMEs present in a commercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 contains equal amounts of 31 fatty acids (% by wt), ranging from C8:0 to C22:6. In case of fatty acids, which were not present in the standard, the inventors took the peak area responses of the most similar FAME. For example, peak area response of FAMEs of 16:1d9 was used for 16:1d7 and FAME response of C22:6 was used for C22:5. The corrected areas were used to calculate the mass of each FAME in the sample by comparison to the internal standard mass. Oil is stored mainly in the form of TAG and its weight was calculated based on FAME weight. Total moles of glycerol was determined by calculating moles of each FAMES and dividing total moles of FAMEs by three. TAG was calculated as the sum of glycerol and fatty acyl moieties using a relation: % oil by weight=100×((41×total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g seed, where 41 and 15 are molecular weights of glycerol moiety and methyl group, respectively.

Analysis of the Sterol Content of Oil Samples

Samples of approximately 10 mg of oil together with an added aliquot of C24:0 monol as an internal standard were saponified using 4 mL 5% KOH in 80% MeOH and heating for 2 h at 80° C. in a Teflon-lined screw-capped glass tube. After the reaction mixture was cooled, 2 mL of Milli-Q water were added and the sterols were extracted into 2 mL of hexane: dichloromethane (4:1 v/v) by shaking and vortexing. The mixture was centrifuged and the sterol extract was removed and washed with 2 mL of Milli-Qwater. The sterol extract was then removed after shaking and centrifugation. The extract was evaporated using a stream of nitrogen gas and the sterols silylated using 200 mL of BSTFA and heating for 2 h at 80° C.

For GC/GC-MS analysis of the sterols, sterol-OTMSi derivatives were dried under a stream of nitrogen gas on a heat block at 40° C. and then re-dissolved in chloroform or hexane immediately prior to GC/GC-MS analysis. The sterol-OTMS derivatives were analysed by gas chromatography (GC) using an Agilent Technologies 6890A GC (Palo Alto, Calif., USA) fitted with an Supelco Equity™-1 fused silica capillary column (15 m×0.1 mm i.d., 0.1 μm film thickness), an FID, a split/splitless injector and an Agilent Technologies 7683B Series auto sampler and injector. Helium was the carrier gas. Samples were injected in splitless mode at an oven temperature of 120° C. After injection, the oven temperature was raised to 270° C. at 10° C. min−1 and finally to 300° C. at 5° C. min−1. Peaks were quantified with Agilent Technologies ChemStation software (Palo Alto, Calif., USA). GC results are subject to an error of ±5% of individual component areas.

GC-mass spectrometric (GC-MS) analyses were performed on a Finnigan Thermoquest GCQ GC-MS and a Finnigan Thermo Electron Corporation GC-MS; both systems were fitted with an on-column injector and Thermoquest Xcalibur software (Austin, Tex., USA). Each GC was fitted with a capillary column of similar polarity to that described above. Individual components were identified using mass spectral data and by comparing retention time data with those obtained for authentic and laboratory standards. A full procedural blank analysis was performed concurrent to the sample batch.

RT-PCR Conditions

Reverse transcription-PCR (RT-PCR) amplification was typically carried out using the Superscript III One-Step RT-PCR system (Invitrogen) in a volume of 25 μL using 10 pmol of the forward primer and 30 pmol of the reverse primer, MgSO4 to a final concentration of 2.5 mM, 400 ng of total RNA with buffer and nucleotide components according to the manufacturer's instructions. Typical temperature regimes were: 1 cycle of 45° C. for 30 minutes for the reverse transcription to occur; then 1 cycle of 94° C. for 2 minutes followed by 40 cycles of 94° C. for 30 seconds, 52° C. for 30 seconds, 70° C. for 1 minute; then 1 cycle of 72° C. for 2 minutes before cooling the reaction mixtures to 5° C.

Production of B. napus Somatic Embryos by Induction with 35S-LEC2

B. napus (cv. Oscar) seeds were sterilized using chlorine gas as described by (Attila Kereszt et al., 2007). Sterilized seeds were germinated on ½ strength MS media (Murashige and Skoog, 1962) with 0.8% agar adjusted to pH 5.8, and grown at 24° C. under fluorescent lighting (50 μE/m2s) with a 18/6 h (light/dark) photoperiod for 6-7 days. Cotyledonary petioles with 2-4 mm stalk length were isolated aseptically from these seedlings and used as explants. Cultures of the transformed A. tumefaciens strain AGL1, one harbouring a seed specific binary vector and a second with a 35S-LEC2 construct were inoculated from single colonies from fresh plates and grown in 10 mL of LB medium with appropriate antibiotics and grown overnight at 28° C. with agitation at 150 rpm. The bacterial cells were collected by centrifugation at 4000 rpm for 5 minutes, washed with MS media containing 2% sucrose and re-suspended in 10 mL of the same medium and grown with antibiotics for selection as appropriate for 4 hours after the addition of acetosyringone to 100 μM. Two hours before addition to the plant tissues, spermidine was added to a final concentration of 1.5 mM and the final density of the bacteria adjusted to OD 600 nm=0.4 with fresh medium. The two bacterial cultures, one carrying the seed specific construct and other carrying 35S-AtLEC2, were mixed in 1:1 to 1:1.5 ratios.

Freshly-isolated B. napus cotyledonary petioles were infected with 20 mL A. tumefaciens cultures for 6 minutes. The cotyledonary petioles were blotted on sterile filter paper to remove excess A. tumefaciens and then transferred to co-cultivation media (MS media with 1 mg/L TDZ, 0.1 mg/L NAA, 100 μM acetosyringone supplemented with L-cysteine (50 mg/L), ascorbic acid (15 mg/L) and MES (250 mg/l)). The plates were sealed with micro-pore tape and incubated in the dark at 24° C. for 48 hrs. The co-cultivated explants were transferred to pre-selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 3 mg/L AgNO3, 250 mg/L cefotaxime and 50 mg/L timentin) and cultured for 4-5 days at 24° C. with a 16 h/8 h photoperiod. The explants were then transferred to selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 3 mg/L AgNO3, 250 mg/L cefotaxime and 50 mg/L timentin) according to the selectable marker gene on the seed specific vector and cultured for 2-3 weeks at 24° C. with a 16 h/8 h photoperiod. Explants with green embryogenic callus were transferred to hormone free MS media (MS with 3 mg/L AgNO3, 250 mg/L cefotaxime, 50 mg/L timentin and the selection agent) and cultured for another 2-3 weeks. Torpedo or cotyledonary stage embryos isolated from surviving explants on the selection medium were analysed for fatty acid composition in their total lipid using GC.

Example 2

Stable Expression of Transgenic DHA Pathways in Arabidopsis thaliana Seeds

Binary Vector Construction

The binary vectors pJP3416-GA7 and pJP3404 each contained seven heterologous fatty acid biosynthesis genes, encoding 5 desaturases and 2 elongases, and a plant selectable marker between the left and right border repeats of the T-DNA present in each vector (FIGS. 2 and 3). SEQ ID NO:1 provides the nucleotide sequence of the T-DNA region of pJP3416-GA7 from the right to left border sequences. Both genetic constructs contained plant codon-optimised genes encoding a Lachancea kluyveri Δ12-desaturase (comprising nucleotides 14143-16648 of SEQ ID NO:1), a Pichia pastoris ω3-desaturase (comprising nucleotides 7654-10156 of SEQ ID NO:1), a Micromonas pusilla Δ6-desaturase (comprising nucleotides 226-2309 of SEQ ID NO:1), Pavlova salina Δ5- and Δ4-desaturases (comprising nucleotides 4524-6485 and 10157-14142 of SEQ ID NO:1, respectively) and Pyramimonas cordata Δ6- and Δ5-elongases (comprising nucleotides 2310-4523 and 17825-19967 of SEQ ID NO:1, respectively). The specific regions of the T-DNA (Orientation: right to left border sequences) region of the binary vector pJP3416-GA7 with respect to SEQ ID NO:1 are as follows:

Nucleotides 1-163: Right border; 480-226, Agrobacterium tumefaciens nopaline synthase terminator (TER_NOS); 1883-489, Micromonas pusilla Δ6-desaturase; 2309-1952, Brassica napus truncated napin promoter (PRO_FP1); 2310-3243, Arabidopsis thaliana FAE1 promoter (PRO_FAE1); 3312-4181, Pyramimonas cordata Δ6-elongase; 4190-4523, Glycine max lectin terminator (TER_Lectin); 4524-4881, PRO_FP1; 4950-6230: Pavlova salina Δ5-desaturase; 6231-6485: TER_NOS; 7653-6486, Nicotiana tabacum Rb7 matrix attachment region (MAR); 8387-7654, Linum usitatissimum conlinin1 terminator (TER_Cnl1); 9638-8388, Pichia pastoris ω3-desaturase; 10156-9707, Linum usitatissimum conlinin1 promoter (PRO_Cnl1); 10157-12189, Linum usitatissimum conlinin1 promoter; 12258-13604, Pavlova salina Δ4-desaturase; 13605-14142, Linum usitatissimum conlinin2 terminator; 14143-14592, PRO_Cnl1; 14661-15914, Lachancea kluyveri Δ12-desaturase; 15915-16648, TER_Cnl1; 17816-16649, MAR; 17825-18758, PRO_FAE1; 18827-19633, Pyramimonas cordata Δ5-elongase; 19634-19967, TER_Lectin; 19990-20527, Cauliflower mosaic virus 35S promoter with duplicated enhancer region; 20537-21088, Streptomyces viridochromogenes phosphinothricin-N-acetyltransferase; 21097-21349, TER_NOS; 21367-21527, Left border.

The seven coding regions in the constructs were each under the control of a seed specific promoter—three different promoters were used, namely the truncated Brassica napus napin promoter (pBnFP1), the Arabidopsis thaliana FAE1 promoter (pAtFAE1) and the Linum usitatissimum conlinin 1 promoter (pLuCnl1). The seven fatty acid biosynthesis genes together coded for an entire DHA synthesis pathway that was designed to convert 18:1Δ9 (oleic acid) through to 22:6Δ4,7,10,13,16,19 (DHA). Both binary vectors contained a BAR plant selectable marker coding region operably linked to a Cauliflower Mosaic Virus (CaMV) 35S promoter with duplicated enhancer region and A. tumefaciens nos3′ polyadenylation region—transcription terminator. The plant selectable marker was situated adjacent to the left border of the T-DNA region, therefore distally located on the T-DNA with respect to the orientation of T-DNA transfer into the plant cells. This increased the likelihood that partial transfer of the T-DNA, which would be likely to not include the selectable marker gene, would not be selected. pJP3416-GA7 and pJP3404 each contained an RiA4 origin of replication from Agrobacterium rhizogenes (Hamilton, 1997).

pJP3416-GA7 was generated by synthesising the DNA region corresponding to nucleotides 226-19975 of SEQ ID NO:1 (GA7 region) and inserting this region into the recipient binary vector pJP3416 at the PspOMI site. Each fatty acid biosynthetic gene on GA7 included a Tobacco Mosaic Virus 5′ untranslated region (5′UTR) sequence which was operably linked to each coding region, between the promoter and the translation initiation ATG, to maximise translation efficiency of the mRNAs produced from the genes. The GA7 construct also included two Nicotiana tabacum Rb7 matrix attachment region (MAR) sequences, as described by Hall et al. (1991). MAR sequences, sometimes termed nuclear attachment regions, are known to bind specifically to the nuclear matrix in vitro and may mediate binding of chromatin to the nuclear matrix in vivo. MARs are thought to function to reduce transgene silencing. In pJP3416-GA7 the MARs were also inserted and positioned within the T-DNA region in order to act as DNA spacers to insulate transgenic expression cassettes. The pJP3416 vector prior to insertion of the GA7 region contained only the plant selectable marker cassette between the borders.

The genetic construct pJP3404 was made by sequential restriction enzyme-based insertions in which gene cassettes were added to the binary vector, pJP3367, which comprised genes for production of SDA in seeds. This construct contained genes encoding the L. kluyveri Δ12-desaturase and P. pastoris ω3-desaturase, both expressed by the B. napus truncated napin promoter (FP1), and the M. pusilla Δ6-desaturase expressed by the A. thaliana FAE1 promoter (FIG. 4). First, the A. thaliana FAD2 intron was flanked by EcoRI sites and cloned into the pJP3367 MfeI site to generate pJP3395. A fragment containing the P. cordata Δ6- and Δ5-elongase cassettes driven by the FAE1 and FP1 promoters, respectively, was cloned into the KasI site of pJP3395 to generate pJP3398. pJP3399 was then generated by replacing the RK2 origin of replication in pJP3398 with a RiA4 origin of replication. The final binary vector, pJP3404, was generated by cloning a SbfI-flanked fragment containing the P. salina Δ5- and Δ4-desaturase cassettes driven by the FP1 and FAE1 promoters, respectively, into the SbfI site of pJP3399.

A. thaliana Transformation and Analysis of Fatty Acid Composition

The chimeric vectors were introduced into A. tumefaciens strain AGL1 and cells from cultures of the transformed Agrobacterium used to treat A. thaliana (ecotypes Columbia and a fad2 mutant) plants using the floral dip method for transformation (Clough and Bent, 1998). After maturation, the T1 seeds from the treated plants were harvested and plated onto MS plates containing PPT for selection of plants containing the BAR selectable marker gene. Surviving, healthy T1 seedlings were transferred to soil. After growth of the plants to maturity and allowing for self-fertilisation, T2 seeds from these plants were harvested and the fatty acid composition of their seed lipid analysed by GC analysis as described in Example 1.

The data for the DHA level in the seed lipids are shown in FIG. 5 (lanes labelled T2) for 13 transformants using pJP3416-GA7 into the Columbia genetic background, and for six transformants using the fad2 mutant. The pJP3416-GA7 construct resulted in the production of slightly higher levels of DHA, as a percentage of total fatty acid content, on average than the pJP3404 construct. Table 4 shows the fatty acid composition of total seed lipid from the T2 lines with the highest DHA levels. The calculated conversion efficiencies for each enzymatic step in the production of DHA from oleic acid in the same seeds are shown in Table 5. Conversion efficiencies were calculated as (% products×100)/(% remaining substrate+% products), thereby expressed as a percentage.

The highest observed level of DHA produced in the pJP3416-GA7 T2 transformed lines was 6.2%, additionally with 0.5% EPA and 0.2% DPA (line #14). These T2 seeds were still segregating for the transgene i.e. were not yet uniformly homozygous. Compiled data from the total seed lipid profiles from independent transgenic seed (Table 4) are shown in Table 6. The level of ω3 fatty acids produced as a result of the transgenes in these seeds (total new ω3 fatty acids, excluding the level of ALA which was produced endogenously in the Columbia background) was 10.7% while the level of ω6 fatty acids (total new ω6 fatty acids but excluding 18:2×9,12) was 1.5%. This represents an extremely favourable ration of new ω3 fatty acids:new ω6 fatty acids, namely 7.3:1.

T2 seeds of selected lines transformed with pJP3416-GA7, namely for lines designated 7, 10, 14, 22 and 34 in the Columbia background and for lines designated 18, 21 and 25 in the fad2 mutant background, were plated onto MS media containing PPT for selection of transgenic seedlings in vitro. Twenty PPT-resistant seedlings for each line were transferred to soil and grown to maturity after self-fertilisation. These plants were highly likely to be homozygous for the selectable marker gene, and therefore for at least one T-DNA insertion in the genome of the plants. T3 seed from these plants were harvested and analysed for fatty acid composition in their seedoil by GC. The data are shown in Table 7. This analysis revealed that the pJP3416-GA7 construct generated higher levels of the ω3 LC-PUFA DHA in T3 seeds of the homozygous plants than in the segregating T2 seed. Up to about 13.9% DHA was observed in the T3 pJP3416-GA7 transformed line designated 22.2 in the Columbia background, increased from about 5.5% in the hemizygous T2 seed, with a sum level of about 24.3% of new ω3 fatty acids as a percentage of the total fatty acids in the seed lipid content. New ω6 fatty acids were at a level of 1.1% of total fatty acids, representing a very favourable ratio of new ω3 fatty acids:new ω6 fatty acids, namely about 22:1. Similarly, transformants in the fad2 mutant background yielded 20.6% as a sum of new ω3 fatty acids, including 11.5% DHA, as a percentage of the total fatty acids in the seed lipid content.


TABLE 4
Fatty acid composition of total seed lipid from independent transgenic T2
Arabidopsis seeds with DHA levels at the higher end of the observed range. ‘Col’
refers to the Columbia ecotype and ‘FAD2’ to the fad2 mutant ecotype. ‘GA7’ refers
to transformation with the T-DNA of the pJP3416-GA7 vector, pJP3404 with the T-
DNA of the pJP3404 vector. 20:1n-9 and 20:1n-11 fatty acids were not resolved in the
GC analysis. “Other minor” fatty acids include 14:0, 16:1n7, 16:1n9, 16:1n13t, 16:2n6,
16:3n3, i18:0, 18:1n5, 20:1n5, 22:0, 22:1n7, 22:1n11/n13, 24:0, 24:1n9.
pJP3404_Col_#1
pJP3404_FAD2_#31
GA7_Col_#7
GA7_Col_#34
GA7_Col_#2
GA7_Col_#10
16:0
9.6
7.8
8.7
8.2
8.7
8.6
18:0
2.9
3.9
3.7
3.9
3.6
3.3
18:1d11
2.2
1.8
2.0
1.9
2.0
2.3
20:0
1.6
2.3
2.0
2.0
2.1
1.6
20:1d13
2.2
1.8
1.6
1.5
1.7
1.6
20:1d9/d11
13.0
15.9
16.1
16.1
16.3
15.0
22:1d13
1.1
1.2
1.1
1.1
1.3
1.0
Other
1.9
1.5
1.5
1.4
1.5
1.3
minor
18:1d9
10.8
14.0
10.6
10.6
10.1
11.1
18:2ω6
28.9
28.3
16.4
16.1
18.2
13.7
18:3ω3
16.6
14.9
29.6
29.6
27.5
32.4
18:3ω6
0.7
0.5
0.1
0.1
0.1
0.1
20:2ω6
1.6
1.5
1.1
1.2
1.3
1.0
20:3ω6
0.0
0.0
0.0
0.0
0.0
0.0
20:4ω6
0.0
0.0
0.0
0.0
0.0
0.0
22:4ω6
1.6
0.6
0.3
0.3
0.3
0.4
22:5ω6
0.1
0.1
0.0
0.0
0.0
0.0
18:4ω3
1.0
0.5
1.2
1.1
1.1
1.5
20:3ω3
0.0
0.0
0.0
0.6
0.0
0.0
20:4ω3
0.4
0.6
0.6
0.7
0.5
0.8
20:5ω3
0.2
0.2
0.3
0.3
0.3
0.3
22:5ω3
0.0
0.2
0.2
0.2
0.2
0.2
22:6ω3
3.6
2.4
3.0
3.1
3.3
3.9
GA7_Col_#22
GA7_Col_#14
GA7_FAD2_#25
GA7_FAD2_#21
GA7_FAD2_#18
16:0
8.3
9.7
7.2
8.5
7.5
18:0
3.4
3.6
3.2
3.9
3.0
18:1d11
2.3
2.7
1.9
2.0
1.8
20:0
1.6
1.8
1.6
2.2
1.5
20:1d13
1.5
1.7
1.5
1.7
1.4
20:1d9/d11
13.9
13.5
18.3
15.9
17.0
22:1d13
1.0
1.0
1.0
1.3
1.2
Other
1.6
1.7
1.6
1.4
1.6
minor
18:1d9
10.0
7.7
26.0
8.2
20.9
18:2ω6
13.7
11.4
6.6
16.6
4.3
18:3ω3
30.4
32.8
21.9
27.7
30.1
18:3ω6
0.2
0.1
0.1
0.2
0.1
20:2ω6
1.0
1.0
0.4
1.4
0.4
20:3ω6
0.0
0.0
0.0
0.0
0.0
20:4ω6
0.0
0.0
0.0
0.0
0.0
22:4ω6
0.5
0.4
0.5
0.4
0.4
22:5ω6
0.0
0.0
0.0
0.0
0.0
18:4ω3
2.7
2.7
1.9
1.8
1.7
20:3ω3
0.6
0.7
0.0
0.8
0.6
20:4ω3
0.8
0.4
1.0
0.8
0.8
20:5ω3
0.7
0.5
0.6
0.4
0.5
22:5ω3
0.2
0.2
0.3
0.3
0.3
22:6ω3
5.5
6.2
4.3
4.4
4.8


TABLE 5
Conversion efficiencies of the individual enzymatic steps for production of DHA from oleic acid, observed in total seed lipid
from independent transgenic seed as for Table 4.
pJP3404_Col_#1
pJP3404_FAD2_#31
GA7_Col_#7
GA7_Col_#34
GA7_Col_#2
GA7_Col_#10
d12-des
69.6%
62.5%
66.4%
66.6%
66.7%
67.5%
d15-des
39.8%
37.8%
66.1%
66.8%
62.3%
72.1%
Omega-6
d6-des
4.5%
2.5%
0.7%
0.7%
0.7%
0.9%
(d9-elo)
3.1%
3.1%
2.2%
2.3%
2.4%
1.8%
d6-elo
71.4%
56.9%
83.3%
83.4%
83.0%
84.7%
d5-des
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
d5-elo
100.0%
97.8%
100.0%
100.0%
100.0%
100.0%
d4-des
6.2%
13.0%
0.0%
0.0%
0.0%
0.0%
Omega-3
d6-des
23.9%
21.0%
15.2%
15.4%
16.4%
17.1%
(d9-elo)
0.0%
0.0%
0.0%
1.8%
0.0%
0.0%
d6-elo
80.6%
86.6%
77.7%
79.6%
79.4%
77.5%
d5-des
93.7%
92.1%
91.7%
91.4%
91.5%
92.6%
d5-elo
93.7%
92.1%
91.7%
91.4%
91.5%
92.6%
d4-des
100.0%
90.6%
94.8%
94.0%
95.3%
94.4%
GA7_Col_#22
GA7_Col_#14
GA7_FAD2_#25
GA7_FAD2_#21
GA7_FAD2_#18
d12-des
70.2%
72.7%
45.9%
69.5%
53.7%
d15-des
72.7%
77.2%
79.7%
66.0%
88.1%
Omega-6
d6-des
1.3%
1.0%
1.6%
1.1%
1.1%
(d9-elo)
1.8%
1.7%
1.2%
2.7%
0.9%
d6-elo
70.3%
74.5%
85.5%
66.1%
88.0%
d5-des
100.0%
100.0%
100.0%
100.0%
100.0%
d5-elo
100.0%
100.0%
100.0%
100.0%
100.0%
d4-des
0.0%
0.0%
0.0%
0.0%
0.0%
Omega-3
d6-des
24.7%
23.6%
27.1%
21.9%
21.0%
(d9-elo)
2.0%
2.2%
0.0%
2.6%
2.1%
d6-elo
72.7%
73.0%
76.7%
77.4%
79.2%
d5-des
89.6%
92.4%
88.0%
91.8%
91.0%
d5-elo
89.6%
92.4%
88.0%
91.8%
91.0%
d4-des
95.8%
96.9%
93.1%
92.9%
94.2%


TABLE 6
Compiled data from the total seed lipid profiles from independent transgenic seed shown in Table 2. Calculations do not
include the ‘minor fatty acids’ in Table 4.
Parameter
pJP3404_Col_#1
pJP3404_FAD2_#31
GA7_Col_#7
GA7_Col_#34
GA7_Col_#2
GA7_Col_#10
total w3 (% of total FA)
21.8
18.8
34.9
35.6
32.9
39.1
total w6 (% of total FA)
32.9
31.0
17.9
17.7
19.9
15.2
w3/w6 ratio
0.66
0.61
1.95
2.01
1.65
2.57
w6/w3 ratio
1.51
1.65
0.51
0.50
0.60
0.39
total novel w3 (% of total FA)
5.2
3.9
5.3
6.0
5.4
6.7
total novel w6 (% of total FA)
4.0
2.7
1.5
1.6
1.7
1.5
novel w3/w6 ratio
1.30
1.44
3.53
3.75
3.18
4.47
novel w6/w3 ratio
0.77
0.69
0.28
0.27
0.31
0.22
OA to EPA efficiency
4.8%
3.5%
4.3%
4.4%
4.7%
5.4%
OA to DHA efficiency
4.5%
3.0%
3.7%
3.8%
4.1%
4.8%
LA to EPA efficiency
6.9%
5.6%
6.6%
6.8%
7.2%
8.1%
LA to DHA efficiency
6.6%
4.8%
5.7%
5.8%
6.3%
7.2%
ALA to EPA efficiency
17.4%
14.9%
10.0%
10.1%
11.6%
11.3%
ALA to DHA efficiency
16.5%
12.8%
8.6%
8.7%
10.0%
10.0%
total saturates
14.1
14.0
14.4
14.1
14.4
13.5
total monounsaturates
29.3
34.7
31.4
31.2
31.4
31.0
total polyunsaturates
54.7
49.8
52.8
53.3
52.8
54.3
total C20
17.4
20
19.7
20.4
20.1
18.7
total C22
6.4
4.5
4.6
4.7
5.1
5.5
C20/C22 ratio
2.72
4.44
4.28
4.34
3.94
3.40
Parameter
GA7_Col_#22
GA7_Col_#14
GA7_FAD2_#25
GA7_FAD2_#21
GA7_FAD2_#18
total w3 (% of total FA)
40.9
43.5
30.0
36.2
38.8
total w6 (% of total FA)
15.4
12.9
7.6
18.6
5.2
w3/w6 ratio
2.66
3.37
3.95
1.95
7.46
w6/w3 ratio
0.38
0.30
0.25
0.51
0.13
total novel w3 (% of total FA)
10.5
10.7
8.1
8.5
8.7
total novel w6 (% of total FA)
1.7
1.5
1.0
2.0
0.9
novel w3/w6 ratio
6.18
7.13
8.10
4.25
9.67
novel w6/w3 ratio
0.16
0.14
0.12
0.24
0.10
OA to EPA efficiency
7.9%
8.8%
6.3%
6.4%
6.7%
OA to DHA efficiency
6.8%
7.9%
5.2%
5.5%
5.8%
LA to EPA efficiency
11.4%
12.2%
13.8%
9.3%
12.7%
LA to DHA efficiency
9.8%
11.0%
11.4%
8.0%
10.9%
ALA to EPA efficiency
15.6%
15.9%
17.3%
14.1%
14.4%
ALA to DHA efficiency
13.4%
14.3%
14.3%
12.2%
12.4%
total saturates
13.3
15.1
12.0
14.6
12.0
total monounsaturates
28.7
26.6
48.7
29.1
42.3
total polyunsaturates
56.3
56.4
37.6
54.8
44.0
total C20
18.5
17.8
21.8
21
20.7
total C22
7.2
7.8
6.1
6.4
6.7
C20/C22 ratio
2.57
2.28
3.57
3.28
3.09


TABLE 7
Fatty acid composition of total seed lipid from independent transgenic T3 and
T4 Arabidopsis progeny seeds obtained from plant lines as in Table 3. The error shown
in the T4 generation denotes the SD of n = 10.
GA7_Col_7.2
GA7_Col_34.2
GA7_Col_10.13
GA7_Col_22.2
GA7_Col_14.19
16:0
9.8
9.0
9.5
11.2
10.4
18:0
4.0
3.8
4.2
3.4
3.5
18:1n7
2.0
1.9
2.2
2.9
2.5
20:0
2.2
1.9
1.7
1.4
2.3
20:1d13
1.4
1.3
1.2
1.6
2.5
20:1d9/11
13.6
14.7
12.4
9.5
13.0
22:1d13
1.2
1.2
0.8
0.6
1.6
Other
1.8
1.5
1.5
2.1
2.6
minor
18:1d9
5.5
6.7
6.8
4.6
6.9
18:2ω6
7.5
7.9
7.4
5.6
14.8
18:3ω3
33.7
33.7
36.1
31.5
26.1
18:3ω6
0.2
0.2
0.2
0.4
0.1
20:2ω6
1.0
1.0
0.7
0.7
1.4
20:3ω6
0
0
0
0
0
20:4ω6
0
0
0
0
0
22:4ω6
0
0
0
0
0
22:5ω6
0
0
0
0
0
18:4ω3
3.1
2.6
3.0
5.3
3.3
20:3ω3
1.4
1.3
1.2
1.3
1.2
20:4ω3
0.7
0.6
0.6
0.9
0.2
20:5ω3
0.9
0.9
0.7
1.9
0.8
22:5ω3
0.7
0.6
0.6
1.0
0.4
22:6ω3
9.5
9.2
9.4
13.9
6.6
T4 Col_22.2
T4 Col_22.2
GA7_FAD2-25.10
GA7_FAD2-21.2
GA7_FAD2-18.14
(mean ± SD)
best line
16:0
8.1
10.7
7.7
10.6 ± 0.9 
12.2
18:0
3.5
3.8
3.3
3.5 ± 0.4
3.6
18:1n7
1.7
2.2
1.6
2.3 ± 0.2
2.6
20:0
1.8
2.0
1.9
1.9 ± 0.3
2.0
20:1d13
1.2
1.4
1.3
1.6 ± 0.2
1.9
20:1d9/11
15.7
12.4
18.4
11.7 ± 1.7 
9.5
22:1d13
1.0
1.1
1.5
0.9 ± 0.1
0.8
Other
1.7
1.9
1.6
1.9 ± 0.1
2.3
minor
18:1d9
11.3
4.2
11.5
4.6 ± 1.0
3.3
18:2ω6
5.8
8.9
5.6
5.3 ± 0.9
4.3
18:3ω3
28.3
28.9
30.8
31.0 ± 1.1 
29.5
18:3ω6
0.3
0.6
0.1
0.4 ± 0.1
0.4
20:2ω6
0.6
1.2
0.6
0.9 ± 0.1
0.9
20:3ω6
0
0
0
20:4ω6
0
0
0
22:4ω6
0
0
0
0.1 ± 0.0
0.1
22:5ω6
0
0
0
18:4ω3
3.7
5.2
2.6
4.8 ± 0.9
5.5
20:3ω3
1.1
1.3
1.3
1.5 ± 0.2
1.7
20:4ω3
1.7
0.9
0.9
0.8 ± 0.2
0.8
20:5ω3
1.2
1.0
0.8
1.5 ± 0.3
1.8
22:5ω3
0.8
0.6
0.5
1.1 ± 0.2
1.5
22:6ω3
10.3
11.5
7.9
13.3 ± 1.6 
15.1

Enzymatic conversion efficiencies for each enzyme step in the pathway for production of DHA from oleic acid are shown in Table 8 for the T3 seeds with the higher DHA levels. The Δ12-desaturase conversion efficiency in seeds of line 22.2 was 81.6% and the ω3-desaturase efficiency was 89.1%, both of them remarkably high and indicating that these fungal (yeast) enzymes were able to function well in developing seeds. The activities of the other exogenous enzymes in the DHA pathway were similarly high for ω3 substrates with the Δ6-desaturase acting at 42.2% efficiency, Δ6-elongase at 76.8%, Δ5-desaturase at 95.0%, Δ5-elongase at 88.7% and Δ4-desaturase at 93.3% efficiency. The Δ6-desaturase activity on the ω6 substrate LA was much lower, with the Δ6-desaturase acting at only 0.7% conversion efficiency on LA. GLA was present at a level of only 0.4% and was the only new ω6 product aside from 20:2ω6 detected in the T3 seeds with the highest DHA content. Compiled data from the total seed lipid profiles from independent transgenic seed (Table 7) are shown in Table 9. This data for the line with the greatest DHA level included a total ω6 FA (including LA) to total ω3 FA (including ALA) ratio of 0.10. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in the lipid of this line was 0.05. Total polyunsaturated fatty acid levels were more than 50% in these lines, and greater than 60% in at least 4 of the lines. Overall conversion efficiencies were calculated to be: OA to EPA=21.8%, OA to DHA=18.0%, LA to EPA=26.9%, LA to DHA=22.2%, ALA to EPA=30.1%, ALA to DHA=24.9%.


TABLE 8
Conversion efficiencies of the individual enzymatic steps for the production of DHA from oleic
acid, observed in total seed lipid from transgenic T3 Arabidopsis seeds as in Table 7.
GA7_Col_7.2
GA7_Col_34.2
GA7_Col_10.13
GA7_Col_22.2
GA7_Col_14.19
d12-des
75.4%
73.1%
75.7%
81.6%
73.4%
d15-des
85.3%
84.4%
86.2%
89.1%
70.2%
Omega-6
d6-des
0.3%
0.3%
0.3%
0.7%
0.3%
(d9-elo)
1.7%
1.7%
1.2%
1.2%
2.6%
d6-elo
d5-des
d5-elo
d4-des
Omega-3
d6-des
30.7%
29.3%
28.2%
42.2%
30.2%
(d9-elo)
2.7%
2.7%
2.3%
2.4%
3.0%
d6-elo
79.0%
81.1%
79.0%
76.8%
70.9%
d5-des
94.0%
94.6%
94.5%
95.0%
97.9%
d5-elo
91.9%
91.7%
93.6%
88.7%
89.5%
d4-des
93.2%
93.7%
94.4%
93.3%
93.7%
GA7_FAD2-
GA7_FAD2-
T4 Col_22.2
T4 Col_22.2
25.10
GA7_FAD2-21.2
18.14
(mean)
best line
d12-des
66.6%
78.5%
63.1%
67.6%
82.7%
d15-des
87.5%
82.2%
87.6%
81.0%
90.9%
Omega-6
d6-des
0.6%
1.0%
0.2%
1.3%
0.7%
(d9-elo)
1.1%
2.0%
1.3%
1.6%
1.5%
d6-elo
d5-des
d5-elo
d4-des
Omega-3
d6-des
38.5%
40.0%
29.2%
41.0%
45.7%
(d9-elo)
2.3%
2.7%
2.9%
2.8%
3.1%
d6-elo
79.2%
73.2%
79.1%
77.5%
77.7%
d5-des
87.8%
93.3%
91.1%
95.0%
95.8%
d5-elo
89.9%
92.2%
91.6%
90.8%
90.2%
d4-des
92.5%
95.0%
93.9%
92.2%
90.9%


TABLE 9
Compiled data from the total seed lipid profiles from independent transgenic seed shown in
Table 5. Calculations do not include the ‘minor fatty acids’ in Table 7.
Parameter
GA7-Col_7.2
GA7-Col_34.2
GA7-Col_10.13
GA7-Col_22.2
GA7-Col_14.19
total w3 (% of total FA)
50.0
48.9
51.6
55.8
38.6
total w6 (% of total FA)
8.7
9.1
8.3
6.7
16.3
w3/w6 ratio
5.75
5.37
6.22
8.33
2.37
w6/w3 ratio
0.17
0.19
0.16
0.12
0.42
total novel w3 (% of total FA)
16.3
15.2
15.5
24.3
12.5
total novel w6 (% of total FA)
1.2
1.2
0.9
1.1
1.5
novel w3/w6 ratio
13.58
12.67
17.22
22.09
8.33
novel w6/w3 ratio
0.07
0.08
0.06
0.05
0.12
OA to EPA efficiency
14.1%
13.3%
13.4%
21.8%
10.2%
OA to DHA efficiency
12.0%
11.4%
11.8%
18.0%
8.6%
LA to EPA efficiency
18.9%
18.4%
17.9%
26.9%
14.2%
LA to DHA efficiency
16.2%
15.9%
15.7%
22.2%
12.0%
ALA to EPA efficiency
22.2%
21.9%
20.7%
30.1%
20.2%
ALA to DHA efficiency
19.0%
18.8%
18.2%
24.9%
17.1%
total saturates
16.0
14.7
15.4
16.0
16.2
total monounsaturates
23.7
25.8
23.4
19.2
26.5
total polyunsaturates
58.7
58.0
59.9
62.5
54.9
total C20
19
19.8
16.8
15.9
19.1
total C22
11.4
11
10.8
15.5
8.6
C20/C22 ratio
1.67
1.80
1.56
1.03
2.22
T4 Col_22.2
T4 Col_22.2
Parameter
GA7-FAD2-25.10
GA7-FAD2-21.2
GA7-FAD2-18.14
(mean ± SD)
best line
total w3 (% of total FA)
47.1
49.4
44.8
54.0
55.9
total w6 (% of total FA)
6.7
10.7
6.3
6.7
5.7
w3/w6 ratio
7.03
4.62
7.11
8.06
9.81
w6/w3 ratio
0.14
0.22
0.14
0.12
0.10
total novel w3 (% of total FA)
18.8
20.5
14.0
23.0
26.4
total novel w6 (% of total FA)
0.9
1.8
0.7
1.4
1.4
novel w3/w6 ratio
20.89
11.39
20.00
16.43
18.86
novel w6/w3 ratio
0.05
0.09
0.05
0.06
0.05
OA to EPA efficiency
15.0%
16.8%
11.2%
20.4%
24.5%
OA to DHA efficiency
12.6%
14.8%
9.6%
17.1%
20.1%
LA to EPA efficiency
22.9%
21.8%
18.0%
26.2%
29.9%
LA to DHA efficiency
19.1%
19.1%
15.5%
21.9%
24.5%
ALA to EPA efficiency
26.1%
26.5%
20.5%
29.4%
32.9%
ALA to DHA efficiency
21.9%
23.3%
17.6%
24.6%
27.0%
total saturates
13.4
16.5
12.9
16.0
17.8
total monounsaturates
30.9
21.3
34.3
21.1
18.1
total polyunsaturates
53.8
60.1
51.1
60.7
61.6
total C20
21.5
18.2
23.3
18
16.6
total C22
12.1
13.2
9.9
15.4
17.5
C20/C22 ratio
1.78
1.38
2.35
1.17
0.95

T3 seeds from the pJP3416-GA7 line 22.2 in the Columbia background, which were progeny from T2 line 22, were sown directly to soil and the fatty acid composition of mature seed from the resultant T3 plants analysed by GC. The average DHA level of these seeds was 13.3%±1.6 (n=10) as a percentage of total fatty acids in the seed lipid. As shown in Table 6 (right hand column), the line with the highest level of DHA contained 15.1% DHA in the total fatty acids of the seed lipid. The enzymatic conversion efficiencies are shown in Table 8 for each step in the production of DHA from oleic acid.

The total ω6 FA (including LA) to ω3 FA (including ALA) ratio in the line with the highest DHA level was 0.102. The new 06 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in the line with the highest DHA level was 0.053. The level of total saturated fatty acids was about 17.8% and the level of monounsaturated fatty acids was about 18.1%. The level of total ω6-fatty acids was about 5.7% and the level of ω3-fatty acids was about 55.9%. Overall conversion efficiencies were calculated to be: OA to EPA=24.5%, OA to DHA=20.1%, LA to EPA=29.9%, LA to DHA=24.5%, ALA to EPA=32.9%, ALA to DHA=27.0%. Total omega-3 fatty acids were found to accumulate to 55.9% of total fatty acids whereas omega-6 fatty acids were 5.7% of the total profile.

Southern blot hybridisation analysis was performed. The results showed that the high-accumulating DHA lines were either single- or double-copy for the T-DNA from the pJP3416-GA7 construct with the exception of transgenic line Columbia#22, which had three T-DNA insertions in the genome of the Arabidopsis plant. The T5 generation seed was also analysed and found to have up to 13.6% DHA in the total seed lipids. The GA7 construct was found to be stable across multiple generations in terms of DHA production capability.

Determination of Oil Content in Transgenic A. thaliana DHA Lines

The oil content of transgenic A. thaliana seeds with various levels of DHA was determined by GC as described in Example 1. The data are shown in FIG. 6, graphing the oil content (% oil by weight of seed) against the DHA content (as a percentage of total fatty acids). Up to 26.5 mg of DHA per gram of seed was observed (Table 10). The oil content of the transgenic Arabidopsis seeds was found to be negatively correlated with DHA content. The amount of DHA per weight of seed was greater in the transformed seeds with a DHA level of about 9% relative to the seeds with about 14% DHA. Whether this would be true for seeds other than Arabidopsis has not been determined.


TABLE 10
Proportion and amount of DHA in GA7-
transformed Arabidopsis seeds.
Oil content
DHA content
DHA content
(% oil per
per weight
(% of TFA)
g seeds)
(mg/g seed)
GA7/col 22.2-1
14.2
14.89
20.2
GA7/col 22.2-2
14.3
15.02
20.5
GA7/col 22.2-3
14.0
15.92
21.2
GA7/col 10.15-1
8.7
30.23
25.06
GA7/col 10.15-2
8.6
31.25
25.77
GA7/col 10.15-3
8.8
31.70
26.49

Example 3

Stable Expression of a Transgenic DHA Pathway in Camelina sativa Seeds

The binary vector pJP3416-GA7 as described above was introduced into A. tumefaciens strain AGL1 and cells from a culture of the transformed Agrobacterium used to treat C. sativa flowering plants using a floral dip method for transformation (Lu and Kang, 2008). After growth and maturation of the plants, the T1 seeds from the treated plants were harvested, sown onto soil and the resultant plants treated by spraying with the herbicide BASTA to select for plants which were transgenic for, and expressing, the bar selectable marker gene present on the T-DNA of pJP3416-GA7. Surviving T1 plants which were tolerant to the herbicide were grown to maturity after allowing them to self-fertilise, and the resultant T2 seed harvested. Five transgenic plants were obtained, only three of which contained the entire T-DNA.

Lipid was extracted from a pool of approximately twenty seeds from each of the three plants that contained the entire T-DNA. Two of the pooled samples contained very low, barely detectable levels of DHA, but the third pool contained about 4.7% DHA (Table 12). Therefore, lipid was extracted from 10 individual T2 seeds from this plant and the fatty acid composition analysed by GC. The fatty acid composition data of the individual seeds for this transformed line is also shown in Table 11. Compiled data from the total seed lipid profiles (Table 11) are shown in Table 12.


TABLE 11
Fatty acid composition of total seed lipids from transgenic T2 Camelina sativa
seeds transformed with the T-DNA from pJP3416-GA7. The fatty acid composition is shown
for a pooled seed batch (FD5.46) and for 10 single seeds ranked (left to right)
from highest to lowest DHA.
FD5.46
Fatty acid
pooled
# 2
# 4
# 8
# 7
# 9
# 1
# 3
# 5
# 6
# 10
14:0
0
0.2
0.2
0.1
0.2
0.2
0.2
0.2
0.1
0.2
0.2
16:0
11.6
12.1
12.3
12.1
13.2
12.3
12.8
11.9
11.4
11.5
11.7
16:1
0.2
0.0
0.1
0.1
0.0
0.2
0.0
0.2
0.2
0.2
0.2
16:3
0.3
0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
18:0
3.7
3.3
3.2
3.2
3.0
3.1
3.2
3.3
3.1
3.2
3.2
18:1
10.8
8.0
8.0
8.6
8.5
9.4
11.0
10.2
8.3
9.4
8.6
18:1d11
1.7
1.3
1.4
1.4
1.7
1.4
1.5
1.3
1.3
1.3
1.3
18:2
24.7
18.2
19.5
19.2
18.5
20.1
23.8
32.2
30.3
29.8
31.6
18:3ω3
27.4
26.7
26.6
27.3
28.9
28.2
27.4
28.3
29.2
29.5
28.2
18:3ω6
0.2
1.4
0.3
0.3
0.4
0.2
0.5
0.0
0.5
0.4
0.6
20:0
1.6
1.4
1.3
1.4
1.2
1.4
1.4
1.8
2.1
1.9
2.0
18:4ω3
2.2
6.8
6.4
5.7
7.2
5.7
4.1
0.0
0.0
0.0
0.0
20:1d11
5.3
4.4
4.6
4.8
3.3
4.1
3.5
4.4
6.1
5.8
5.5
20:1iso
0.4
0.3
0.3
0.3
0.3
0.3
0.0
0.5
0.6
0.5
0.5
20:2ω6
0.8
0.8
0.9
0.8
0.6
0.8
0.7
1.3
1.5
1.4
1.4
20:3ω3
0.6
0.8
0.8
0.8
0.7
0.8
0.7
0.6
0.7
0.7
0.6
22:0
0.4
0.5
0.5
0.5
0.4
0.5
0.5
0.6
0.6
0.6
0.6
20:4ω3
0.2
0.3
0.3
0.3
0.4
0.4
0.5
0.0
0.0
0.0
0.0
22:1
1.1
1.1
1.2
1.1
0.5
0.9
0.8
1.6
2.2
1.9
2.0
20:5ω3
0.7
1.3
1.6
1.5
1.6
1.1
1.7
0.0
0.0
0.0
0.1
22:2ω6
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.3
0.2
0.2
22:4ω6 + 22:3ω3
0.3
0.2
0.3
0.3
0.0
0.3
0.0
0.4
0.6
0.5
0.5
24:0
0.3
0.3
0.3
0.3
0.0
0.3
0.0
0.4
0.4
0.4
0.4
24:1
0.3
0.4
0.4
0.3
0.0
0.3
0.0
0.5
0.6
0.5
0.5
22:5ω3
0.3
1.1
1.2
1.1
1.1
0.9
0.8
0.0
0.0
0.0
0.0
22:6ω3
4.7
9.0
8.5
8.3
8.3
7.1
4.9
0.0
0.0
0.0
0.0


TABLE 12
Compiled data from the total seed lipid profiles from transgenic seed shown in
Table 11. Calculations do not include the ‘minor fatty acids’ in Table 11.
FD5.46
Parameter
pooled
# 2
# 4
# 8
# 7
# 9
# 1
# 3
# 5
# 6
# 10
total w3 (% of total FA)
36.1
46
45.4
45
48.2
44.2
40.1
28.9
29.9
30.2
28.9
total w6 (% of total FA)
25.8
20.4
20.7
20.3
19.5
21.1
25
33.7
32.6
31.8
33.8
w3/w6 ratio
1.40
2.25
2.19
2.22
2.47
2.09
1.60
0.86
0.92
0.95
0.86
w6/w3 ratio
0.71
0.44
0.46
0.45
0.40
0.48
0.62
1.17
1.09
1.05
1.17
total novel w3 (% of total FA)
8.1
18.5
18
16.9
18.6
15.2
12
0
0
0
0.1
total novel w6 (% of total FA)
1.1
2.2
1.2
1.1
1
1
1.2
1.5
2.3
2
2.2
novel w3/w6 ratio
7.36
8.41
15.00
15.36
18.60
15.20
10.00
0.05
novel w6/w3 ratio
0.14
0.12
0.07
0.07
0.05
0.07
0.10
22.00
OA to EPA efficiency
8.2%
15.6%
15.5%
15.1%
15.1%
12.8%
10.5%
0.0%
0.0%
0.0%
0.1%
OA to DHA efficiency
6.7%
12.3%
11.6%
11.5%
11.4%
10.0%
7.0%
0.0%
0.0%
0.0%
0.0%
LA to EPA efficiency
9.2%
17.2%
17.1%
16.7%
16.2%
13.9%
11.4%
0.0%
0.0%
0.0%
0.2%
LA to DHA efficiency
7.6%
13.6%
12.9%
12.7%
12.3%
10.9%
7.5%
0.0%
0.0%
0.0%
0.0%
ALA to EPA efficiency
15.8%
24.8%
24.9%
24.2%
22.8%
20.6%
18.5%
0.0%
0.0%
0.0%
0.3%
ALA to DHA efficiency
13.0%
19.6%
18.7%
18.4%
17.2%
16.1%
12.2%
0.0%
0.0%
0.0%
0.0%
total saturates
17.6
17.8
17.8
17.6
18
17.8
18.1
18.2
17.7
17.8
18.1
total monounsaturates
19.8
15.5
16
16.6
14.3
16.6
16.8
18.7
19.3
19.6
18.6
total polyunsaturates
62.5
66.6
66.4
65.6
67.7
65.6
65.1
63
63.1
62.5
63.2
total C20
9.6
9.3
9.8
9.9
8.1
8.9
8.5
8.6
11
10.3
10.1
total C22
5.4
10.3
10
9.7
9.4
8.3
5.7
0.6
0.9
0.7
0.7
C20/C22 ratio
1.78
0.90
0.98
1.02
0.86
1.07
1.49
14.33
12.22
14.71
14.43

DHA was present in six of the 10 individual seeds. The four other seeds did not have DHA and were presumed to be null segregants which did not have the T-DNA, based on hemizygosity of the T-DNA insertion in the parental plant. Extracted lipid from the single seed with the highest level of DHA had 9.0% DHA while the sum of the percentages for EPA, DPA and DHA was 11.4%. The sum of the percentages for the new ω3 fatty acids produced in this seed as a result of the transformation (SDA, ETrA, ETA, EPA, DPA, DHA) was 19.3% whilst the corresponding sum for the new ω6 fatty acids (GLA, EDA, DGLA, ARA and any ω6 elongation products) was 2.2%—only GLA and EDA were detected as new ω6 fatty acids. The total ω6 FA (including LA) to ω3 FA (including ALA) ratio was found to be 0.44. The new 06 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in the seed with the highest DHA level was 0.12. The level of total saturated fatty acids was about 17.8% and the level of monounsaturated fatty acids was about 15.5%. The level of total ω6-fatty acids was about 20.4% and the level of ω3-fatty acids was about 46%. Overall conversion efficiencies were calculated to be: OA to EPA=15.6%, OA to DHA=12.3%, LA to EPA=17.2%, LA to DHA=13.6%, ALA to EPA=24.8%, ALA to DHA=19.6%.

Homozygous seed from this line was obtained in the T4 generation. Up to 10.3% DHA was produced in event FD5-Δ6-18-110 with an average of 7.3% DHA observed across the entire T4 generation.

Homozygous seed was planted out across several glasshouses to generate a total of over 600 individual plants. Oil is being extracted from the seed using a variety of methods including soxhlet, acetone and hexane extractions.

Since the number of independently transformed lines of C. sativa obtained as described above was low, further experiments to transform C. sativa with pJP3416-GA7 are performed. The inventors predict that DHA levels of greater than 10% as a percentage of total fatty acids in seed oil will be achieved in further transformed lines, and plants which are homozygous for the T-DNA to 20% DHA. Twenty C. sativa GA7_modH events were generated and seed is being analysed for DHA content. Three GA7_modB events were generated and analysis of the T1 seed from event CMD17.1 revealed a pooled seed DHA content of 9.8%. The highest single seed DHA value was found to be 13.5%.

Example 4

Stable Expression of Transgenic DHA Pathways in Brassica napus Seeds

B. napus Transformation and Analysis of Fatty Acid Composition Using Single Vector

The binary vector pJP3416-GA7 was used to generate transformed Brassica napus plants and seeds from the plants. The vector pJP3416-GA7 as described above was introduced into Agrobacterium tumefaciens strain AGL1 via standard electroporation procedures. Cultures of the transgenic Agrobacterium cells were grown overnight at 28° C. in LB medium with agitation at 150 rpm. The bacterial cells were collected by centrifugation at 4000 rpm for 5 minutes, washed with Winans AB medium (Winans, 1988) and re-suspended in 10 mL of Winans AB medium (pH 5.2) and growth continued overnight in the presence of kanamycin (50 mg/L), rifampicin (25 mg/L) and 100 μM acetosyringone. Two hours before infection of the Brassica cells, spermidine (120 mg/L) was added and the final density of the bacteria adjusted to an OD 600 nm of 0.3-0.4 with fresh AB media. Freshly isolated cotyledonary petioles from 8-day old Brassica napus seedlings grown on ½ MS (Murashige and Skoog, 1962) or hypocotyl segments preconditioned by 3-4 days on MS media with 1 mg/L thidiazuron (TDZ) and 0.1 mg/L α-naphthaleneacetic acid (NAA) were infected with 10 mL Agrobacterium cultures for 5 minutes. The explants infected with Agrobacterium were then blotted on sterile filter paper to remove the excess Agrobacterium and transferred to co-cultivation media (MS media with 1 mg/L TDZ, 0.1 mg/L NAA and 100 μM acetosyringone) supplemented with or without different antioxidants (L-cysteine 50 mg/L and ascorbic 15 mg/L). All the plates were sealed with parafilm and incubated in the dark at 23-24° C. for 48 hrs.

The treated explants were then washed with sterile distilled water containing 500 mg/L cefotaxime and 50 mg/L timentin for 10 minutes, rinsed in sterile distilled water for 10 minutes, blotted dry on sterile filter paper, transferred to pre-selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 20 mg/L adenine sulphate (ADS), 1.5 mg/L AgNO3, 250 mg/L cefotaxime and 50 mg/L timentin) and cultured for five days at 24° C. with a 16 h/8 h photoperiod. They were then transferred to selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 20 mg/L ADS, 1.5 mg/L AgNO3, 250 mg/L cefotaxime and 50 mg/L timentin) with 1.5 mg/L glufosinate ammonium as the agent for selection of transformed cells, and cultured for 4 weeks at 24° C. with 16 h/8 h photoperiod with a biweekly subculture on to the same media. Explants with green callus were transferred to shoot initiation media (MS containing 1 mg/L kinetin, 20 mg/L ADS, 1.5 mg/L AgNO3, 250 mg/L cefotaxime, 50 mg/L timentin and 1.5 mg/L glufosinate ammonium) and cultured for another 2-3 weeks. Shoots emerging from the resistant explants were transferred to shoot elongation media (MS media with 0.1 mg/L gibberelic acid, 20 mg/L ADS, 1.5 mg/L AgNO3, 250 mg/L cefotaxime and 1.5 mg/L glufosinate ammonium) and cultured for another two weeks. Healthy shoots 2-3 cm long were selected and transferred to rooting media (½ MS containing 1 mg/L NAA, 20 mg/L ADS, 1.5 mg/L AgNO3 and 250 mg/L cefotaxime) and cultured for 2-3 weeks. Well established shoots with roots were transferred to pots containing seedling raising mix and grown in a growth cabinet for two weeks and subsequently transferred to a glasshouse. Approximately 40 (T0) plants transformed with the GA7 construct were obtained by this method.

Plants were grown to maturity after being allowed to self-fertilise. Seeds obtained from transformed plants were analysed for fatty acid composition in their seedoil as described in Example 1. Data for a transformed line with the highest DHA level are shown in Table 13. DHA levels on average were significantly lower in the seedoil of the B. napus seeds transformed with the T-DNA from pJP3416-GA7 than in A. thaliana seeds (Example 2) or Camelina seeds (Example 3) transformed with the same construct. The highest level of DHA in approximately 40 lines was found to be 1.52% with the majority of the transgenic lines having detectable DHA. It was noted that there was a substantial accumulation of ALA, about 35% of the total fatty acids, in these seeds which was not being converted efficiently to SDA or following products in the pathway.

Fatty acid profile analysis of single B. napus seeds from a T1 event, CT125-2, was performed to better determine the amount of DHA produced in transgenic seeds. Seeds were found to contain between 0% (null seeds) and 8.5% DHA (Table 13).

Some of the seeds from the plant line CT116 as well as other transgenic lines showing DHA production were sown to produce progeny plants. RT-PCR was performed on total RNA isolated from developing embryos from these plants in order to determine why the GA7 construct performed poorly for DHA production relative to transgenic A. thaliana and C. sativa having the same construct, and poorly relative to the combination of the genes on pJP3115 and pJP3116 (below). RT-PCR was performed on total RNA using a one-step RT-PCR kit (Invitrogen) and gene-specific primers targeting each transgene. This confirmed that each of the genes in the GA7 construct was expressed well in the B. napus transformants except for the Δ6-desaturase which was poorly expressed in the majority of transformed seeds. The other genes from this construct functioned well in both B. napus and A. thaliana seeds, for example the Δ12- and Δ15-desaturases which functioned to produce increased levels of LA and ALA in the seeds whilst decreasing oleic acid levels. A representative RT-PCR gel is shown in FIG. 7 which clearly shows the low expression of the Δ6-desaturase relative to the other transgenes from pJP3416-GA7.

Transgenic plants and seed which are homozygous for the transgenes are generated by planting out progeny from the lines with the highest DHA.


TABLE 13
Fatty acid composition as a percentage of total fatty
acids in seedoil from independent T1 Brassica napus
seed transformed with pJP3416-GA7, lines CT116-11
and CT-125-2 compared to wild-type (untransformed)
control. 22:6ω3 is DHA. Data from single CT125-2
B. napus seeds is denoted by ‘SS’.
CT116-
CT125-
CT125-2
CT125-2
CT125-2
Control
11
2
#2 SS
#3 SS
#10 SS
14:0
0.1
0.2
0.1
0.1
0.1
0.1
16:0
4.3
7.2
5.2
6.5
4.7
7.7
16:1
0.2
0.5
0.4
0.3
0.3
0.8
16:3
0.2
0.2
0.2
0.1
0.2
0.2
18:0
2.1
2.2
2.4
2.3
2.3
2.8
18:1d9
59.1
27.0
38.1
34.0
19.3
14.8
18:1d11
3.7
6.6
4.2
4.4
4.3
9.6
18:2
19.7
14.1
16.6
13.9
10.2
10.2
18:3ω3
8.3
35.2
27.7
34.1
49.5
37.9
20:0
0.6
0.5
0.6
0.4
0.3
0.7
18:4ω3
0.0
0.9
0.3
0.5
0.6
2.6
20:1d11
1.2
1.1
1.0
1.0
0.8
0.6
20:1is0
0.2
0.1
0.2
20:2ω6
0.1
0.1
0.1
0.1
0.1
0.1
20:3ω3
1.3
0.7
0.8
1.6
0.9
22:0
0.3
0.4
0.3
0.1
0.1
0.4
20:4ω3
0.1
0.3
0.4
0.6
0.5
22:1
20:5ω3
0.1
0.3
22:3ω3
0.1
24:0
0.2
0.4
0.3
0.1
0.1
0.3
24:1
0.1
0.3
0.1
0.1
0.2
0.1
22:5ω3
0.1
0.1
0.1
0.1
0.5
22:6ω3
1.52
1.2
1.3
2.7
8.5

B. napus Transformation and Analysis of Fatty Acid Composition Using Two Vectors

In another experiment in B. napus and as an alternative format for introducing the transgenes, the binary vectors pJP3115 and pJP3116 as described in WO 2010/057246 were used to separately generate transformed B. napus plants and transformed seeds were obtained from the plants. The T-DNA on pJP3115 comprised chimeric genes encoding the Crepis palestina Δ12-desaturase, Micromonas pusilla Δ6-desaturase, Pyramimonas cordata Δ6-elongase and Pavlova salina Δ5-desaturase and the T-DNA on pJP3116 contained chimeric genes encoding Perilla frutescens Δ15-desaturase, Pyramimonas cordata Δ5-elongase and Pavlova salina Δ4-desaturase. The two T-DNAs, when present together and expressed in developing seeds, formed a 7-gene pathway for producing DHA from endogenously produced oleic acid. These vectors were introduced into Agrobacterium tumefaciens strain AGL1 via standard electroporation procedures and the transformed cells used independently to transform B. napus using the method as described above to generate stably transformed T0 plants. 29 pJP3115 and 19 pJP3116 transformants were obtained and these plants were grown to maturity and seeds obtained after self-fertilisation were analysed for fatty acid composition in their seedoil. Transformation with the T-DNA from pJP3115 was expected to result in EPA production from endogenously produced ALA whilst transformation with the T-DNA from pJP3116 was expected to result in increased ALA production from LA. Several plants were identified which displayed these phenotypes. The majority of events displayed a decreased OA/increased LA phenotype due to Δ12 desaturation with a low level of EPA production. Up to 2.6% EPA was observed in pJP31115 transgenic pooled seed. Similarly, the majority of pJP3116 events were found to have an elevated ALA phenotype due to Δ15-desaturase activity. Up to 18.5% ALA was found in pooled seed transformed with the T-DNA from pJP3116.

T1 plants from the lines with the highest levels of EPA and ALA were crossed and the progeny seed (F1) from 24 recovered events analysed for DHA content. DHA was found in 17 of these events with up to 1.9% DHA found in pooled seed from these events. Single-seed analysis was performed to determine the range of DHA production—the data are shown in Table 14. A large range of DHA levels were observed in the crossed progeny, probably due to the hemizygous nature of the T-DNAs in the parental plants, so that some seeds did not receive both T-DNAs. Up to 6.7% DHA was observed in total seed lipid.


TABLE 14
Fatty acid composition as a percentage of total fatty acids in seedoil from B. napus
F1 single seeds that were from a cross of plants transgenic for the T-DNA from
pJP3115 with plants transgenic for the T-DNA from pJP3116. B1, B2 and B4
designate events. 0.0 = not detectable by the GC method.
B1.1
B1.2
B1.3
B1.4-g
B1.5-g
B2.1
B2.2
B2.3g
B2.4g
B2.5g
B3.1
B3.2
14:0
0.1
0.1
0.1
0.2
0.2
0.1
0.1
0.2
0.2
0.1
0.1
0.1
16:0
6.6
6.4
4.5
12.3
7.9
5.1
5.0
10.1
8.5
6.8
5.3
7.2
16:1
0.4
0.5
0.2
1.0
0.6
0.4
0.4
0.6
1.1
0.5
0.5
0.6
16:3
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
0.1
0.1
0.2
18:0
2.3
2.6
2.2
1.6
2.9
2.9
3.4
2.2
1.8
2.9
3.4
2.4
18:1
34.1
39.3
46.9
14.9
20.7
41.6
46.3
14.4
23.4
38.3
43.6
32.0
18:1d11
4.6
5.8
2.7
6.8
6.2
3.8
4.9
5.9
8.7
4.5
5.5
5.1
18:2
33.6
30.7
30.4
29.2
34.4
31.7
27.7
33.2
23.9
33.3
27.9
33.4
18:3ω6
0.2
0.3
0.1
0.4
0.4
0.2
0.2
0.7
0.1
0.2
0.2
0.3
18:3ω3
10.3
7.1
7.7
18.7
14.9
8.2
5.9
14.8
28.1
6.3
7.3
10.0
20:0
0.6
0.7
0.6
0.5
0.7
0.8
0.9
0.6
0.4
0.7
0.9
0.7
18:4ω3
0.2
0.1
0.1
0.8
0.5
0.2
0.2
0.8
0.0
0.2
0.2
0.2
20:1d11
1.0
1.1
1.1
0.7
0.8
1.1
1.1
0.5
0.9
1.1
1.1
0.9
20:1iso
0.1
0.1
0.0
0.1
0.1
0.1
0.1
0.1
0.3
0.1
0.1
0.1
20:2ω6
0.4
0.3
0.2
0.5
0.5
0.4
0.3
0.4
0.5
0.5
0.3
0.5
20:3ω6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
20:4ω6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
20:3ω3
1.8
1.6
1.1
2.8
2.1
1.1
1.0
2.7
0.7
1.4
0.9
1.6
22:0
0.3
0.4
0.3
0.3
0.4
0.4
0.5
0.3
0.3
0.4
0.5
0.4
20:4ω3
0.3
0.2
0.2
0.4
0.4
0.1
0.1
0.5
0.0
0.2
0.1
0.2
22:1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
20:5ω3
0.0
0.0
0.0
0.1
0.1
0.0
0.0
0.2
0.0
0.0
0.0
0.0
22:2ω6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
22:4ω6
0.1
0.2
0.1
0.2
0.2
0.1
0.1
0.4
0.2
0.2
0.1
0.2
24:0
0.3
0.4
0.2
0.2
0.3
0.3
0.3
0.3
0.4
0.4
0.4
0.3
22:5ω6
0.1
0.2
0.1
0.2
0.3
0.1
0.1
0.5
0.0
0.2
0.1
0.2
24:1
0.2
0.2
0.2
0.3
0.3
0.2
0.2
0.3
0.3
0.2
0.2
0.2
22:5ω3
0.7
0.7
0.3
2.1
1.6
0.3
0.4
3.2
0.0
0.5
0.4
1.2
22:6ω3
1.4
1.0
0.5
5.5
3.9
0.8
0.7
6.7
0.0
1.1
0.8
2.0


TABLE 15
Compiled data from the total seed lipid profiles from transgenic seed shown in
Table 14. Calculations do not include the ‘minor fatty acids’ in Table 14.
Parameter
B1.1
B1.2
B1.3
B1.4-g
B1.5-g
B2.1
B2.2
B2.3g
B2.4g
B2.5g
B3.1
total w3 (% of total FA)
4.6
3.9
2.3
12.1
9
2.7
2.6
14.8
0.8
3.6
2.6
total w6 (% of total FA)
44.5
38.5
38.5
48.8
50.3
40.5
34.1
49.4
52.7
40.5
35.7
w3/w6 ratio
0.10
0.10
0.06
0.25
0.18
0.07
0.08
0.30
0.02
0.09
0.07
w6/w3 ratio
9.67
9.87
16.74
4.03
5.59
15.00
13.12
3.34
65.88
11.25
13.73
total novel w3 (% of total FA)
2.6
2
1.1
8.9
6.5
1.4
1.4
11.4
0
2
1.5
total novel w6 (% of total FA)
10.5
7.5
7.9
19.1
15.4
8.4
6.1
15.8
28.3
6.7
7.5
novel w3/w6 ratio
0.25
0.27
0.14
0.47
0.42
0.17
0.23
0.72
0.00
0.30
0.20
novel w6/w3 ratio
4.04
3.75
7.18
2.15
2.37
6.00
4.36
1.39
3.35
5.00
OA to EPA efficiency
2.5%
2.1%
0.9%
10.1%
6.9%
1.3%
1.3%
12.8%
1.9%
1.4%
OA to DHA efficiency
1.7%
1.2%
0.6%
7.2%
4.8%
0.9%
0.8%
8.5%
1.3%
1.0%
LA to EPA efficiency
4.3%
4.0%
2.0%
12.6%
9.4%
2.5%
3.0%
15.7%
3.6%
3.1%
LA to DHA efficiency
2.9%
2.4%
1.2%
9.0%
6.6%
1.9%
1.9%
10.4%
2.5%
2.1%
ALA to EPA efficiency
47.7%
44.7%
36.4%
68.1%
65.9%
44.0%
45.8%
72.1%
47.1%
50.0%
ALA to DHA efficiency
31.8%
26.3%
22.7%
48.7%
45.9%
32.0%
29.2%
47.9%
32.4%
33.3%
total saturates
10.2
10.6
7.9
15.1
12.4
9.6
10.2
13.7
11.6
11.3
10.6
total monounsaturates
40.4
47
51.1
23.8
28.7
47.2
53
21.8
34.7
44.7
51
total polyunsaturates
49.2
42.5
40.9
61
59.4
43.3
36.8
64.3
53.7
44.2
38.4
total C20
4.2
4
3.2
5.1
4.7
3.6
3.5
5.1
2.8
4
3.4
total C22
2.6
2.5
1.3
8.3
6.4
1.7
1.8
11.1
0.5
2.4
1.9
C20/C22 ratio
1.62
1.60
2.46
0.61
0.73
2.12
1.94
0.46
5.60
1.67
1.79

Compiled data from the total lipid profiles (Table 14) are shown in Table 15. From the data in Table 15, the total ω6 FA (including LA) to ω3 FA (including ALA) ratio in the seed with the highest level of DHA was 3.34. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio was 1.39. The level of total saturated fatty acids was about 13.7% and the level of monounsaturated fatty acids was about 21.8%. The level of total ω6-fatty acids was about 46.4% and the level of ω3-fatty acids was about 14.8%. Overall conversion efficiencies were calculated to be: OA to EPA=12.8%, OA to DHA=8.5%, LA to EPA=15.7%, LA to DHA=10.4%, ALA to EPA=72.1%, ALA to DHA=47.9%. The reduced efficiency of the 06 fatty acids to ω3 fatty acids conversion observed in this experiment with the combination of the pJP3115 and pJP3116 was thought to be due to a lower efficiency of the plant Δ15-desaturase compared to the fungal Δ15/ω3 desaturase (Examples 2 and 3) when combined with the genes for conversion of ALA to DHA.

Progeny from DHA-containing lines which are homozygous for all of the introduced transgenes are generated for analysis.

Example 5

Modifications to T-DNAs Encoding DHA Pathways in Plant Seeds

In order to improve the DHA production level in B. napus beyond the levels described in Example 4, the binary vectors pJP3416-GA7-modA, pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE and pJP3416-GA7-modF were constructed as follows. These binary vectors were variants of the pJP3416-GA7 construct described in Example 2 and were designed to further increase the synthesis of DHA in plant seeds, particularly by improving Δ6-desaturase and Δ6-elongase functions. SDA had been observed to accumulate in some seed transformed with the GA7 construct due to a relatively low elongation efficiency compared to the Δ5-elongase, so amongst other modifications, the two elongase gene positions were switched in the T-DNA.

The two elongase coding sequences in pJP3416-GA7 were switched in their positions on the T-DNA to yield pJP3416-GA7-modA by first cloning a new P. cordata Δ6-elongase cassette between the SbfI sites of pJP3416-GA7 to replace the P. cordata Δ5-elongase cassette. This construct was further modified by exchanging the FP1 promoter driving the M. pusilla Δ6-desaturase with a conlinin Cnl2 promoter (pLuCnl2) to yield pJP3416-GA7-modB. This modification was made in an attempt to increase the Δ6-desaturase expression and thereby enzyme efficiency. It was thought that the Cnl2 promoter might yield higher expression of the transgene in B. napus than the truncated napin promoter. pJP3416-GA7-modC was produced by adding a second M. pusilla Δ6-desaturase cassette with slightly different codon usage (SEQ ID NO:15) and driven by the FP1 promoter, which was inserted at the PmeI site just inside the right border of pJP3416-GA7-modB. The second Δ6-desaturase cassette was added to both pJP3416-GA7-modB and pJP3416-GA7-modF in order to increase the Δ6-desaturase expression level and extend the time period during seed development for expression of Δ6-desaturase by the use of multiple promoters. Different codon usages were used in the two nucleotide sequences to result in the translation of the same protein sequence without risking co-suppression from similar coding regions within the same T-DNA. pJP3416-GA7-modD and pJP3416-GA7-modE were similar variants in which a third MAR sequence, corresponding to nucleotides 16649-17816 of SEQ ID NO: 1, was added to pJP3416-GA7 and pJP3416-GA7-modB, respectively, at the PmeI site. pJP3416-GA7-modF was produced by adding a second M. pusilla Δ6-desaturase cassette containing the native Δ6-desaturase nucleotide sequence and driven by the FP1 promoter at the PmeI site at the right border of pJP3416-GA7-modB. pJP3416-GA7-modG was made by first replacing the M. pusilla Δ6-desaturase cassette with a Cnl2:P. cordata Δ5-elongase cassette by restriction cloning at the AscI-PacI sites. pJP3416-GA7-modG was then made by replacing the original FAE1:P. cordata Δ5-elongase cassette with a FAE1:M. pusilla Δ6-desaturase cassette by restriction cloning at the SbfI sites. The nucleotide sequences of the T-DNAs from each of these genetic constructs are shown as: pJP3416-GA7-modB (SEQ ID NO:2), pJP3416-GA7-modC (SEQ ID NO:3), pJP3416-GA7-modD (SEQ ID NO:4), pJP3416-GA7-modE (SEQ ID NO:5), pJP3416-GA7-modF (SEQ ID NO:6) and pJP3416-GA7-modG (SEQ ID NO:7).

The binary vectors pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE, pJP3416-GA7-modF and pJP3416-GA7-modG are used to generate transformed Brassica somatic embryos and Brassica napus, Camelina sativa and Arabidopsis thaliana plants and progeny seeds. Data for pJP3416-GA7-modB are shown in the next Example.

Eight transgenic pJP3416-GA7-modB A. thaliana events and 15 transgenic pJP3416-GA7-modGA. thaliana events were generated. Between 3.4% and 7.2% DHA in pooled pJP3416-GA7-modB seed was observed and between 0.6 and 4.1% DHA in pooled T2 pJP3416-GA7-modG seed was observed. Several of the highest pJP3416-GA7-modB events were sown out on selectable media and surviving seedlings taken to the next generation. Seed is being analysed for DHA content. Since the pooled T1 seeds represented populations that were segregating for the transgenes and included any null segregants, it is expected that the homozygous seeds from progeny plants will have increased levels of DHA, up to 20% of the total fatty acid content in the seed oil. The other modified constructs were used to transform A. thaliana. Although only a small number of transformed lines were obtained, none yielded higher levels of DHA than the modB construct.

The pJP3416-GA7-modB construct was also used to generate transformed B. napus plants of cultivar Oscar and in a breeding line designated NX005. Ten independent transformed plants (T0) were obtained so far for the Oscar transformation, and 20 independent lines for NX005. Seed (T1 seed) was harvested from these transgenic lines. Pools of seed were tested for levels of DHA in the seed oil, and two lines which showed the highest levels were selected, these were designated lines CT132.5 (in cultivar Oscar) and CT133.15 (in NX005). Twenty seeds from CT132.5 and 11 seeds from CT133.15 were imbibed and, after two days, oil was extracted from a half cotyledon from each of the individual seeds. The other half cotyledons with embryonic axes were kept and cultured on media to maintain the specific progeny lines. The fatty acid composition in the oil was determined; the data is shown in Table 16 for CT132.5. The DHA level in ten of the 20 seeds analysed was in the range of 7-20% of the total fatty acid content as determined by the GC analysis. Other seeds had less than 7% DHA and may have contained a partial (incomplete) copy of the T-DNA from pJP3416-GA7-modB. The transgenic line appeared to contain multiple transgene insertions that were genetically unlinked. The seeds of transgenic line CT133.15 exhibited DHA levels in the range 0-5%. Seeds with no DHA were likely to be null segregants. These data confirmed that the modB construct performed well for DHA production in canola seed.

The pJP3416-GA7-modB and pJP3416-GA7-modF constructs were also used to generate transformed Camelina sativa plants. At least 24 independent transformed plants (T0) were obtained and examined in more detail by progeny analysis. Seed (T1 seed) was harvested from these transgenic lines. Pools of seed were tested for levels of DHA in the seed oil, and 6 lines which showed the highest levels of DHA (between 6% and 9%) were selected. The DHA levels in 20 T1 seeds from each line were analysed-most seeds exhibited DHA levels in the range of 6-14% of the total fatty acid content as determined by the GC analysis. The fatty acid composition in the oil was determined; the data is shown in Table 17 for several transgenic seeds. These data confirmed that the modB and modF constructs both performed well for DHA production in Camelina seed.


TABLE 16
Fatty acid profiles of half cotyledons of germinating T1 transgenic B. napus
seeds containing the modB construct. Up to 18.1% DHA was observed with
numerous samples containing greater than 10% DHA.
Seed
14:0
16:0
16:1d3?
16:1
16:3
18:0
18:1
18:1d11
18:2
18:3n6
18:3n3
20:0
18:4n3
C20:1d11
1
0.1
4.2
0.1
0.1
0.2
1.8
29.9
2.5
9.9
0.1
38.4
0.5
0.8
1.0
2
0.1
4.7
0.1
0.1
0.2
4.0
23.0
2.3
7.4
0.3
29.3
1.0
4.3
1.1
3
0.1
3.7
0.2
0.1
0.2
1.8
55.1
1.9
4.7
0.2
15.2
0.8
1.8
1.4
4
0.1
4.6
0.2
0.2
0.2
2.9
22.1
1.8
6.6
0.4
26.5
1.0
7.2
1.0
5
0.1
4.0
0.1
0.1
0.2
1.7
27.4
2.1
8.1
0.3
26.4
0.6
2.8
1.0
6
0.1
3.5
0.1
0.1
0.2
1.6
59.8
2.0
4.3
0.1
18.5
0.6
0.5
1.3
7
0.1
6.0
0.3
0.3
0.3
1.7
16.6
2.6
23.9
1.0
23.2
0.6
5.4
0.8
8
0.1
4.9
0.1
0.1
0.2
2.7
12.9
1.4
11.7
0.3
34.3
0.9
5.0
0.9
9
0.1
3.9
0.1
0.1
0.1
2.4
41.6
1.7
21.5
0.0
23.4
0.7
0.0
1.2
10
0.1
3.7
0.2
0.1
0.1
2.1
30.9
1.7
19.2
0.4
23.6
0.7
2.1
1.1
11
0.1
5.7
0.4
0.3
0.2
3.8
41.2
2.4
26.7
2.1
7.2
1.3
0.3
1.2
12
0.1
4.6
0.0
0.1
0.2
2.4
25.5
1.7
16.1
0.3
28.9
0.8
3.9
1.1
13
0.1
4.3
0.1
0.1
0.1
4.2
19.4
1.6
9.2
0.1
45.5
1.0
0.2
1.1
14
0.1
6.3
0.2
0.2
0.2
4.0
10.5
2.3
8.4
0.3
31.1
1.3
3.9
0.8
15
0.1
5.1
0.1
0.2
0.2
3.3
16.8
2.4
11.2
0.3
28.8
1.0
4.5
0.9
16
0.1
4.4
0.1
0.1
0.2
4.0
16.2
1.5
11.6
0.2
33.5
0.9
2.8
1.1
17
0.2
7.2
0.2
0.2
0.2
4.9
15.0
2.1
8.9
0.3
25.9
1.4
5.1
0.9
18
0.1
4.0
0.1
0.1
0.2
2.3
64.8
1.2
7.2
0.1
12.5
1.0
3.5
1.5
19
0.1
3.9
0.1
0.1
0.2
4.6
36.9
1.7
7.1
0.2
28.6
1.2
1.8
1.2
20
0.1
4.8
0.1
0.1
0.2
6.0
18.5
1.2
12.8
0.2
34.8
1.4
2.4
1.1
Seed
20:1d13
C20:2n6
C20:3n3
C22:0
20:4n3
20:5n3
22:3n3
C24:0
C24:1
22:5n3
C22:6n3
 1
0.0
0.1
2.1
0.3
2.8
0.3
0.1
0.2
0.2
0.5
3.9
 2
0.0
0.1
1.9
0.4
6.9
1.0
0.0
0.3
0.1
1.7
9.5
 3
0.0
0.1
0.3
0.5
11.3
0.0
0.0
0.3
0.2
0.0
0.0
 4
0.0
0.1
0.8
0.5
11.2
1.9
0.0
0.2
0.2
1.7
8.7
 5
0.0
0.1
1.5
0.3
7.6
1.5
0.0
0.1
0.1
1.8
12.2
 6
0.0
0.0
0.7
0.3
6.0
0.0
0.0
0.2
0.1
0.0
0.0
 7
0.0
0.2
0.6
0.4
2.6
1.1
0.0
0.3
0.3
1.7
9.9
 8
0.0
0.2
2.4
0.5
4.1
1.3
0.0
0.2
0.2
1.8
13.8
 9
0.0
0.1
2.2
0.4
0.0
0.0
0.1
0.3
0.2
0.0
0.0
10
0.0
0.1
1.5
0.4
3.6
0.6
0.0
0.2
0.1
0.7
6.9
11
0.0
0.2
0.3
0.8
4.8
0.0
0.0
0.6
0.3
0.0
0.0
12
0.0
0.1
1.9
0.4
3.9
0.6
0.0
0.2
0.0
1.1
6.2
13
0.0
0.1
5.2
0.4
2.6
0.3
0.2
0.2
0.1
0.4
3.4
14
0.0
0.1
2.3
0.6
4.6
1.8
0.1
0.3
0.2
2.5
18.1
15
0.0
0.1
2.1
0.6
3.2
1.5
0.1
0.3
0.1
1.8
15.1
16
0.0
0.2
3.7
0.4
4.6
0.7
0.1
0.3
0.1
1.3
12.1
17
0.0
0.0
1.6
0.8
4.9
2.1
0.0
0.6
0.3
2.2
15.0
18
0.0
0.1
0.0
0.7
0.0
0.0
0.0
0.5
0.2
0.0
0.0
19
0.0
0.1
1.4
0.5
4.3
0.4
0.0
0.4
0.1
0.8
4.3
20
0.0
0.1
3.4
0.6
3.2
0.4
0.1
0.3
0.1
0.7
7.6


TABLE 17
Fatty acid profiles of T1 transgenic C. sativa seeds containing the modB or modF constructs
C14:0
C16:0
C16:1
C18:0
C18:1
C18:1d11
C18:2
C18:3n6
C18:3n3
C20:0
123-8 
0.1
7.3
0.0
5.2
7.9
1.0
7.7
0.7
29.9
2.3
123-12 
0.1
8.3
0.0
5.3
7.2
1.2
8.7
0.9
27.2
2.5
5-8
0.1
8.3
0.1
3.5
9.4
1.3
8.1
1.1
29.0
1.0
5-9
0.1
8.1
0.0
3.5
9.4
1.2
8.4
1.2
29.2
1.0
17-10
0.1
8.7
0.1
4.1
8.4
1.3
5.5
1.2
26.1
1.6
17-26
0.1
8.8
0.1
5.5
5.0
1.3
7.6
0.9
27.8
2.7
18:4n3
C20:1d11
20:1d13
C20:2n6
C20:3n6
C20:4n6
C20:3n3
C22:0
20:4n3
123-8 
6.0
7.1
0.4
0.7
0.0
0.0
0.9
0.4
1.3
123-12 
5.7
6.9
0.5
0.7
0.0
0.1
0.9
0.5
1.5
5-8
9.3
7.9
0.4
0.6
0.0
0.0
0.8
0.2
0.4
5-9
9.0
8.1
0.3
0.6
0.0
0.0
0.8
0.2
0.5
17-10
11.8
7.2
0.3
0.0
0.4
0.03
0.8
0.3
0.4
17-26
10.1
6.2
0.3
0.0
0.7
0.03
1.1
0.6
0.5
C22:1
20:5n3
C22:2n6
22:3n3
C24:0
C24:1
22:5n3
C22:6n3
123-8 
1.0
4.6
0.0
0.1
0.2
0.3
1.5
13.3
123-12 
1.2
5.0
0.0
0.1
0.2
0.4
1.5
13.2
5-8
0.8
3.4
0.0
0.1
0.2
0.4
0.9
12.6
5-9
0.8
3.5
0.0
0.1
0.1
0.3
0.9
12.6
17-10
0.7
5.5
0.0
0.0
0.2
0.3
1.3
13.5
17-26
1.0
4.7
0.1
0.1
0.3
0.4
1.0
13.1

The inventors considered that, in general, the efficiency of rate-limiting enzyme activities in the DHA pathway can be greater in multicopy T-DNA transformants compared to single-copy T-DNA transformants, or can be increased by inserting into the T-DNA multiple genes encoding the enzyme which might be limiting in the pathway. Evidence for the possible importance of multi-copy transformants was seen in the Arabidopsis seeds transformed with the GA7 construct (Example 2), where the highest yielding DHA event had three T-DNAs inserted into the host genome. The multiple genes can be identical, or preferably are different variants that encode the same polypeptide, or are under the control of different promoters which have overlapping expression patterns. For example, increased expression could be achieved by expression of multiple Δ6-desaturase coding regions, even where the same protein is produced. In pJP3416-GA7-modF and pJP3416-GA7-modC, for instance, two versions of the M. pusilla Δ6-desaturase were present and expressed by different promoters. The coding sequences had different codon usage and therefore different nucleotide sequences, to reduce potential silencing or co-suppression effects but resulting in the production of the same protein.

Example 6

Activity of Seed-Specific Constructs in Somatic Embryos

In order to establish a rapid assay system which was predictive of expression of genetic constructs in seeds under the control of seed-specific promoters, a somatic embryo system was set up for Brassica napus. This used a vector to express the LEC2 transcription factor which is involved in initiation of somatic embryogenesis. As a demonstration, the binary vectors 35S:LEC2 and pJP107 (Petrie et al., 2010a and b) were introduced into Agrobacterium tumefaciens strain AGL1 via standard electroporation and the Agrobacterium transformants used to co-transform Brassica napus by co-cultivation. The T-DNA region of pJP107 contained genes encoding the Isochrysis galbana Δ9-elongase, P. salina Δ8-desaturase and P. salina Δ5-desaturase with each gene expressed by a seed-specific promoter. A control transformation used the 35S:LEC2 vector alone. 35S:LEC2 expression resulted in the generation of somatic embryos in tissue culture directly from the transformed B. napus callus tissue as described in Example 1.

Fatty acid analysis showed that the seed-specific genes on the T-DNA of the construct pJP107 were expressed in the transgenic somatic embryos in the presence of the co-transformed LEC2 gene and functioned to produce ARA (20:4Δ5,8,11,14) from LA and EPA (20:5Δ5,8,11,14,17) from ALA. The data for three co-transformed somatic embryos are shown in Table 18 and the fatty acid composition of each compared to the fatty acid composition of seed oil from Brassica napus seed which was transgenic for, and expressing, the T-DNA of pJP107 (Petrie et al., 2010a and b). Similar total percentages of ARA and the intermediate fatty acids EDA (20:2ω6) and DGLA (20:3ω6), as well as conversion efficiencies, were observed in somatic embryo tissue when compared with stably-transformed seed profiles. Similar results were observed in the fatty acid compositions of the stable T2 transgenic seed and somatic embryos: ω6 fatty acids were at a level of 26.6% and 25.6% (on average), respectively, whilst ARA levels were found to be 9.7% and 10.6% (on average), respectively.

When 35S:LEC2 alone was introduced and the somatic embryos analysed in a time-course, the fatty acid profile was found to change to a more embryo-like profile with 18:3Δ9,12,15 decreasing and 18:1Δ9 increasing in an inversely correlated manner (FIG. 8). These results indicated that the somatic embryos were indeed becoming seed-like in character and the genes on the T-DNA from pJP107 were expressed. This demonstrated that the somatic embryo system allowed a rapid characterisation of transgenic seed-specific constructs in B. napus without requiring the full process of producing a transgenic plant and, from that, mature seed.


TABLE 18
Fatty acid composition of lipid obtained from Brassica napus somatic embryos generated by co-transforming
pJP107 with 35S:LEC2, compared to the control untransformed (WT) and T2 seeds transformed with pJP107.
Individual enzymatic conversion efficiencies are shown in brackets after the relevant enzymatic steps.
D9-Elo is Δ9-elongase, D8-Des is Δ8-desaturase and D5-Des is Δ5-desaturase.
T2 pJP107
WT
transgenic seed
LEC2:#45
LEC2:#57
LEC2:#58
18:1Δ9
57.2
45.7
3.8
2.5
1.9
18:2Δ9, 12
19.1
8.7
10
10.6
10
18:3Δ9, 12, 15
10.2
4.1
22.5
27.5
24.2
20:2Δ11, 14
7.1 ± 1.9 (67% D9-elo)
5.2 (61.8% D9-elo)
3.7 (56.7% D9-elo)
4.6 (61.8% D9-elo)
20:3Δ8, 11, 14
1.1 ± 0.2 (60% D8-des)
 0.4 (67% D8-des)
0.2 (73% D8-des) 
 0.4 (73% D8-des)
20:4Δ5, 8, 11, 14
9.7 ± 0.9 (90% D5-des)
10.6 (98% D5-des)
10 (96% D5-des)
11.2 (97% D5-des)
20:3Δ11, 14, 17
4.0 ± 0.8
9.9
5.5
7.3
20:4Δ8, 11, 14, 17
0.3 ± 0.1
0.4
0.3
0.4
20:5Δ5, 8, 11, 14, 17
2.4 ± 0.2
7.6
6.4
7.9
Total new
24.6
34.1
26.1
31.8

Using the same system to generate somatic embryos, Brassica napus cells were transformed separately with pJP3416-GA7-modB and pJP3416-GA7-modD. 42 embryos were obtained, 18 for modB and 24 for modD. Total lipid was extracted from the embryos and analysed for fatty acid composition. The embryos contained between 0% and up to 16.9% DHA (Table 19). The results with 0% DHA was presumed to be due to integration of only a partial T-DNA or an insertion into a transcriptionally silent region of the genome. The total ω3 FA (including ALA) to total ω6 FA (including LA) ratio was found to be 2.3 for embryo #270 and 11.96 for embryo #284. The total ω6 FA (including LA) to total ω3 FA (including ALA) ratio was 0.08 for #284. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio was 0.03 for #284. Overall conversion efficiencies were calculated to be: (for embryos #270, #284) OA to EPA=14.0%, 29.8%; OA to DHA=9.7%, 24.2%; LA to EPA=15.4%, 30.7%; LA to DHA=10.7%, 25.0%; ALA to EPA=22.1%, 33.3%; ALA to DHA=15.3%, 27.0%. These efficiencies were similar, or greater than in the case of #284, to those observed for the T3 pJP3416-GA7 Arabidopsis lines which indicated that the pJP3416-GA7-modB vector was capable of functioning well in B. napus cells. The SDA level was below 3.0%, indicating that the Δ6-elongase was performing even better than the GA7 construct. The individual enzyme efficiencies achieved in #284 were: Δ12-desaturase, 97.4%; ω3-desaturase, 92.3%; Δ6-desaturase, 38.2%; Δ6-elongase, 88.2%; Δ5-desaturase, 98.8%; Δ5-elongase, 94.1%; and Δ4-desaturase, 86.3%. Total saturates were 21.2%, total monounsaturates were 10.2%, total polyunsaturates were 68.6%.

The inventors believe this was the highest level of DHA achieved in B. napus cells to date, except for further data described below. This also demonstrated that the modification in pJP3416-GA7-modB relative to pJP3416-GA7 was effective in increasing the level of expression of the Δ6-desaturase gene. The binary vectors pJP3416-GA7, pJP3416-GA7-modA, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE and pJP3416-GA7-modF as described above are co-transformed with 35S:LEC2 to generate transformed B. napus somatic embryos. Up to 7.0% DHA was observed in modD embryos, 9.9% in modE embryos, 8.3% in modF embryos and 3.6% in a small number of modG embryos.


TABLE 19
Fatty acid composition of oil from Brassica napus somatic
embryos #270 and #284 generated by co-transforming
the seed-specific DHA acid construct pJP3416-GA7-modB
with 35S:LEC2, and #286 and #289 (pJP3416-GA7-modD).
#270
#284
#286
#289
14:0
0.3
0.2
0.2
0.2
16:0
14.0
15.7
17.2
16.6
16:1d9
0.7
0.4
0.8
0.8
16:3
0.5
0.6
1.1
1.3
18:0
2.6
2.4
2.5
2.5
18:1d9
6.6
1.8
1.5
1.1
18:1d11
6.3
6.8
6.5
6.7
18:2
18.9
4.5
10.0
9.8
18:3ω6
0.7
0.8
0.3
0.3
18:3ω3
33.0
37.2
42.0
41.5
20:0
0.9
0.9
0.8
0.8
18:4ω3
1.9
2.8
3.6
4.5
20:1d11
0.2
0.1
0.1
0.1
20:2ω6
0.1
0.1
0.1
0.2
20:3ω3
0.5
0.0
0.5
0.6
22:0
0.8
1.5
0.6
0.7
20:4ω3
0.2
0.9
0.7
0.7
20:5ω3
0.7
0.2
0.3
0.3
22:2ω6
0.0
1.2
0.0
0.0
22:3ω3
0.0
0.1
0.0
0.1
24:0
0.8
1.0
1.0
1.0
24:1
0.8
1.0
0.7
0.9
22:5ω3
2.4
2.7
3.2
3.0
22:6ω3
7.0
16.9
6.1
6.4

Example 7

Analysis of TAG from Transgenic A. thaliana Seeds Producing DHA

The positional distribution of DHA on the TAG from the transformed A. thaliana seed was determined by NMR. Total lipid was extracted from approximately 200 mg of seed by first crushing them under hexane before transferring the crushed seed to a glass tube containing 10 mL hexane. The tube was warmed at approximately 55° C. in a water bath and then vortexed and centrifuged. The hexane solution was removed and the procedure repeated with a further 4×10 mL. The extracts were combined, concentrated by rotary evaporation and the TAG in the extracted lipid purified away from polar lipids by passage through a short silica column using 20 mL of 7% diethyl ether in hexane. Acyl group positional distributions on the purified TAG were determined quantitatively as previously described (Petrie et al., 2010a and b).

The analysis showed that the majority of the DHA in the total seed oil was located at the sn-1/3 positions of TAG with little found at the sn-2 position (FIG. 9). This was in contrast to TAG from ARA producing seeds which demonstrated that 50% of the ARA (20:4Δ5,8,11,14) was located at the sn-2 position of transgenic canola oil whereas only 33% would be expected in a random distribution (Petrie et al., 2012).

Positional distribution of DHA in the TAG from the B. napus seeds transformed with pJP3416-GA7 or with the combination of pJP3115 and pJP3116 is determined by essentially the same method.

The total lipid from transgenic A. thaliana seeds was also analysed by triple quadrupole LC-MS to determine the major DHA-containing triacylglycerol (TAG) species (FIG. 10). The most abundant DHA-containing TAG species was found to be DHA-18:3-18:3 (TAG 58:12; nomenclature not descriptive of positional distribution) with the second-most abundant being DHA-18:3-18:2 (TAG 58:11). Tri-DHA TAG (TAG 66:18) was observed in total seed oil, albeit at low but detectable levels. Other major DHA-containing TAG species included DHA-34:3 (TAG 56:9), DHA-36:3 (TAG 58:9), DHA-36:4 (TAG 58:10), DHA-36:7 (TAG 58:13) and DHA-38:4 (TAG 60:10). The identities of the two major DHA-containing TAG were further confirmed by Q-TOF MS/MS.

Example 8

Predicting DHA Production in B. napus Seeds

Efficient production of DHA in Arabidopsis seeds at a 15% level using the GA7 genetic construct was demonstrated in Example 2. The same construct in Brassica napus seeds produced only about 1.5% DHA in many (but not all) of the transformants, primarily due to the poor expression of the Δ6-desaturase gene of GA7 in this species (Example 4). Based on the realisation that modifications to the GA7 construct would overcome the Δ6-desaturase gene expression problem (see Example 5, as demonstrated in Example 6), calculations were performed to determine the likely fatty acid profile of B. napus transgenic seeds expressing the genes from a variant of pJP3416-GA7, where each transgene-encoded enzyme was performing as efficiently as was observed in A. thaliana with the GA7 construct. The predicted fatty acid compositions for three calculations (#1, #2, #3) are shown in Table 20. This was based on a wild-type (non-transformed) fatty acid composition for B. napus that included 59% oleic acid, 20% LA and 8% ALA. The three predicted partial fatty acid profiles shown in the lower half of the table were based on the conversion efficiencies for each enzymatic step shown in the upper half of the table. In prediction #2, a combination of Δ12-desaturation at 75% efficiency, Δ15-desaturation at 75%, Δ6-desaturation at 35%, Δ6-elongation at 80%, Δ5-desaturation at 90%, Δ5-elongation at 90% and Δ4-desaturation at 90% would result in the production of approximately 10% DHA in a typical canola transgenic seed. These efficiencies were all lower or about equal to the individual efficiencies seen in Arabidopsis, so prediction #2 represented a conservative estimate. The conversion efficiencies listed in #3 were approximations based on the efficient conversions seen in A. thaliana transformed with pJP3416-GA7. DHA was predicted to be produced at about 15% of the total fatty acid content in seedoil produced in B. napus seed, a result that mirrored the most efficient production levels observed in A. thaliana. Insertion of multiple T-DNAs in the homozygous state is expected to raise the DHA level to 20% in B. napus.


TABLE 20
Predicted fatty acid composition for selected fatty acids as a percentage
of total fatty acid content in seed oil from Brassica napus transformed
with a DHA pathway construct, based on observed enzymatic efficiencies
in transgenic Arabidopsis. The enzymes are listed in order in the
pathway for producing DHA from oleic acid. des = desaturase, elo =
elongase. Predicted fatty acid compositions #1, #2 and
#3 are based on the efficiencies in the upper half of the Table.
Enzyme
#1
#2
#3
d12-des
70%
75%
80%
d15-des
70%
75%
80%
d6-des (ω3)
30%
35%
40%
d6-elo
80%
80%
90%
d5-des
80%
90%
90%
d5-elo
80%
90%
90%
d4-des
80%
90%
90%
Fatty acid
WT
#1
#2
#3
18:1d9
59%
26%
22%
18%
18:2ω6
20%
19%
17%
14%
18:3ω6
 1%
 2%
 3%
18:3ω3
 8%
30%
32%
34%
18:4ω3
 3%
 3%
 2%
20:4ω3
 2%
 1%
 2%
20:5ω3
 2%
 1%
 2%
22:5ω3
 1%
 1%
 2%
22:6ω3
 5%
10%
15%

Example 9

Stable Expression of a Transgenic EPA Pathway in Plant Leaf

Binary Vector Construction

A binary vector, pORE04+11ABGBEC_Cowpea_EPA_insert (SEQ ID NO:8), was designed for introduction of a T-DNA into plants for the synthesis of EPA in leaf tissues. It contained chimeric genes encoding the enzymes: M. pusilla Δ6-desaturase (SEQ ID NO:16), P. cordata Δ6-elongase (SEQ ID NO:25) and P. salina Δ5-desaturase (SEQ ID NO:30), each under the control of the CaMV 35S and A. thaliana rubisco small subunit (SSU) promoters (FIG. 9). The binary vector was constructed by synthesising the region 199-10878 of SEQ ID 2 and cloning this into the recipient binary vector pORE04 (Coutu et al., 1997) at the BsiWI and KasI sites. The three fatty acid biosynthesis genes coded for the enzymes required to convert ALA, 18:3Δ9,12,15 to EPA, 20:5Δ5,8,11,14,17.

Transient Expression of EPA Construct in N. benthamiana Leaf Cells

To test that the construct was correct and would express the genes efficiently in leaf tissues, the chimeric vector pORE04+11ABGBEC_Cowpea_EPA_insert was introduced into A. tumefaciens strain AGL1. The chimeric vector 35S:p19 was also introduced into A. tumefaciens strain AGL1 as described in Example 1. Cells from cultures of these infiltrated into leaf tissue of Nicotiana benthamiana plants in a 24° C. growth room. Several direct comparisons were infiltrated with the samples being compared located on either side of the same leaf. Experiments were performed in triplicate. Following infiltration, the plants were grown for a further five days before leaf discs were taken for fatty acid profile analysis by GC as described in Example 1. GC analysis revealed that the EPA vector was functioning to produce EPA in Nicotiana benthamiana leaf (Table 21) with the highest level of EPA found to be 10.7% of total leaf lipids.

Nicotiana tabacum Stable Transformation

The chimeric vector pORE04+11ABGBEC_Cowpea_EPA_insert was used to stably transform Nicotiana tabacum. The vector was introduced into A. tumefaciens strain AGL1 via standard electroporation procedure. The transformed cells were grown on solid LB media supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L) and incubated at 28° C. for two days. A single colony was used to initiate fresh culture. Following 48 h vigorous culture, the cells were collected by centrifugation at 2,000×g and the supernatant was removed. The cells were resuspended in fresh solution containing 50% LB and 50% MS medium at the density of OD600=0.5.


TABLE 21
Fatty acid composition of total leaf lipid from transgenic
Nicotiana benthamiana (transient) and Nicotiana tabacum
(stable primary transformant) events with the highest
EPA levels from each experiment.
14:0
0.1
0.1
16:0
18.5
17.8
16:1w13t
2.2
3.8
16:1d9
0.1
0
16:3
6.2
5.7
18:0
3.4
3.2
18:1d11
0.3
0.3
20:0
0.5
0.5
22:0
0.2
0.3
24:0
0.1
0.4
18:1
2.9
1.6
18:2ω6
12.6
14.5
Omega-6
18:3ω6
2.3
2.9
20:2ω6
0.0
0.0
20:3ω6
0.1
0.0
20:4ω6
0.3
0.7
Omega-3
18:3ω3
37.1
32.4
18:4ω3
1.6
1.9
20:3ω3
0.1
0.3
20:4ω3
0.3
1.1
20:5ω3
10.7
12.1
22:5ω3
0.3
0.4

Leaf samples of N. tabacum cultivar W38 grown in vitro were excised and cut into square sections around 0.5-1 cm2 in size with a sharp scalpel while immersed in the A. tumefaciens solution. The wounded N. tabacum leaf pieces submerged in A. tumefaciens were allowed to stand at room temperature for 10 minutes prior to being blotted dry on a sterile filter paper and transferred onto MS plates without supplement. Following a co-cultivation period of two days at 24° C., the explants were washed three times with sterile, liquid MS medium, then blotted dry with sterile filter paper and placed on the selective MS agar supplemented with 1.0 mg/L benzylaminopurine (BAP), 0.25 mg/L indoleacetic acid (IAA), 50 mg/L kanamycin and 250 mg/L cefotaxime. The plates were incubated at 24° C. for two weeks to allow for shoot development from the transformed N. tabacum leaf pieces.

To establish rooted transgenic plants in vitro, healthy green shoots were cut off and transferred into 200 mL tissue culture pots containing MS agar medium supplemented with 25 μg/L IAA, 50 mg/L kanamycin and 250 mg/L cefotaxime. Transgenic shoots were transferred to soil after rooting and grown to maturity in the glasshouse. Sufficiently large leaf discs were taken from 21 mature transgenic plants from and analysed for fatty acid profile as described in Example 1. All transgenic samples were found to contain EPA (Table 21) with the highest level of EPA in a hemizygous primary transformant found to be 12.1% of total leaf lipids. The leaf samples also contained a small amount (<0.5%) of DPA in their lipid, which resulted from elongation of the EPA by a low level of Δ5-elongation activity of the Δ6-elongase. The total ω3 FA (including ALA) to ω6 FA (including LA) ratio was found to be 2.7. Overall conversion efficiencies were calculated to be: OA to EPA=18.4%, LA to EPA=18.9%, ALA to EPA=25.9%. The production of 12.1% EPA is notable especially since the events were hemizygous primary transformants. The ALA to EPA efficiency in particular is close to that observed in stable seed transformants. It is worth noting that the construct did not contain a Δ12 or Δ15-desaturase to increase the conversion of OA and LA to ALA. Increased efficiencies would be expected with addition of these activities.

Seed from hemizygous transformants is being harvested and sown out to generate homozygous plants.

Seed set in the top EPA lines appeared normal and seed from lines #10 and #17 germinated well to establish the T2 generation. The ratio of EPA to null (no EPA) lines indicated that event #28 was single-locus and the T3 generation of this line was therefore also established. Fatty acid profile analysis of the T3 population indicated that the transgenes were homozygous with no null events found and a stable amount of EPA. The average amount of EPA in the total leaf lipids in the entire T3 population was found to be 9.4%±0.3 (Table 22).


TABLE 22
Representative fatty acid profiles of total leaf lipids from wildtype (WT) and
independent transgenic or transiently-transformed lines (EPA). Species are Nicotiana
benthamiana (transient transformation), N. tabacum (a stably transformed T3
population), Vigna unguiculata (stably transformed T1 event). The errors denote
standard deviation of multiple samples. Apparent conversion efficiencies shown at the
bottom describe the ω3 pathway and are calculated as the sum of product FAs/sum of
substrate + product FAs.
WT
EPA
WT
EPA
WT
EPA
16:0
 17.7 ± 0.1
18.7 ± 0.2 
15.0 ± 0.6 
16.5 ± 0.5 
18.0
18.2 ± 0.2 
16:1ω13t
  3.2 ± 0.1
2.2 ± 0  
3.5 ± 0.1
3.0 ± 0.3
3.8
2.0 ± 0.9
16:3
  6.8 ± 0.1
6.2 ± 0.1
5.2 ± 0.5
5.4 ± 0.3
18:0
3.1 ± 0
3.5 ± 0.3
2.2 ± 0.2
2.6 ± 0.1
1.8
4.5 ± 0.4
Minor
1.4 ± 0
1.4 ± 0.1
3.1 ± 0.4
2.5 ± 0.3
2.3
2.5 ± 0.4
OA
  1.7 ± 0.1
2.7 ± 0.2
1.6 ± 0.3
2.1 ± 0.3
2.0
4.3 ± 1.3
LA
 12.5 ± 0.4
12.7 ± 0.2 
17.0 ± 1.1 
18.0 ± 0.9 
13.4
18.2 ± 3.0 
ALA
 53.3 ± 0.2
37.2 ± 0.2 
52.2 ± 1.9 
34.0 ± 0.6 
58.6
38.2 ± 0  
Omega-6
GLA
2.3 ± 0.1
2.3 ± 0.3
0.6 ± 0.2
20:2ω6
0.1 ± 0
0.1 ± 0  
0.1 ± 0  
0.1 ± 0  
DGLA
0.1 ± 0
0.1 ± 0  
ARA
0.3 ± 0  
0.7 ± 0.1
0.2 ± 0  
Omega-3
SDA
1.5 ± 0.1
1.6 ± 0.1
1.5 ± 0  
20:3ω3
0.1 ± 0
0.1 ± 0  
0.1 ± 0  
0.3 ± 0  
0.1 ± 0
1.5 ± 0.1
ETA
0.4 ± 0  
1.1 ± 0.1
0.3 ± 0.2
EPA
10.2 ± 0.5 
9.4 ± 0.3
7.1 ± 0.2
DPA
0.3 ± 0.1
0.4 ± 0  
0.8 ± 0.1
Omega-3
Δ6-des
25%
27%
20%
conversion
Δ6-elo
88%
87%
85%
Δ5-des
97%
90%
96%
Δ5-elo
3%
4%
10%

Leaf samples of homozygous T3 N. tabacum plants were subjected to further biochemical analysis. Total lipids were extracted from freeze-dried leaf material and fractionated by thin-layer chromatography (TLC). EPA was found to be present in N. tabacum TAG at up to 30.1% as well as in the polar lipids at 6.3% (Table 23). It was interesting to note that the EPA produced by the transgenic pathway was present in all of the lipid fractions assessed including TAG, MGDG, DGDG, SQDG, PG, PC, PE, PI and PS. All lipid pools contained low levels of novel intermediate or ω6 LC-PUFA fatty acids with the TAG ratio of novel ω3 to ω6 fatty acids being 10:1.

Stable Transformation of Cowpea

The chimeric vector pORE04+11ABGBEC-Cowpea-EPA-insert was transformed into cowpea (Vigna unguiculata) as follows. Mature dry seeds are the preferred starting material although seeds harvested from immature pods at maximum fresh weight of seeds can also be used. Dry seeds are threshed by hand to avoid cracking of seed coats and thus reduce contamination with microorganisms.

Dry seeds or immature pods are submerged in 70% ethanol for 2 min and then treated for 30 min in 20% commercial bleach (8.4 g/L sodium hypochlorite final concentration). The seeds are then washed several times with sterile water. Immature seeds are removed aseptically from pods while mature seeds are imbibed overnight. Two different explants can be used for multiple shoot production, ie the embryonic axis and the cotyledon itself, preferably the cotyledon with the bisected embryonic axis attached. The shoot and root tips are removed from the axis before wounding at the cotyledonary node, i.e. the point of attachment of the axis to the cotyledon. From an initial comparison of 19 cultivars and lines, it is now clear that most lines of cowpea can be transformed, the only caveat being that different tissue culture conditions need to be optimised for each line.


TABLE 23
Analysis of young and mature (young|mature) leaf lipid fractions triacylglycerol (TAG), total polar lipid (PL),
monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG),
phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) from transgenic Nicotiana tabacum
leaf samples. The errors denote standard deviation of multiple samples. Up to 30% EPA was observed in leaf TAG with EPA also distributed
throughout the polar lipids. Differences between yound and mature leaf profiles were also observed for several fatty acids.
Chloroplastidic
Extra-chloroplastidic
TAG
PL
MGDG
DGDG
SQDG
PG
PC
PE
PI
PS
16:0
 9.8|18.3
17.8|23.8
3.1|3.2
18.0|16.8
48.3|50.0
21.0|26.4
22.9|30.0
24.0|30.5
38.7|43.3
31.9|36.2
16:1ω13t
0|0
3.4|3.1
0|0
0|0
0|0
34.0|32.0
0|0
0|0
0|0
1.0|1.4
16:3
0.2|0.9
5.6|6.4
14.8|19.4
1.2|1.8
0.4|1.2
0|0
0|0
0|0
0|0
0|0
18:0
7.3|3.7
2.9|3.9
1.1|1.2
3.5|3.5
5.4|7.1
4.7|6.9
6.6|9.1
11.0|11.4
9.4|9.3
20.2|19.4
Minor
2.5|2.9
1.4|2.4
1.0|0.4
0.8|1.0
1.9|2.1
1.0|1.5
1.4|1.6
4.9|4.1
6.5|7.7
2.5|3.7
OA
5.5|0.8
2.8|1.1
0.8|0.3
1.8|1.0
2.7|1.3
5.3|4.9
8.1|2.9
2.5|1.1
2.5|0.8
4.9|2.3
LA
27.7|13.7
17.3|12.3
8.0|6.8
 9.2|10.5
11.7|8.9 
17.1|13.2
39.2|25.2
37.9|28.5
22.0|13.4
24.4|17.1
ALA
 9.6|17.2
39.0|34.4
60.3|51.9
61.2|58.6
23.7|21.5
15.7|14.1
 7.3|18.2
 5.5|10.5
 7.6|10.0
 4.8|10.5
Omega-6
GLA
2.5|3.0
1.5|2.1
2.1|3.0
1.1|1.8
1.4|1.9
0.2|0  
1.8|2.5
1.7|2.7
0.8|0.9
1.1|1.3
20:2ω6
0|0
0.1|1.1
0|0
0|0
0|0
0|0
0|0
0.5|0  
0|0
0|0
DGLA
0|0
0|0
0|0
0|0
0|0
0|0
0|0
0|0
0|0
0|0
ARA
0.6|0.9
0.1|0.2
0.2|0.4
0|0
0|0
0|0
0.3|0.3
0.4|0.4
0.4|0.6
  0|0.2
Omega-3
SDA
4.0|7.6
1.6|2.0
1.7|2.0
0.6|0.7
1.2|1.2
0|0
2.1|3.6
1.3|2.0
0.8|0.8
0.9|1.6
20:3ω3
0.2|0.3
0.1|0.2
0|0
0.2|0.3
0|0
  0|0.1
0.2|0  
0.3|0.4
0|0
0|0
ETA
0.9|0.2
0.2|0.3
  0|0.2
  0|0.3
0|0
0|0
0.2|0  
0.4|0.2
0.1|0.2
0|0
EPA
28.8|30.1
6.1|6.3
 6.9|11.2
2.3|3.6
3.4|4.6
1.0|0.8
9.7|6.4
9.1|7.8
11.2|12.8
8.4|6.2
DPA
0.4|0.5
0|0
  0|0.1
0|0
0|0
0|0
0.3|0.4
0.5|0.4
  0|0.2
  0|0.1

The selectable marker genes, bar or NptII can be used for transformation. The Agrobacterium tumefaciens strain AGL1 is the preferred strain for cowpea transformation. Agrobacterium containing the pORE04+11ABGBEC-Cowpea-EPA-insert vector is cultured overnight at 28° C. on a shaker at 180 rpm and the suspension is centrifuged at 8000 g for 10 min and re-suspended in Medium 1 (MS-basic medium diluted one in ten and containing 30 g/l sucrose, 20 mM 2-MES, adjusted to pH 5.6 prior to autoclaving, supplemented with filter sterilized MS-vitamins, 100 mg/l myo-inositol, 1.7 mg/l BAP, 0.25 mg/l GA3, 0.2 mM acetosyringone, 250 mg/l Na-thiosulphate, 150 mg/l dithiothreitol and 0.4 g/l L-cysteine). The explants are submerged without shaking in the bacterial suspension for one hour following wounding in the meristematic regions with a scalpel. The treated explants are then blotted on sterile filter paper and transferred to solidified Medium 2 (Medium 1 containing 0.8% agar) overlayed with filter paper. After four days of co-cultivation, explants are transferred to Medium 3 (full strength MS medium, supplemented with 100 mg/l myo-inositol, 150 mg/l timentin, 30 g/L sucrose, 3 mM MES, 1.7 mg/L BAP, 5 mg/L PPT or 25-50 mg/L geneticin or 150 mg/L kanamycin, 0.8 g/L agar and adjusted to pH 5.6) for shoot initiation and selection of transformed shoots. After two weeks the first shoots are visible. The cotyledons are removed from the cotyledonary node region and cultures are transferred to fresh Medium 3. Cultures are transferred to fresh Medium 3 every two weeks following removal of dead and dying tissue. The first four subcultures are on kanamycin selection followed by alternating with geneticin and kanamycin. After six sub-cultures, the surviving green shoots are transferred to Medium 4 (Medium 3 without BAP but supplemented with 0.5 mg/l GA3, 50 mg/l asparagine, 0.1 mg/l 3-indoleacetic acid (IAA), 150 mg/l timentin, and either PPT (10 mg/l), geneticin (50 mg/L) or kanamycin (150 mg/L), for shoot elongation. The shoots are sub-cultured every two weeks until single shoots are more than 1 cm long. These larger shoots are transferred from petri dishes to culture jars (80 mm height) for further growth under selection.

The majority of the regenerated shoots can be rooted in vitro, and the rooted plants are transferred to soil and allowed to establish in a high humidity chamber for 14-21 days before transfer to ambient greenhouse conditions.

To enhance gene transfer to cowpea, co-culture media is supplemented with thiol compounds. The addition of L-cysteine, dithiothreitol, and sodium thiosulfate reduces browning of wounded tissue.

Large numbers of cowpea explants can be processed in a simplified protocol. In brief, the protocol consists of the following steps: imbibition of sterilized mature seeds overnight in water, explants are derived by longitudinally bisecting the seed as a result of which, the split embryonic axis (with shoot and root apices removed) is still attached to the cotyledon, infection with Agrobacterium strain AGL1 aided by local wounding in the meristematic regions, co-culture on medium containing thiol compounds over 4 days at 25° C. in light, shoot initiation and elongation on medium containing selective agents, shoots are rooted in vitro and transferred to greenhouse conditions for flowering and seed setting, PCR or enzyme analysis of putative transgenic plants, and screening of next generation progeny by PCR or enzyme activity.

The progeny of transgenic T0 plants are normal in phenotype. The transgenes are transmitted to the progeny and homozygous T2 plants are identified by screening their T3 progeny for enzyme activity or by PCR.

Using this transformation system about 10 transgenic plants are produced per 1000 explants, which is similar to the transformation frequency for other legumes. Depending on the cultivar or line to be transformed, this protocol requires 5-8 months from explant preparation to harvested T1 seeds.

The transformation system is used to introduce the pORE04+11ABGBEC-Cowpea-EPA-insert binary vector into regenerated, transformed cowpea plants.

Modifications to the pORE04+11ABGBEC-Cowpea-EPA-insert binary vector are made in which genes encoding a Δ5-elongase and Δ4-desaturase are added, to provide a genetic construct which confers the ability to further convert the produced EPA to DHA. The construct is transformed into plants for production of DHA in vegetative tissues.

EPA was found to be present in the small number of events surviving chemical selection. The highest line contained 7.1%±0.2 EPA in the total leaf lipids. The rate of transformation was lower than usually experienced for cowpea with only six lines confirmed transgenic. It is, as yet, unknown what caused this effect although it is interesting to note that a larger than usual proportion of transgenic events contained incomplete T-DNA regions. It is possible that the large construct size contributed to the reduced efficiency. The apparent conversion efficiencies of each of the three transgenic enzymes were also calculated (Table 22). Results were broadly similar in all three species with good conversion to EPA after initial Δ6-desaturation of the native ALA. Some Δ5-elongation of EPA to DPA was noted despite the absence of a specific Δ5-elongase. The P. cordata Δ6-elongase has previously been shown to have a low level of Δ9-elongase activity (i.e. 18:3Δ9,12,15 to 20:3Δ11,14,17 conversion) although no Δ5-elongase activity was detected in a yeast assay.

Example 10

Testing Variations of Δ12-Desaturase Genes

Binary Vector Construction

In an attempt to test and compare a series of chimeric Δ12-desaturase genes, several binary vectors were made which were used to transform A. thaliana and B. napus. The binary vectors pJP3365, pJP3366, pJP3367, pJP3368 and pJP3369 each contained genes that encoded the P. pastoris ω3-desaturase (SEQ ID NO:12) and M. pusilla Δ6-desaturase (SEQ ID NO:16) enzymes, and one of a series of Δ12-desaturases. The Δ12-desaturases were from Cryptococcus neoformans (Accession No. XP_570226 in pJP3365), a version of the Cryptococcus neoformans Δ12-desaturase which contained a L151M mutation in an attempt to increase gene activity (in pJP3366), Lachancea kluyveri (SEQ ID NO:10 in pJP3367), Synechocystis PCC6803 (Accession No. BAA18169 in pJP3368) and Crepis palaestina (Accession No. CAA76157, Lee et al., 1998, in pJP3369). The Crepis desaturase was the only plant desaturase in the series; the others were fungal enzymes. The vectors were made by inserting a plant codon-optimised protein coding region, except for the Crepis palestina Δ12-desaturase which was wildtype, for each Δ12-desaturase into the NotI site of the vector pJP3364 (see FIG. 12), in the orientation operably linked to the FP1 promoter to provide for seed-specific expression of each desaturase. The vector pJP3364 already contained the chimeric genes encoding the P. pastoris ω3-desaturase and M. pusilla Δ6-desaturase, each under the control of seed-specific promoters (FIG. 12). The combination of the three fatty acid biosynthesis enzymes, namely Δ12-desaturase, ω3-desaturase and Δ6-desaturase, was designed to assemble a pathway to convert oleic acid (18:1Δ9) to SDA (18:4Δ6,9,12,15). Assays were therefore carried out to measure the level of SDA production in transformed seeds.

A. thaliana and B. napus Transformation and Analysis

The chimeric binary vectors were introduced into A. tumefaciens strain AGL1 and cells from cultures of the transformed Agrobacterium used to transform fad2 mutant A. thaliana plants using the floral dip method for transformation (Clough and Bent, 1998). After maturation, the T1 seeds from the treated plants were harvested and plated on MS plates containing kanamycin for selection of plantlets having the NptII selectable marker gene present on the T-DNA of each chimeric vector. Surviving T1 seedlings were transferred to soil. After allowing the plants to self-fertilise and growing them to maturity, the T2 seeds from these plants were harvested and the fatty acid composition of seed lipids analysed by GC.

The chimeric vector pJP3367 was also used to transform B. napus by the method described in Example 4 to generate 12 transgenic events. SDA was found to range from 0.6% to 2.2% in pooled seed of the plants, and nine individual seeds from the transgenic plant with the highest SDA transgenic plant were analysed for fatty acid composition. Fatty acid composition data from such analysis is shown in Table 24.

The data showed that the Δ12-desaturase activity expressed from each of the T-DNAs in both A. thaliana and B. napus were unexpectedly low, providing an enzyme conversion efficiency of about 20% rather than the 70-80% seen with the same expression cassette in the GA7 construct (Examples 2 and 3). The reason for this relatively poor expression of the Δ12-desaturase genes from these vectors is unclear but could be related to the position of the genes in the construct as a whole.

In contrast, RT-PCR expression analysis demonstrated that the P. pastoris ω3-desaturase and M. pusilla Δ6-desaturase genes on the T-DNAs were relatively well expressed in the transformed seed. Table 24 includes the Δ6-desaturase conversion efficiencies in the transformed seeds, which ranged from about 11% to about 25% in the one B. napus transformed line. This was considerably higher than the Δ6-desaturase conversion efficiency of about 7% seen in the B. napus seeds transformed with the GA7 construct (Example 4).


TABLE 24
Fatty acid composition as a percentage of total fatty acids in seed oil from
single seeds from a T1 Brassica napus plant transformed with the T-DNA from
pJP3367. SDA (18:4ω3) is shown in bold.
CT110-
CT110-
Sample
3#1
3#2
CT110-3#3
CT110-3#4
CT110-3#5
CT110-3#6
CT110-3#7
CT110-3#8
CT110-3#9
C14:0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
C16:0
4.3
4.2
4.1
4.5
3.8
4.3
4.0
5.0
4.7
16:1d7
0.1
0.1
0.1
0.1
0.0
0.1
0.1
0.1
0.1
C16:1d9
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.3
16:3
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
C18:0
1.9
1.9
1.3
1.8
2.1
1.8
2.4
3.1
2.2
C18:1
58.1
59.4
55.5
59.1
62.1
56.0
57.2
52.0
53.2
C18:1d11
3.5
3.6
3.0
3.2
2.9
3.6
3.2
4.4
3.5
C18:2
18.4
17.1
19.2
17.3
17.4
18.7
19.0
20.3
20.2
C18:3ω6
0.3
0.2
0.3
0.2
0.2
0.2
0.2
0.2
0.3
C18:3ω3
8.2
9.0
11.1
8.6
7.5
10.2
9.8
9.3
9.8
C20:0
0.5
0.5
0.4
0.5
0.6
0.5
0.6
0.7
0.6
C20:1d11
1.1
1.1
1.2
1.2
1.2
1.1
1.2
1.1
1.1
20:1iso
0.03
0.03
0.03
0.03
0.01
0.03
0.02
0.03
0.02
C20:2ω6
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
C22:0
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.2
C24:0
0.1
0.1
0.1
0.2
0.1
0.1
0.2
0.3
0.2
C24:1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Δ6-des %
22.9
17.9
20.3
22.8
15.8
20.2
11.7
20.9
24.9

Therefore, to take advantage of the higher Δ6-desaturase conversion efficiencies conferred by the T-DNA from pJP3367, B. napus plants transformed with this T-DNA were crossed to plants transformed with the T-DNA from pJP3416-GA7 (Example 4) to produce progeny plants and seeds carrying both T-DNAs. The fatty acid composition of oil extracted from F1 seeds is analysed by GC for DHA content and other fatty acid contents. Increased DHA levels are observed as a consequence of increased expression of the Δ6-desaturase. Plants which are homozygous for both T-DNAs are produced and should produce higher levels of DHA.

Example 11

Increasing Accumulation of Fatty Acids by Using Silencing Suppressor Proteins

Binary Vector Construction

WO 2010/057246 describes the use of silencing suppressor proteins (SSP) to increase transgene expression in the seeds of plants. To demonstrate that the use of such proteins could enhance and stabilise the production of LC-PUFA in oilseeds over several generations, several SSP were selected for testing, namely V2 (Accession No. GU178820.1), p19 (Accession No. AJ288943.1), p38 (Accession No. DQ286869.1) and P0PE (Accession No. L04573.1). p19 is a suppressor protein from Tomato Bushy Stunt Virus (TBSV) which binds to 21 nucleotide long siRNAs before they guide Argonaute-guided cleavage of homologous RNA (Voinnet et al., 2003). V2, a suppressor protein from Tomato Yellow Leaf Curl Virus (TYLCV), binds to the plant protein SGS3 (Glick et al., 2008), a protein thought to be required for the production of double stranded RNA intermediates from ssRNA substrates (Beclin et al., 2002), or binds to dsRNA structures that have a 5′ overhangs (Fukunaga et al., 2009). p38 is a suppressor protein from Turnip Crinkle Virus (TCV) which interferes with plant silencing mechanisms by binding to Dicer and Argonaute proteins (Azevedo et al., 2010). P0 proteins such as P0PE and RPV-P0, from poleroviruses, target Argonaut proteins for enhanced degradation (Baumberger et al., 2007; Bortolamiol et al., 2007, Fusaro et al., 2012). Genetic constructs were therefore prepared for expression of these SSP in plant seed in combination with a set of fatty acid biosynthesis genes for production of ARA (20:4Δ5,8,11,14) from LA (18:1Δ9,12), as follows.

The fatty acid biosynthesis genes encoding the Isochrysis galbana Δ9-elongase and the Pavlova salina 48- and Δ5-desaturases and the bacterial selection marker were obtained on a single DNA fragment from pJP3010 by digestion with PmeI and AvrII giving rise to a 9560 bp fragment. The Δ9-elongase coding region on this fragment was joined to an A. thaliana FAE1 promoter (pAtFAE1) and a conlinin transcription termination/polyadenylation region (LuCnl2-3′). The desaturase coding regions were each joined to a truncated napin FP1 promoter (pBnFP1) and a nos3′ transcription termination/polyadenylation region. The three fatty acid biosynthesis genes on this fragment were oriented and spaced in the same manner as in pJP107 (Petrie et al., 2012) and encoded the same proteins as pJP107. The DNA fragment also comprised a pFP1:GFiP:nos3′ gene from pCW141 (see WO2010/057246) which encoded a green fluorescent protein (GFP). This screenable marker gene was used as a visual seed-specific marker, allowing simple and non-destructive identification and thereby selection of transgenic seed comprising and expressing the gene.

The PmeI-AvrII fragment was inserted into the PmeI-AvrII site of each of a series of five vectors, each containing a different SSP gene (WO2010/057246), resulting in the genetic constructs designated pFN045, pFN046, pFN047, pFN048 and pFN049. These comprise the genes encoding the SSPs P0PE, p38, p19, 35S:V2 and V2, respectively. Each of the SSP genes was under the control of the FP1 promoter and ocs3′ transcription termination/polyadenylation region except in the construct pFN048 where the V2 coding region was under the control of the constitutive CaMV 35S promoter. The SSP gene in each case was within the T-DNA region of the constructs, adjacent to the right border (RB) of the T-DNA. A sixth construct, pFN050 which lacked any SSP coding sequence, was made by digesting pFN045 with AhdI and NheI followed by recircularisation with DNA ligase to delete the FP1:P0PE gene. Each of the six constructs comprised an NptII selectable marker gene within the T-DNA and adjacent to the left border of the T-DNA. All of the constructs had an RK2 origin of replication for maintenance of the plasmids in Agrobacterium.

Transformation of A. thaliana with ARA Expression Vectors in Combination with SSPs

To transform the genotype MC49 of Arabidopsis, which is a fad2/fae1 double mutant with high linoleic acid levels in its seed lipid, plants were treated by the floral dip method (Clough and Bent, 1998) with A. tumefaciens strain GV3101 separately transformed with each of the six constructs pFN045-pFN050. The treated plants were grown to maturity and T1 seeds harvested from them were plated on MS media containing kanamycin to select for transformed T1 plants. Screening for GFP expression in the seed was also used as a visual marker for transformed T1 seeds. The seedlings which survived on MS/Kan plates or which were obtained from GFP-positive seeds were transferred to soil and grown to maturity for T2 seeds. The numbers of transformed plants obtained were 5, 14, 32, 8, 23 and 24 for the transformations with pFN045, pFN046, pFN047, pFN048, pFN049 and pFN050, respectively. It was discovered at this stage that the gene encoding p38 in pFN046 was not functional and therefore plants transformed with the vector pFN046 were considered as additional controls i.e. essentially the same as for pFN050.

About 100 pooled T2 seeds were taken from each transformed plant for fatty acid composition determination of seed lipid by FAME preparation and GC analysis. Six T2 seedlings from each transgenic line were also grown to produce T3 seeds.

The fatty acid composition in total lipid extracted from the T2 seeds was determined using GC. The analysis showed a range of levels of ARA and the intermediates EDA (20:2ω6) and DGLA (20:3ω6) in the T2 populations. The data for ARA is shown in FIGS. 13 and 14.

FIG. 13 shows a box-plot analysis of the ARA level in the lipid of the populations of the T2 seeds. It was evident that the median (50th percentile) level of ARA in the populations of seeds which contained the FP1:p19 and 35S:V2 genes in addition to the ARA biosynthetic genes was significantly higher than in seeds containing the defective FP1:p38 gene or the control T-DNA from pFP050 which did not contain an SSP gene. The average ARA levels for seeds transformed with genes encoding p19 and V2 were greater than for seeds transformed with the p38 gene or those without an SSP (FIG. 14). One FP1:p19 and two FP1:V2 lines exhibited about 19%, 20% and 23% ARA, respectively. These were outliers and therefore not included in the calculations for the box-plot analysis. Fewer plants transformed with the T-DNAs comprising the genes FP1:P0PE and 35S:V2 survived compared to the other constructs; it is thought that these genes could be detrimental to plant health in the MC49 background.

Not only were the ARA levels significantly different among the constructs, the levels in seed lipid of the first intermediate of the pathway from LA to ARA, namely EDA (20:2ω6), was observed to be lower in lines expressing either V2 or p19 than in seeds lacking an SSP or containing the p38 construct (FIG. 15). In T3 seeds, one population containing the construct expressing p19 exhibited 38% ARA as a percentage of total fatty acids in the seed lipid.

A range of transgenic T3 lines were progressed to the T4 generation. The levels of ARA in the T4 seeds expressing V2 were either the same as compared to the previous generation, or indeed exhibited increased levels compared to their T3 parents (FIG. 16). The lines expressing p19 showed more varied ARA levels. The ARA level was decreased in some lines while in others it was the same or increased compared to the T3 parents. In contrast, the lines containing the defective p38 gene or lacking an SSP generally showed a decline in the level of ARA and an increase in the levels of intermediates (FIG. 18). In some of these lines, ARA was reduced to about 1% and levels of EDA had increased to about 20%. The mean levels of ARA in T4 seeds were higher for lines expressing p19 and V2 compared to lines expressing p38 or lacking an SSP (FIG. 17).

This experiment showed that the expression of an SSP in seeds of a transgenic plant along with additional genes for a LC-PUFA biosynthetic pathway not only increased the level of production of the desired fatty acid in the first generation of progeny, but also stabilised the level of the fatty acid production in later generations such as the third or fourth generation of progeny. The increased fatty acid production was accompanied by decreased levels of intermediate fatty acids in the biosynthetic pathway. The SSP's p19 and V2 expressed from seed-specific promoters were preferred. The construct designed to express the p38 SSP was defective and no useful data were obtained with this construct. The V2 SSP and its homologs from other viruses are thought to be particularly preferred because they allow maximal expression of the biosynthetic pathway genes and the simultaneous silencing of other genes in the same cells in the developing seed.

Example 12

Assaying Sterol Content and Composition in Oils

The phytosterols from 12 vegetable oil samples purchased from commercial sources in Australia were characterised by GC and GC-MS analysis as O-trimethylsilyl ether (OTMSi-ether) derivatives as described in Example 1. Sterols were identified by retention data, interpretation of mass spectra and comparison with literature and laboratory standard mass spectral data. The sterols were quantified by use of a 5β(H)-Cholan-24-ol internal standard. The basic phytosterol structure and the chemical structures of some of the identified sterols are shown in FIG. 19 and Table 25.

The vegetable oils analysed were from: sesame (Sesamum indicum), olive (Olea europaea), sunflower (Helianthus annus), castor (Ricinus communis), canola (Brassica napus), safflower (Carthamus tinctorius), peanut (Arachis hypogaea), flax (Linum usitatissimum) and soybean (Glycine max). In decreasing relative abundance, across all of the oil samples, the major phytosterols were: β-sitosterol (range 28-55% of total sterol content), Δ5-avenasterol (isofucosterol) (3-24%), campesterol (2-33%), Δ5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%) and Δ7-avenasterol (0.1-5%). Several other minor sterols were identified, these were: cholesterol, brassicasterol, chalinasterol, campestanol and eburicol. Four C29:2 and two C30:2 sterols were also detected, but further research is required to complete identification of these minor components. In addition, several other unidentified sterols were present in some of the oils but due to their very low abundance, the mass spectra were not intense enough to enable identification of their structures.

The sterol contents expressed as mg/g of oil in decreasing amount were: canola oil (6.8 mg/g), sesame oil (5.8 mg/g), flax oil (4.8-5.2 mg/g), sunflower oil (3.7-4.1 mg/g), peanut oil (3.2 mg/g), safflower oil (3.0 mg/g), soybean oil (3.0 mg/g), olive oil (2.4 mg/g), castor oil (1.9 mg/g). The % sterol compositions and total sterol content are presented in Table 26.


TABLE 25
IUPAC/systematic names of identified sterols.
Sterol
No.
Common name(s)
IUPAC/Systematic name
1
cholesterol
cholest-5-en-3β-ol
2
brassicasterol
24-methylcholesta-5,22E-
dien-3β-ol
3
chalinasterol/24-
24-methylcholesta-5,24(28)E-
methylene cholesterol
dien-3β-ol
4
campesterol/24-
24-methylcholest-5-en-3β-ol
methylcholesterol
5
campestanol/24-
24-methylcholestan-3β-ol
methylcholestanol
7
Δ5-stigmasterol
24-ethylcholesta-5,22E-
dien-3β-o l
9
ergost-7-en-3β-ol
24-methylcholest-7-en-3β-ol
11
eburicol
4,4,14-trimthylergosta-8,24(28)-
dien-3β-ol
12
β-sitosterol/24-
24-ethylcholest-5-en-3β-ol
ethylcholesterol
13
D5-avenasterol/
24-ethylcholesta-5,24(28)Z-
isofucosterol
dien-3β-ol
19
D7-stigmasterol/
24-ethylcholest-7-en-3β-ol
stigmast-7-en-3b-ol
20
D7-avenasterol
24-ethylcholesta 7,24(28)-
dien-3β-ol

Among all the seed oil samples, the major phytosterol was generally β-sitosterol (range 30-57% of total sterol content). There was a wide range amongst the oils in the proportions of the other major sterols: campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%), Δ7-stigmasterol (1-18%). Oils from different species had a different sterol profile with some having quite distinctive profiles. In the case of canola oil, it had the highest proportion of campesterol (33.6%), while the other species samples generally had lower levels, e.g. up to 17% in peanut oil. Safflower oil had a relatively high proportion of Δ7-stigmasterol (18%), while this sterol was usually low in the other species oils, up to 9% in sunflower oil. Because they were distinctive for each species, sterol profiles can therefore be used to help in the identification of specific vegetable or plant oils and to check their genuineness or adulteration with other oils.


TABLE 26
Sterol content and composition of assayed plant oils.
Sun-
Saf-
flower
flower
Sterol
Sterol common
Sun-
cold-
Saf-
cold-
Flax
Flax
number*
name
Sesame
Olive
flower
pressed
Castor
Canola
flower
pressed
Peanut
(linseed)
(linseed)
Soybean
1
cholesterol
0.2
0.8
0.2
0.0
0.1
0.3
0.2
0.1
0.2
0.4
0.4
0.2
2
brassicasterol
0.1
0.0
0.0
0.0
0.3
0.1
0.0
0.0
0.0
0.2
0.2
0.0
3
chalinasterol/24-
1.5
0.1
0.3
0.1
1.1
2.4
0.2
0.1
0.9
1.5
1.4
0.8
methylene
cholesterol
4
campesterol/24-
16.2
2.4
7.4
7.9
8.4
33.6
12.1
8.5
17.4
15.7
14.4
16.9
methylcholesterol
5
campestanol/24-
0.7
0.3
0.3
0.1
0.9
0.2
0.8
0.8
0.3
0.2
0.2
0.7
methylcholestanol
6
C29:2*
0.0
0.0
0.1
0.2
0.0
0.1
0.5
0.5
0.0
1.2
1.3
0.1
7
Δ5-stigmasterol
6.4
1.2
7.4
7.2
18.6
0.7
7.0
4.6
6.9
5.1
5.8
17.6
8
unknown
0.5
1.3
0.7
0.6
0.8
0.7
0.7
1.3
0.4
0.7
0.6
1.3
9
ergost-7-en-3β-ol
0.1
0.1
1.9
1.8
0.2
0.4
2.7
4.0
1.4
1.4
1.4
1.0
10
unknown
0.0
1.3
0.9
0.8
1.2
0.9
1.8
0.7
1.2
0.7
0.5
0.7
11
eburicol
1.6
1.8
4.1
4.4
1.5
1.0
1.9
2.9
1.2
3.5
3.3
0.9
12
β-sitosterol/24-
55.3
45.6
43.9
43.6
37.7
50.8
40.2
35.1
57.2
29.9
28.4
40.2
ethylcholesterol
13
Δ5-avenasterol/
8.6
16.9
7.2
4.1
19.3
4.4
7.3
6.3
5.3
23.0
24.2
3.3
isofucosterol
14
triterpenoid
0.0
2.4
0.9
1.1
0.0
0.0
1.6
1.9
0.0
0.0
0.0
0.9
alcohol
15
triterpenoid
0.0
0.0
0.7
0.6
0.0
0.0
2.8
1.8
0.0
0.0
0.3
0.0
alcohol
16
C29:2*
0.0
0.5
0.7
0.7
1.5
1.2
2.8
1.9
2.0
1.0
0.7
0.5
17
C29:2*
1.0
0.9
2.3
2.4
0.6
0.4
1.3
1.9
0.9
1.0
1.0
1.0
18
C30:2*
0.0
0.0
0.0
0.0
1.9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
19
Δ7-stigmasterol/
2.2
7.1
9.3
10.9
2.3
0.9
10.5
18.3
1.1
7.9
8.7
5.6
stigmast-7-en-3β-
ol
20
Δ7-avenasterol
1.3
0.1
4.0
3.6
0.6
0.2
2.0
4.7
0.7
0.4
0.4
0.6
21
unknown
0.7
7.1
0.9
0.8
0.0
0.4
0.3
0.4
0.0
3.0
3.6
0.0
22
unknown
0.3
0.0
0.3
0.9
0.0
0.0
1.2
1.3
0.2
0.1
0.0
0.3
23
unknown
0.2
0.2
0.3
0.3
0.2
0.1
0.3
0.2
0.2
0.1
0.2
0.5
24
unknown
0.0
3.1
0.9
1.3
0.6
0.4
0.2
0.4
0.7
1.7
1.9
0.8
25
unknown
0.9
0.4
0.3
0.5
0.3
0.1
0.5
0.7
0.3
0.1
0.1
0.6
26
C30:2
2.2
6.0
4.6
5.7
1.4
0.6
1.0
1.2
1.2
1.2
1.1
5.2
27
unknown
0.0
0.4
0.4
0.3
0.3
0.2
0.1
0.2
0.3
0.1
0.0
0.3
Sum
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
Total sterol (mg/g
5.8
2.4
4.1
3.7
1.9
6.8
3.2
3.0
3.2
4.8
5.2
3.0
oil)
C29:2* and and C30:2* denotes a C29 sterol with two double bonds and a C30 sterol with two double bonds, respectively

Two samples each of sunflower and safflower were compared, in each case one was produced by cold pressing of seeds and unrefined, while the other was not cold-pressed and refined. Although some differences were observed, the two sources of oils had similar sterol compositions and total sterol contents, suggesting that processing and refining had little effect on these two parameters. The sterol content among the samples varied three-fold and ranged from 1.9 mg/g to 6.8 mg/g. Canola oil had the highest and castor oil the lowest sterol content.

Example 13

Increasing Accumulation of DHA at the Sn-2 TAG Position

The present inventors considered that DHA accumulation at the sn-2 position in TAG could be increased by co-expressing an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) together with the DHA biosynthesis pathway such as conferred by the GA7 construct or its variants. Preferred LPAATs are those which can act on polyunsaturated C22 fatty acyl-CoA as substrate, resulting in increased insertion of the polyunsaturated C22 chain at the sn-2 position of LPA to form PA, relative to the endogenous LPAAT. Cytoplasmic LPAAT enzymes often display varied substrate preferences, particularly where the species synthesises and accumulates unusual fatty acids in TAG. A LPAAT2 from Limnanthes douglasii was shown to use erucoyl-CoA (C22:1-CoA) as a substrate for PA synthesis, in contrast to an LPAAT1 from the same species that could not utilise the C22 substrate (Brown et al., 2002).

Known LPAATs were considered and a number were selected for testing, including some which were not expected to increase DHA incorporation at the sn-2 position, as controls. The known LPAATs included: Arabidopsis thaliana LPAAT2: (SEQ ID NO: 63, Accession No. ABG48392, Kim et al., 2005), Limnanthes alba LPAAT (SEQ ID NO: 64, Accession No. AAC49185, Lassner et al., 1995), Saccharomyces cerevisiae Slc1p (SEQ ID NO: 65, Accession No. NP_010231, Zou et al., 1997), Mortierella alpina LPAAT1 (SEQ ID NO: 67, Accession No. AED33305; U.S. Pat. No. 7,879,591) and Brassica napus LPAATs (SEQ ID NO: 68 and SEQ ID NO:69, Accession Nos ADC97479 and ADC97478 respectively). These were chosen to cover three groups of LPAAT enzymes: 1) control plant seed LPAATs with typically low activity on unusual long-chain polyunsaturated fatty acids (including the Arabidopsis and Brassica LPAATs), 2. LPAATs that had previously been demonstrated to act on C22 fatty acids by using C22 acyl-CoA as substrate, in this case erucic acid C22:1 (including the Limnanthes and Saccharomyces LPAATs), 3. LPAATs which the inventors considered likely to be able to utilise long-chain polyunsaturated fatty acids such as EPA and DHA as substrates (including the Mortierella LPAAT).

The Arabidopsis LPAAT2 (also designated LPAT2) is an endoplasmic reticulum-localised enzyme shown to have activity on C16 and C18 substrates, however activity on C20 or C22 substrates was not tested (Kim et al., 2005). Limnanthes alba LPAAT2 was demonstrated to insert a C22:1 acyl chain into the sn-2 position of PA, although the ability to use DHA as a substrate was not tested (Lassner et al., 1995). The selected S. cerevisiae LPAAT Slc1p was shown to have activity using 22:1-CoA in addition to 18:1-CoA as substrates, indicating a broad substrate specificity with respect to chain length (Zou et al., 1997). Again, DHA-CoA and other LC-PUFAs were not tested as substrates. The Mortierella LPAAT had previously been shown to have activity on EPA and DHA fatty acid substrates in transgenic Yarrowia lipolytica (U.S. Pat. No. 7,879,591).

Additional LPAATs were identified by the inventors. Micromonas pusilla is a microalga that produces and accumulates DHA in its oil, although the positional distribution of the DHA on TAG in this species has not been confirmed. The Micromonas pusilla LPAAT (SEQ ID NO: 66, Accession No. XP_002501997) was identified by searching the Micromonas pusilla genomic sequence using the Arabidopsis LPAAT2 as a BLAST query sequence. Several candidate sequences emerged and the sequence XP_002501997 was synthesised for testing as a likely LPAAT enzyme with activity on C22 LC-PUFA. The Ricinus communis LPAAT was annotated as a putative LPAAT in the castor genome sequence (Chan et al., 2010). Four candidate LPAATs from the castor genome were synthesised and tested in crude leaf lysates of infiltrated N. benthamiana leaf tissue. The candidate sequence described here showed LPAAT activity.

A number of candidate LPAATs were aligned with known LPAATs on a phylogenetic tree (FIG. 20). It was noted that the putative Micromonas LPAAT did not cluster with the putative C22 LPAATs but was a divergent sequence.

As an initial test of various LPAATs for their ability to use DHA-CoA as substrate, chimeric genetic constructs are made for constitutive expression of exogenous LPAATs in N. benthamiana leaves, each under the control of the 35S promoter, as follows: 35S:Arath-LPAAT2 (Arabidopsis ER LPAAT); 35S:Ricco-LPAAT2; 35S:Limal-LPAAT (Limnanthes alba LPAAT); 35S:Sacce-Slc1p (S. cerevisiae LPAAT); 35S:Micpu-LPAAT (Micromonas pusilla LPAAT); 35S:Moral-LPAAT1 (Mortierella alpina LPAAT). A 35S:p19 construct lacking an exogenous LPAAT is used as a control in the experiment. Each of these constructs is introduced via Agrobacterium into N. benthamiana leaves as described in Example 1, and 5 days after infiltration, the treated leaf zones are excised and ground to make leaf lysates. Each lysate includes the exogenous LPAAT as well as the endogenous enzymes for synthesizing LPA. In vitro reactions are set up by separately adding 14C-labelled-OA, -LA or -ALA (C18 substrates), -ARA (a C20 substrate) and -DHA (C22) to the lysates, in triplicate. Reactions are incubated at 25° C. and the level of incorporation of the 14C labelled fatty acids into PA determined by TLC. The ability of each LPAAT to use DHA relative to ARA and the C18 fatty acids is calculated. The meadowfoam, Mortierella and Saccharomyces LPAATs were found to have activity on DHA substrate, with radiolabelled PA appearing for these but not the other LPAATs. All LPAATs were confirmed active by a similar oleic acid feed.

To test LPAAT activity in seeds, several of the protein coding sequences or LPAATs are inserted into a binary vector under the control of a conlinin (pLuCnl1) promoter. The resultant genetic constructs containing the chimeric genes, Cnl1:Arath-LPAAT (negative control), Cnl1:Limal-LPAAT, Cnl:Sacce-Slc1p, and Cnl1:Moral-LPAAT, respectively, are then used transform B. napus and A. thaliana plants to generate stable transformants expressing the LPAATs in a seed-specific manner. The transformed plants having the Cnl1:LPAAT constructs are crossed with plants expressing the GA7 construct or its variants (Example 5) which produce DHA in the seed to result in increased incorporation of DHA at the sn-2 position of TAG. The constructs are also used to transform B. napus, C. sativa and A. thaliana plants that already contain the GA7 construct and variants thereof (Examples 2 to 5) to generate progeny carrying both the parental and LPAAT genetic constructs. Increased incorporation of DHA at the sn-2 position of TAG is expected relative to the incorporation in plants lacking the LPAAT encoding transgenes. Oil content is also improved in the seeds, particularly for seeds producing higher levels of DHA, counteracting the trend seen in Arabidopsis seed as described in Example 2.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from U.S. 61/660,392 filed 15 Jun. 2012, U.S. 61/663,344 filed 22 Jun. 2012, U.S. 61/697,676 filed 6 Sep. 2012 and U.S. 61/782,680 filed 14 Mar. 2013, the entire contents of each of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

This application incorporates herein by reference U.S. 61/660,392 filed 15 Jun. 2012, U.S. 61/663,344 filed 22 Jun. 2012 and U.S. 61/697,676 filed 6 Sep. 2012.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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<160> NUMBER OF SEQ ID NOS: 72

<210> SEQ ID NO: 1

<211> LENGTH: 21527

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pJP3416-GA7 nucleotide sequence.

<400> SEQENCE: 1

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgccccgcgg aaagcttgcg gccgcccgat ctagtaacat 240

agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc 300

gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat 360

gcattacatg ttaattatta cgtgcttaac gtaattcaac agaaattata tgataatcat 420

cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc 480

ggcgcgcctc attagtgagc cttctcagcc tttccgttaa cgtagtagtg ctgtcccacc 540

ttatcaaggt tagagaaagt agccttccaa gcaccgtagt aagagagcac cttgtagttg 600

agtccccact tcttagcgaa aggaacgaat cttctgctaa cctcaggctg tctgaattga 660

ggcatatcag ggaagaggtg gtggataacc tgacagttaa ggtatcccat aagccagttc 720

acgtatcctc tagaaggatc gatatcaacg gtgtgatcaa cagcgtagtt aacccaagaa 780

aggtgcttat cagatggaac aacagggagg tgagtatgag aagtagagaa gtgagcgaaa 840

aggtacatgt aagcgatcca gtttccgaaa gtgaaccacc agtaagcaac aggccaagag 900

tatccagtag caagcttgat aacagcggtt ctaacaacat gagaaacgag catccaagaa 960

gcctcttcgt agttcttctt acggagaact tgtctagggt ggagaacgta gatccagaaa 1020

gcttgaacaa gaagtccaga ggtaacagga acgaaagtcc aagcttgaag tctagcccaa 1080

gctctagaga atcctctagg tctgttatcc tcaacagcag tgttgaagaa agccacagca 1140

ggagtggtat caagatccat atcgtgtcta accttttgag gggtagcatg gtgcttgtta 1200

tgcatctggt tccacatctc accagaagta gaaagtccga atccacaagt catagcctga 1260

agtctcttgt ccacgtaaac agatccggta agagagttat gtccaccctc atgttgaacc 1320

catccacatc tagctccgaa gaaagcaccg taaacaacag aagcaatgat agggtatcca 1380

gcgtacataa gagcagttcc aagagcgaat gtagcaagaa gctcgagaag tctgtaagcc 1440

acatgggtga tagaaggctt gaagaatcca tctctctcaa gctcagcacg ccatctagcg 1500

aaatcctcaa gcataggagc atcctcagac tcagatctct tgatctcagc aggtctagaa 1560

ggcaaagctc taagcatctt ccaagccttg agagaacgca tgtggaattc tttgaaagcc 1620

tcagtagcat cagcaccagt gttagcaagc atgtagaaga tcacagatcc accagggtgc 1680

ttgaagttag tcacatcgta ctcaacgtcc tcaactctaa cccatctagt ctcgaaagta 1740

gcagcaagct catgaggctc aagagtctta agatcaacag gagcagtaga agcatcctta 1800

gcatcaagag cctcagcaga agatttagac ctggtaagtg gagatctagg agaagatctt 1860

ccatcagtct taggagggca catggtatgg taattgtaaa tgtaattgta atgttgtttg 1920

ttgtttgttg ttgttggtaa ttgttgtaaa agatcctcgt gtatgttttt aatcttgttt 1980

gtatcgatga gttttggttt gagtaaagag tgaagcggat gagttaattt ataggctata 2040

aaggagattt gcatggcgat cacgtgtaat aatgcatgca cgcatgtgat tgtatgtgtg 2100

tgctgtgaga gagaagctct taggtgtttg aagggagtga caagtggcga agaaaaacaa 2160

ttctccgcgg ctgcatgcta tgtgtaacgt gtagctaatg ttctggcatg gcatcttatg 2220

aacgattctt tttaaaaaca aggtaaaaac ttaacttcat aaaattaaaa aaaaaaacgt 2280

ttactaagtt ggtttaaaag gggatgagac tagtagattg gttggttggt ttccatgtac 2340

cagaaggctt accctattag ttgaaagttg aaactttgtt ccctactcaa ttcctagttg 2400

tgtaaatgta tgtatatgta atgtgtataa aacgtagtac ttaaatgact aggagtggtt 2460

cttgagaccg atgagagatg ggagcagaac taaagatgat gacataatta agaacgaatt 2520

tgaaaggctc ttaggtttga atcctattcg agaatgtttt tgtcaaagat agtggcgatt 2580

ttgaaccaaa gaaaacattt aaaaaatcag tatccggtta cgttcatgca aatagaaagt 2640

ggtctaggat ctgattgtaa ttttagactt aaagagtctc ttaagattca atcctggctg 2700

tgtacaaaac tacaaataat atattttaga ctatttggcc ttaactaaac ttccactcat 2760

tatttactga ggttagagaa tagacttgcg aataaacaca ttcccgagaa atactcatga 2820

tcccataatt agtcagaggg tatgccaatc agatctaaga acacacattc cctcaaattt 2880

taatgcacat gtaatcatag tttagcacaa ttcaaaaata atgtagtatt aaagacagaa 2940

atttgtagac ttttttttgg cgttaaaaga agactaagtt tatacgtaca ttttatttta 3000

agtggaaaac cgaaattttc catcgaaata tatgaattta gtatatatat ttctgcaatg 3060

tactattttg ctattttggc aactttcagt ggactactac tttattacaa tgtgtatgga 3120

tgcatgagtt tgagtataca catgtctaaa tgcatgcttt gtaaaacgta acggaccaca 3180

aaagaggatc catacaaata catctcatag cttcctccat tattttccga cacaaacaga 3240

gcattttaca acaattacca acaacaacaa acaacaaaca acattacaat tacatttaca 3300

attaccatac catggaattc gcccagcctc ttgttgctat ggctcaagag caatacgctg 3360

ctatcgatgc tgttgttgct cctgctatct tctctgctac tgattctatc ggatggggac 3420

ttaagcctat ctcttctgct actaaggact tgcctcttgt tgagtctcct acacctctca 3480

tcctttcttt gcttgcttac ttcgctatcg ttggatctgg actcgtttac agaaaggttt 3540

tccctagaac cgtgaaggga caagatccat tccttttgaa ggctcttatg cttgctcaca 3600

acgtgttcct tatcggactt tctctttaca tgtgcctcaa gcttgtgtac gaggcttacg 3660

ttaacaagta ctctttctgg ggaaacgctt acaaccctgc tcaaactgag atggctaagg 3720

ttatctggat cttctacgtg agcaagatct acgagttcat ggataccttc atcatgctcc 3780

tcaagggaaa tgttaaccag gttagcttcc ttcacgttta ccatcacgga tctatctctg 3840

gaatctggtg gatgattact tacgctgctc ctggtggtga tgcttacttc tctgctgctc 3900

ttaactcttg ggttcacgtg tgtatgtaca cctactattt tatggctgcc gtgcttccta 3960

aggacgagaa aactaagaga aagtacctct ggtggggaag ataccttact caaatgcaga 4020

tgttccagtt cttcatgaac cttctccagg ctgtttacct tctctactct tcatctcctt 4080

accctaagtt tatcgctcag ctcctcgtgg tgtacatggt tactcttctc atgcttttcg 4140

gaaacttcta ctacatgaag caccacgcta gcaagtgatg aggcgcgccg ggccgccgcc 4200

atgtgacaga tcgaaggaag aaagtgtaat aagacgactc tcactactcg atcgctagtg 4260

attgtcattg ttatatataa taatgttatc tttcacaact tatcgtaatg catgtgaaac 4320

tataacacat taatcctact tgtcatatga taacactctc cccatttaaa actcttgtca 4380

atttaaagat ataagattct ttaaatgatt aaaaaaaata tattataaat tcaatcactc 4440

ctactaataa attattaatt attatttatt gattaaaaaa atacttatac taatttagtc 4500

tgaatagaat aattagattc tagtctcatc cccttttaaa ccaacttagt aaacgttttt 4560

ttttttaatt ttatgaagtt aagtttttac cttgttttta aaaagaatcg ttcataagat 4620

gccatgccag aacattagct acacgttaca catagcatgc agccgcggag aattgttttt 4680

cttcgccact tgtcactccc ttcaaacacc taagagcttc tctctcacag cacacacata 4740

caatcacatg cgtgcatgca ttattacacg tgatcgccat gcaaatctcc tttatagcct 4800

ataaattaac tcatccgctt cactctttac tcaaaccaaa actcatcgat acaaacaaga 4860

ttaaaaacat acacgaggat cttttacaac aattaccaac aacaacaaac aacaaacaac 4920

attacaatta catttacaat taccatacca tgcctccaag ggactcttac tcttatgctg 4980

ctcctccttc tgctcaactt cacgaagttg atactcctca agagcacgac aagaaagagc 5040

ttgttatcgg agatagggct tacgatgtta ccaacttcgt taagagacac cctggtggaa 5100

agatcattgc ttaccaagtt ggaactgatg ctaccgatgc ttacaagcag ttccatgtta 5160

gatctgctaa ggctgacaag atgcttaagt ctcttccttc tcgtcctgtt cacaagggat 5220

actctccaag aagggctgat cttatcgctg atttccaaga gttcaccaag caacttgagg 5280

ctgagggaat gttcgagcct tctcttcctc atgttgctta cagacttgct gaggttatcg 5340

ctatgcatgt tgctggtgct gctcttatct ggcatggata cactttcgct ggaatcgcta 5400

tgcttggagt tgttcaggga agatgtggat ggcttatgca tgagggtgga cattactctc 5460

tcactggaaa cattgctttc gacagagcta tccaagttgc ttgttacgga cttggatgtg 5520

gaatgtctgg tgcttggtgg cgtaaccagc ataacaagca ccatgctact cctcaaaagc 5580

ttcagcacga tgttgatctt gatacccttc ctctcgttgc tttccatgag agaatcgctg 5640

ctaaggttaa gtctcctgct atgaaggctt ggctttctat gcaagctaag cttttcgctc 5700

ctgttaccac tcttcttgtt gctcttggat ggcagcttta ccttcatcct agacacatgc 5760

tcaggactaa gcactacgat gagcttgcta tgctcggaat cagatacgga cttgttggat 5820

accttgctgc taactacggt gctggatacg ttctcgcttg ttaccttctt tacgttcagc 5880

ttggagctat gtacatcttc tgcaacttcg ctgtttctca tactcacctc cctgttgttg 5940

agcctaacga gcatgctact tgggttgagt acgctgctaa ccacactact aactgttctc 6000

catcttggtg gtgtgattgg tggatgtctt accttaacta ccagatcgag caccaccttt 6060

acccttctat gcctcaattc agacacccta agatcgctcc tagagttaag cagcttttcg 6120

agaagcacgg acttcactac gatgttagag gatacttcga ggctatggct gatactttcg 6180

ctaaccttga taacgttgcc catgctcctg agaagaaaat gcagtaatga gatcgttcaa 6240

acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca 6300

tataatttct gttgaattac gttaagcacg taataattaa catgtaatgc atgacgttat 6360

ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa 6420

acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag 6480

atcggtcgat taaaaatccc aattatattt ggtctaattt agtttggtat tgagtaaaac 6540

aaattcgaac caaaccaaaa tataaatata tagtttttat atatatgcct ttaagacttt 6600

ttatagaatt ttctttaaaa aatatctaga aatatttgcg actcttctgg catgtaatat 6660

ttcgttaaat atgaagtgct ccatttttat taactttaaa taattggttg tacgatcact 6720

ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt cttccatatt catatgtcaa 6780

aatctatcaa aattcttata tatctttttc gaatttgaag tgaaatttcg ataatttaaa 6840

attaaataga acatatcatt atttaggtat catattgatt tttatactta attactaaat 6900

ttggttaact ttgaaagtgt acatcaacga aaaattagtc aaacgactaa aataaataaa 6960

tatcatgtgt tattaagaaa attctcctat aagaatattt taatagatca tatgtttgta 7020

aaaaaaatta atttttacta acacatatat ttacttatca aaaatttgac aaagtaagat 7080

taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc ggcaaaaccg 7140

aaccaatcca aaccgatata gttggtttgg tttgattttg atataaaccg aaccaactcg 7200

gtccatttgc acccctaatc ataatagctt taatatttca agatattatt aagttaacgt 7260

tgtcaatatc ctggaaattt tgcaaaatga atcaagccta tatggctgta atatgaattt 7320

aaaagcagct cgatgtggtg gtaatatgta atttacttga ttctaaaaaa atatcccaag 7380

tattaataat ttctgctagg aagaaggtta gctacgattt acagcaaagc cagaatacaa 7440

agaaccataa agtgattgaa gctcgaaata tacgaaggaa caaatatttt taaaaaaata 7500

cgcaatgact tggaacaaaa gaaagtgata tattttttgt tcttaaacaa gcatcccctc 7560

taaagaatgg cagttttcct ttgcatgtaa ctattatgct cccttcgtta caaaaatttt 7620

ggactactat tgggaacttc ttctgaaaat agtgatagaa cccacacgag catgtgcttt 7680

ccatttaatt ttaaaaacca agaaacatac atacataaca ttccatcagc ctctctctct 7740

ttttattacg gttaatgact taaaacacat cttattatcc catccttaac acctagcagt 7800

gtctttatac gatctcatcg atcaccactt caaaaccatg cagactgctg ctgcccctgg 7860

agctggcatc ggctaggctg ggtgccgcac tgtcccggaa ggtccctagc gacttgttta 7920

gattgatggg accacctctc aacttcctgc tgctgtccct gctgctggat gtcctgcctc 7980

atctggccga ttgcacgctc cagtcccctg catgtgcact cgctcctcaa ttgcttaaga 8040

tcatcgcagc agctatcgaa gtgctggctc tgttgccctc ctccacggcc ttggttgtag 8100

tagtagctgc cgccgccctt ctggactttt tcccacagga accgccgaat aattcgatag 8160

aaccacacga gcatgtgctt tcatttattt taaaaaccaa gaaacataca taacatttca 8220

tcagcctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctcttta 8280

ttacagctgt tacactaact taaaacacat tcatctcatt attattatta ttatccatcc 8340

ttaacaccta gcagtgtctt tgtacgatct cataatcgat caccccttca tcaggtatcc 8400

ttaggcttca ctccaacgtt gttgcagtta cggaacatgt acacaccatc atggttctca 8460

acgaactggc aagatctcca agttttccaa aggctaaccc acatgttctc atcggtgtgt 8520

ctgtagtgct ctcccataac tttcttgatg cactcggtag cttctctagc atggtagaat 8580

gggatccttg aaacgtagtg atggagcaca tgagtctcga tgatgtcatg gaagatgatt 8640

ccgaggattc cgaactctct atcgatagta gcagcagcac ccttagcgaa agtccactct 8700

tgagcatcgt aatgaggcat agaagaatcg gtgtgctgaa ggaaggtaac gaaaacaagc 8760

cagtggttaa caaggatcca aggacagaac catgtgatga aagtaggcca gaatccgaaa 8820

accttgtaag cggtgtaaac agaagtgagg gtagcaagga ttccaagatc agaaagaacg 8880

atgtaccagt agtccttctt atcgaaaaca gggctagaag gccagtagtg agacttgaag 8940

aacttagaaa caccagggta aggttgtcca gtagcgttag tagcaaggta aagagaaagt 9000

cctccaagct gttggaacaa gagagcgaaa acagagtaga taggagtttc ctcagcgata 9060

tcgtgaaggc tggtaacttg gtgcttctct ttgaattcct cggcggtgta aggaacgaaa 9120

accatatctc tggtcatgtg tccagtagcc ttatggtgct tagcatgaga gaacttccag 9180

ctgaagtaag gaaccataac aagagagtgg agaacccatc caacggtatc gttaacccat 9240

ccgtagttag agaaagcaga atgtccacac tcatgtccaa ggatccagat tccgaatccg 9300

aaacaagaga tagagaacac gtaagcagac caagcagcga atctaaggaa ttcgttaggg 9360

agaagaggga tgtaggtaag tccaacgtaa gcgatagcag agatagccac gatatctctc 9420

accacgtaag acatagactt cacgagagat ctctcgtaac agtgcttagg gatagcgtca 9480

aggatatcct tgatggtgta atctggcacc ttgaaaacgt ttccgaaggt atcgatagcg 9540

gtcttttgct gcttgaaaga tgcaacgttt ccagaacgcc taacggtctt agtagatccc 9600

tcaaggatct cagatccaga cacggtaacc ttagacatgg tatggtaatt gtaaatgtaa 9660

ttgtaatgtt gtttgttgtt tgttgttgtt ggtaattgtt gtaaaatttt tggtggtgat 9720

tggttcttta aggtgtgaga gtgagttgtg agttgtgtgg tgggtttggt gagattgggg 9780

atggtgggtt tatatagtgg agactgagga atggggtcgt gagtgttaac tttgcatggg 9840

ctacacgtgg gttcttttgg gcttacacgt agtattattc atgcaaatgc agccaataca 9900

tatacggtat tttaataatg tgtgggaata caatatgccg agtattttac taattttggc 9960

aatgacaagt gtacatttgg attatcttac ttggcctctc ttgctttaat ttggattatt 10020

tttattctct taccttggcc gttcatattc acatccctaa aggcaagaca gaattgaatg 10080

gtggccaaaa attaaaacga tggatatgac ctacatagtg taggatcaat taacgtcgaa 10140

ggaaaatact gattctctca agcatacgga caagggtaaa taacatagtc accagaacat 10200

aataaacaaa aagtgcagaa gcaagactaa aaaaattagc tatggacatt caggttcata 10260

ttggaaacat cattatccta gtcttgtgac catccttcct cctgctctag ttgagaggcc 10320

ttgggactaa cgagaggtca gttgggatag cagatcctta tcctggacta gcctttctgg 10380

tgtttcagag tcttcgtgcc gccgtctaca tctatctcca ttaggtctga agatgactct 10440

tcacaccaac gacgtttaag gtctctatcc tactcctagc ttgcaatacc tggcttgcaa 10500

tacctggagc atcgtgcacg atgattggat actgtggagg aggagtgttt gctgatttag 10560

agctcccggt tgggtgattt gacttcgatt tcagtttagg cttgttgaaa tttttcaggt 10620

tccattgtga agcctttaga gcttgagctt ccttccatgt taatgccttg atcgaatact 10680

cctagagaaa agggaagtcg atctctgagt attgaaatcg aagtgcacat tttttttcaa 10740

cgtgtccaat caatccacaa acaaagcaga agacaggtaa tctttcatac ttatactgac 10800

aagtaatagt cttaccgtca tgcataataa cgtctcgttc cttcaagagg ggttttccga 10860

catccataac gacccgaagc ctcatgaaag cattagggaa gaacttttgg ttcttcttgt 10920

catggccttt ataggtgtca gccgagctcg ccaattcccg tccgactggc tccgcaaaat 10980

attcgaacgg caagttatgg acttgcaacc ataactccac ggtattgagc aggacctatt 11040

gtgaagactc atctcatgga gcttcagaat gtggttgtca gcaaaccaat gaccgaaatc 11100

catcacatga cggacgtcca gtgggtgagc gaaacgaaac aggaagcgcc tatctttcag 11160

agtcgtgagc tccacaccgg attccggcaa ctacgtgttg ggcaggcttc gccgtattag 11220

agatatgttg aggcagaccc atctgtgcca ctcgtacaat tacgagagtt gttttttttg 11280

tgattttcct agtttctcgt tgatggtgag ctcatattct acatcgtatg gtctctcaac 11340

gtcgtttcct gtcatctgat atcccgtcat ttgcatccac gtgcgccgcc tcccgtgcca 11400

agtccctagg tgtcatgcac gccaaattgg tggtggtgcg ggctgccctg tgcttcttac 11460

cgatgggtgg aggttgagtt tgggggtctc cgcggcgatg gtagtgggtt gacggtttgg 11520

tgtgggttga cggcattgat caatttactt cttgcttcaa attctttggc agaaaacaat 11580

tcattagatt agaactggaa accagagtga tgagacggat taagtcagat tccaacagag 11640

ttacatctct taagaaataa tgtaacccct ttagacttta tatatttgca attaaaaaaa 11700

taatttaact tttagacttt atatatagtt ttaataacta agtttaacca ctctattatt 11760

tatatcgaaa ctatttgtat gtctcccctc taaataaact tggtattgtg tttacagaac 11820

ctataatcaa ataatcaata ctcaactgaa gtttgtgcag ttaattgaag ggattaacgg 11880

ccaaaatgca ctagtattat caaccgaata gattcacact agatggccat ttccatcaat 11940

atcatcgccg ttcttcttct gtccacatat cccctctgaa acttgagaga cacctgcact 12000

tcattgtcct tattacgtgt tacaaaatga aacccatgca tccatgcaaa ctgaagaatg 12060

gcgcaagaac ccttcccctc catttcttat gtggcgacca tccatttcac catctcccgc 12120

tataaaacac ccccatcact tcacctagaa catcatcact acttgcttat ccatccaaaa 12180

gatacccact tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattaca 12240

tttacaatta ccataccatg ccacctagcg ctgctaagca aatgggagct tctactggtg 12300

ttcatgctgg tgttactgac tcttctgctt tcaccagaaa ggatgttgct gatagacctg 12360

atctcaccat cgttggagat tctgtttacg atgctaaggc tttcagatct gagcatcctg 12420

gtggtgctca tttcgtttct ttgttcggag gaagagatgc tactgaggct ttcatggaat 12480

accatagaag ggcttggcct aagtctagaa tgtctagatt ccacgttgga tctcttgctt 12540

ctactgagga acctgttgct gctgatgagg gataccttca actttgtgct aggatcgcta 12600

agatggtgcc ttctgtttct tctggattcg ctcctgcttc ttactgggtt aaggctggac 12660

ttatccttgg atctgctatc gctcttgagg cttacatgct ttacgctgga aagagacttc 12720

tcccttctat cgttcttgga tggcttttcg ctcttatcgg tcttaacatc cagcatgatg 12780

ctaaccatgg tgctttgtct aagtctgctt ctgttaacct tgctcttgga ctttgtcagg 12840

attggatcgg aggatctatg atcctttggc ttcaagagca tgttgttatg caccacctcc 12900

acactaacga tgttgataag gatcctgatc aaaaggctca cggtgctctt agactcaagc 12960

ctactgatgc ttggtcacct atgcattggc ttcagcatct ttaccttttg cctggtgaga 13020

ctatgtacgc tttcaagctt ttgttcctcg acatctctga gcttgttatg tggcgttggg 13080

agggtgagcc tatctctaag cttgctggat acctctttat gccttctttg cttctcaagc 13140

ttaccttctg ggctagattc gttgctttgc ctctttacct tgctccttct gttcatactg 13200

ctgtgtgtat cgctgctact gttatgactg gatctttcta cctcgctttc ttcttcttca 13260

tctcccacaa cttcgagggt gttgcttctg ttggacctga tggatctatc acttctatga 13320

ctagaggtgc tagcttcctt aagagacaag ctgagacttc ttctaacgtt ggaggacctc 13380

ttcttgctac tcttaacggt ggactcaact accaaattga gcatcacttg ttccctagag 13440

ttcaccatgg attctaccct agacttgctc ctcttgttaa ggctgagctt gaggctagag 13500

gaatcgagta caagcactac cctactatct ggtctaacct tgcttctacc ctcagacata 13560

tgtacgctct tggaagaagg cctagatcta aggctgagta atgacaagct tatgtgacgt 13620

gaaataataa cggtaaaata tatgtaataa taataataat aaagccacaa agtgagaatg 13680

aggggaaggg gaaatgtgta atgagccagt agccggtggt gctaattttg tatcgtattg 13740

tcaataaatc atgaattttg tggtttttat gtgttttttt aaatcatgaa ttttaaattt 13800

tataaaataa tctccaatcg gaagaacaac attccatatc catgcatgga tgtttcttta 13860

cccaaatcta gttcttgaga ggatgaagca tcaccgaaca gttctgcaac tatccctcaa 13920

aagctttaaa atgaacaaca aggaacagag caacgttcca aagatcccaa acgaaacata 13980

ttatctatac taatactata ttattaatta ctactgcccg gaatcacaat ccctgaatga 14040

ttcctattaa ctacaagcct tgttggcggc ggagaagtga tcggcgcggc gagaagcagc 14100

ggactcggag acgaggcctt ggaagatctg agtcgaacgg gcagaatcag tattttcctt 14160

cgacgttaat tgatcctaca ctatgtaggt catatccatc gttttaattt ttggccacca 14220

ttcaattctg tcttgccttt agggatgtga atatgaacgg ccaaggtaag agaataaaaa 14280

taatccaaat taaagcaaga gaggccaagt aagataatcc aaatgtacac ttgtcattgc 14340

caaaattagt aaaatactcg gcatattgta ttcccacaca ttattaaaat accgtatatg 14400

tattggctgc atttgcatga ataatactac gtgtaagccc aaaagaaccc acgtgtagcc 14460

catgcaaagt taacactcac gaccccattc ctcagtctcc actatataaa cccaccatcc 14520

ccaatctcac caaacccacc acacaactca caactcactc tcacacctta aagaaccaat 14580

caccaccaaa aattttacaa caattaccaa caacaacaaa caacaaacaa cattacaatt 14640

acatttacaa ttaccatacc atgagcgctg ttaccgttac tggatctgat cctaagaaca 14700

gaggatcttc tagcaacacc gagcaagagg ttccaaaagt tgctatcgat accaacggaa 14760

acgtgttctc tgttcctgat ttcaccatca aggacatcct tggagctatc cctcatgagt 14820

gttacgagag aagattggct acctctctct actacgtgtt cagagatatc ttctgcatgc 14880

ttaccaccgg ataccttacc cataagatcc tttaccctct cctcatctct tacacctcta 14940

acagcatcat caagttcact ttctgggccc tttacactta cgttcaagga cttttcggaa 15000

ccggaatctg ggttctcgct catgagtgtg gacatcaagc tttctctgat tacggaatcg 15060

tgaacgattt cgttggatgg acccttcact cttaccttat ggttccttac ttcagctgga 15120

agtactctca tggaaagcac cataaggcta ctggacacat gaccagagat atggttttcg 15180

ttcctgccac caaagaggaa ttcaagaagt ctaggaactt cttcggtaac ctcgctgagt 15240

actctgagga ttctccactt agaacccttt acgagcttct tgttcaacaa cttggaggat 15300

ggatcgctta cctcttcgtt aacgttacag gacaacctta ccctgatgtt ccttcttgga 15360

aatggaacca cttctggctt acctctccac ttttcgagca aagagatgct ctctacatct 15420

tcctttctga tcttggaatc ctcacccagg gaatcgttct tactctttgg tacaagaaat 15480

tcggaggatg gtcccttttc atcaactggt tcgttcctta catctgggtt aaccactggc 15540

tcgttttcat cacattcctt cagcacactg atcctactat gcctcattac aacgctgagg 15600

aatggacttt cgctaagggt gctgctgcta ctatcgatag aaagttcgga ttcatcggac 15660

ctcacatctt ccatgatatc atcgagactc atgtgcttca ccactactgt tctaggatcc 15720

cattctacaa cgctagacct gcttctgagg ctatcaagaa agttatggga aagcactaca 15780

ggtctagcga cgagaacatg tggaagtcac tttggaagtc tttcaggtct tgccaatacg 15840

ttgacggtga taacggtgtt ctcatgttcc gtaacatcaa caactgcgga gttggagctg 15900

ctgagaagta atgaaggggt gatcgattat gagatcgtac aaagacactg ctaggtgtta 15960

aggatggata ataataataa taatgagatg aatgtgtttt aagttagtgt aacagctgta 16020

ataaagagag agagagagag agagagagag agagagagag agagagagag agagaggctg 16080

atgaaatgtt atgtatgttt cttggttttt aaaataaatg aaagcacatg ctcgtgtggt 16140

tctatcgaat tattcggcgg ttcctgtggg aaaaagtcca gaagggccgc cgcagctact 16200

actacaacca aggccgtgga ggagggcaac agagccagca cttcgatagc tgctgcgatg 16260

atcttaagca attgaggagc gagtgcacat gcaggggact ggagcgtgca atcggccaga 16320

tgaggcagga catccagcag cagggacagc agcaggaagt tgagaggtgg tcccatcaat 16380

ctaaacaagt cgctagggac cttccgggac agtgcggcac ccagcctagc cgatgccagc 16440

tccaggggca gcagcagtct gcatggtttt gaagtggtga tcgatgagat cgtataaaga 16500

cactgctagg tgttaaggat gggataataa gatgtgtttt aagtcattaa ccgtaataaa 16560

aagagagaga ggctgatgga atgttatgta tgtatgtttc ttggttttta aaattaaatg 16620

gaaagcacat gctcgtgtgg gttctatctc gattaaaaat cccaattata tttggtctaa 16680

tttagtttgg tattgagtaa aacaaattcg aaccaaacca aaatataaat atatagtttt 16740

tatatatatg cctttaagac tttttataga attttcttta aaaaatatct agaaatattt 16800

gcgactcttc tggcatgtaa tatttcgtta aatatgaagt gctccatttt tattaacttt 16860

aaataattgg ttgtacgatc actttcttat caagtgttac taaaatgcgt caatctcttt 16920

gttcttccat attcatatgt caaaatctat caaaattctt atatatcttt ttcgaatttg 16980

aagtgaaatt tcgataattt aaaattaaat agaacatatc attatttagg tatcatattg 17040

atttttatac ttaattacta aatttggtta actttgaaag tgtacatcaa cgaaaaatta 17100

gtcaaacgac taaaataaat aaatatcatg tgttattaag aaaattctcc tataagaata 17160

ttttaataga tcatatgttt gtaaaaaaaa ttaattttta ctaacacata tatttactta 17220

tcaaaaattt gacaaagtaa gattaaaata atattcatct aacaaaaaaa aaaccagaaa 17280

atgctgaaaa cccggcaaaa ccgaaccaat ccaaaccgat atagttggtt tggtttgatt 17340

ttgatataaa ccgaaccaac tcggtccatt tgcaccccta atcataatag ctttaatatt 17400

tcaagatatt attaagttaa cgttgtcaat atcctggaaa ttttgcaaaa tgaatcaagc 17460

ctatatggct gtaatatgaa tttaaaagca gctcgatgtg gtggtaatat gtaatttact 17520

tgattctaaa aaaatatccc aagtattaat aatttctgct aggaagaagg ttagctacga 17580

tttacagcaa agccagaata caaagaacca taaagtgatt gaagctcgaa atatacgaag 17640

gaacaaatat ttttaaaaaa atacgcaatg acttggaaca aaagaaagtg atatattttt 17700

tgttcttaaa caagcatccc ctctaaagaa tggcagtttt cctttgcatg taactattat 17760

gctcccttcg ttacaaaaat tttggactac tattgggaac ttcttctgaa aatagtcctg 17820

caggctagta gattggttgg ttggtttcca tgtaccagaa ggcttaccct attagttgaa 17880

agttgaaact ttgttcccta ctcaattcct agttgtgtaa atgtatgtat atgtaatgtg 17940

tataaaacgt agtacttaaa tgactaggag tggttcttga gaccgatgag agatgggagc 18000

agaactaaag atgatgacat aattaagaac gaatttgaaa ggctcttagg tttgaatcct 18060

attcgagaat gtttttgtca aagatagtgg cgattttgaa ccaaagaaaa catttaaaaa 18120

atcagtatcc ggttacgttc atgcaaatag aaagtggtct aggatctgat tgtaatttta 18180

gacttaaaga gtctcttaag attcaatcct ggctgtgtac aaaactacaa ataatatatt 18240

ttagactatt tggccttaac taaacttcca ctcattattt actgaggtta gagaatagac 18300

ttgcgaataa acacattccc gagaaatact catgatccca taattagtca gagggtatgc 18360

caatcagatc taagaacaca cattccctca aattttaatg cacatgtaat catagtttag 18420

cacaattcaa aaataatgta gtattaaaga cagaaatttg tagacttttt tttggcgtta 18480

aaagaagact aagtttatac gtacatttta ttttaagtgg aaaaccgaaa ttttccatcg 18540

aaatatatga atttagtata tatatttctg caatgtacta ttttgctatt ttggcaactt 18600

tcagtggact actactttat tacaatgtgt atggatgcat gagtttgagt atacacatgt 18660

ctaaatgcat gctttgtaaa acgtaacgga ccacaaaaga ggatccatac aaatacatct 18720

catagcttcc tccattattt tccgacacaa acagagcatt ttacaacaat taccaacaac 18780

aacaaacaac aaacaacatt acaattacat ttacaattac cataccatgg cctctatcgc 18840

tatccctgct gctcttgctg gaactcttgg atacgttacc tacaatgtgg ctaaccctga 18900

tatcccagct tctgagaaag ttcctgctta cttcatgcag gttgagtact ggggacctac 18960

tatcggaact attggatacc tcctcttcat ctacttcgga aagcgtatca tgcagaacag 19020

atctcaacct ttcggactca agaacgctat gctcgtttac aacttctacc agaccttctt 19080

caacagctac tgcatctacc ttttcgttac ttctcatagg gctcagggac ttaaggtttg 19140

gggaaacatc cctgatatga ctgctaactc ttggggaatc tctcaggtta tctggcttca 19200

ctacaacaac aagtacgttg agcttctcga caccttcttc atggtgatga ggaagaagtt 19260

cgaccagctt tctttccttc acatctacca ccacactctt ctcatctggt catggttcgt 19320

tgttatgaag cttgagcctg ttggagattg ctacttcgga tcttctgtta acaccttcgt 19380

gcacgtgatc atgtactctt actacggact tgctgctctt ggagttaact gtttctggaa 19440

gaagtacatc acccagatcc agatgcttca gttctgtatc tgtgcttctc actctatcta 19500

caccgcttac gttcagaata ccgctttctg gcttccttac cttcaactct gggttatggt 19560

gaacatgttc gttctcttcg ccaacttcta ccgtaagagg tacaagtcta agggtgctaa 19620

gaagcagtga taagggccgc cgccatgtga cagatcgaag gaagaaagtg taataagacg 19680

actctcacta ctcgatcgct agtgattgtc attgttatat ataataatgt tatctttcac 19740

aacttatcgt aatgcatgtg aaactataac acattaatcc tacttgtcat atgataacac 19800

tctccccatt taaaactctt gtcaatttaa agatataaga ttctttaaat gattaaaaaa 19860

aatatattat aaattcaatc actcctacta ataaattatt aattattatt tattgattaa 19920

aaaaatactt atactaattt agtctgaata gaataattag attctagcct gcagggcggc 19980

cgcggatccc atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac 20040

tggcgaacag ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa 20100

catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga 20160

ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca 20220

ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa 20280

atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc 20340

caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 20400

ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca 20460

ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agagaacacg 20520

ggggactgaa ttaaatatga gccctgagag gcgtcctgtt gaaatcagac ctgctactgc 20580

tgctgatatg gctgctgttt gtgatatcgt gaaccactac atcgagactt ctaccgttaa 20640

cttcagaact gagcctcaaa ctcctcaaga gtggatcgat gatcttgaga gactccaaga 20700

tagataccct tggcttgttg ctgaggttga gggtgttgtt gctggaatcg cttacgctgg 20760

accttggaag gctagaaacg cttacgattg gactgttgag tctaccgttt acgtttcaca 20820

cagacatcag agacttggac ttggatctac cctttacact caccttctca agtctatgga 20880

agctcaggga ttcaagtctg ttgttgctgt tatcggactc cctaacgatc cttctgttag 20940

acttcatgag gctcttggat acactgctag aggaactctt agagctgctg gatacaagca 21000

cggtggatgg catgatgttg gattctggca aagagatttc gagcttcctg ctcctcctag 21060

acctgttaga ccagttactc agatctgaat ttgcgtgatc gttcaaacat ttggcaataa 21120

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 21180

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 21240

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 21300

gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatca ctagtgatgt 21360

acggttaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 21420

cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc tccccgaccg 21480

gcagctcggc acaaaatcac cactcgatac aggcagccca tcagtcc 21527

<210> SEQ ID NO: 2

<211> LENGTH: 23512

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pGA7- mod_B nucleotide sequence

<400> SEQENCE: 2

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgccccgcgg aaagcttgcg gccgcggtac cgcccgttcg 240

actcagatct tccaaggcct cgtctccgag tccgctgctt ctcgccgcgc cgatcacttc 300

tccgccgcca acaaggcttg tagttaatag gaatcattca gggattgtga ttccgggcag 360

tagtaattaa taatatagta ttagtataga taatatgttt cgtttgggat ctttggaacg 420

ttgctctgtt ccttgttgtt cattttaaag cttttgaggg atagttgcag aactgttcgg 480

tgatgcttca tcctctcaag aactagattt gggtaaagaa acatccatgc atggatatgg 540

aatgttgttc ttccgattgg agattatttt ataaaattta aaattcatga tttaaaaaaa 600

cacataaaaa ccacaaaatt catgatttat tgacaatacg atacaaaatt agcaccaccg 660

gctactggct cattacacat ttccccttcc cctcattctc actttgtggc tttattatta 720

ttattattac atatatttta ccgttattat ttcacgtcac ataagcttgt taattaatca 780

ttagtgagcc ttctcagcct ttccgttaac gtagtagtgc tgtcccacct tatcaaggtt 840

agagaaagta gccttccaag caccgtagta agagagcacc ttgtagttga gtccccactt 900

cttagcgaaa ggaacgaatc ttctgctaac ctcaggctgt ctgaattgag gcatatcagg 960

gaagaggtgg tggataacct gacagttaag gtatcccata agccagttca cgtatcctct 1020

agaaggatcg atatcaacgg tgtgatcaac agcgtagtta acccaagaaa ggtgcttatc 1080

agatggaaca acagggaggt gagtatgaga agtagagaag tgagcgaaaa ggtacatgta 1140

agcgatccag tttccgaaag tgaaccacca gtaagcaaca ggccaagagt atccagtagc 1200

aagcttgata acagcggttc taacaacatg agaaacgagc atccaagaag cctcttcgta 1260

gttcttctta cggagaactt gtctagggtg gagaacgtag atccagaaag cttgaacaag 1320

aagtccagag gtaacaggaa cgaaagtcca agcttgaagt ctagcccaag ctctagagaa 1380

tcctctaggt ctgttatcct caacagcagt gttgaagaaa gccacagcag gagtggtatc 1440

aagatccata tcgtgtctaa ccttttgagg ggtagcatgg tgcttgttat gcatctggtt 1500

ccacatctca ccagaagtag aaagtccgaa tccacaagtc atagcctgaa gtctcttgtc 1560

cacgtaaaca gatccggtaa gagagttatg tccaccctca tgttgaaccc atccacatct 1620

agctccgaag aaagcaccgt aaacaacaga agcaatgata gggtatccag cgtacataag 1680

agcagttcca agagcgaatg tagcaagaag ctcgagaagt ctgtaagcca catgggtgat 1740

agaaggcttg aagaatccat ctctctcaag ctcagcacgc catctagcga aatcctcaag 1800

cataggagca tcctcagact cagatctctt gatctcagca ggtctagaag gcaaagctct 1860

aagcatcttc caagccttga gagaacgcat gtggaattct ttgaaagcct cagtagcatc 1920

agcaccagtg ttagcaagca tgtagaagat cacagatcca ccagggtgct tgaagttagt 1980

cacatcgtac tcaacgtcct caactctaac ccatctagtc tcgaaagtag cagcaagctc 2040

atgaggctca agagtcttaa gatcaacagg agcagtagaa gcatccttag catcaagagc 2100

ctcagcagaa gatttagacc tggtaagtgg agatctagga gaagatcttc catcagtctt 2160

aggagggcac atggtatggt aattgtaaat gtaattgtaa tgttgtttgt tgtttgttgt 2220

tgttggtaat tgttgtaaaa ttaattaagt gggtatcttt tggatggata agcaagtagt 2280

gatgatgttc taggtgaagt gatgggggtg ttttatagcg ggagatggtg aaatggatgg 2340

tcgccacata agaaatggag gggaagggtt cttgcgccat tcttcagttt gcatggatgc 2400

atgggtttca ttttgtaaca cgtaataagg acaatgaagt gcaggtgtct ctcaagtttc 2460

agaggggata tgtggacaga agaagaacgg cgatgatatt gatggaaatg gccatctagt 2520

gtgaatctat tcggttgata atactagtgc attttggccg ttaatccctt caattaactg 2580

cacaaacttc agttgagtat tgattatttg attataggtt ctgtaaacac aataccaagt 2640

ttatttagag gggagacata caaatagttt cgatataaat aatagagtgg ttaaacttag 2700

ttattaaaac tatatataaa gtctaaaagt taaattattt ttttaattgc aaatatataa 2760

agtctaaagg ggttacatta tttcttaaga gatgtaactc tgttggaatc tgacttaatc 2820

cgtctcatca ctctggtttc cagttctaat ctaatgaatt gttttctgcc aaagaatttg 2880

aagcaagaag taaattgatc aatgccgtca acccacacca aaccgtcaac ccactaccat 2940

cgccgcggag acccccaaac tcaacctcca cccatcggta agaagcacag ggcagcccgc 3000

accaccacca atttggcgtg catgacacct agggacttgg cacgggaggc ggcgcacgtg 3060

gatgcaaatg acgggatatc agatgacagg aaacgacgtt gagagaccat acgatgtaga 3120

atatgagctc accatcaacg agaaactagg aaaatcacaa aaaaaacaac tctcgtaatt 3180

gtacgagtgg cacagatggg tctgcctcaa catatctcta atacggcgaa gcctgcccaa 3240

cacgtagttg ccggaatccg gtgtggagct cacgactctg aaagataggc gcttcctgtt 3300

tcgtttcgct cacccactgg acgtccgtca tgtgatggat ttcggtcatt ggtttgctga 3360

caaccacatt ctgaagctcc atgagatgag tcttcacaat aggtcctgct caataccgtg 3420

gagttatggt tgcaagtcca taacttgccg ttcgaatatt ttgcggagcc agtcggacgg 3480

gaattggcga gctcggctga cacctataaa ggccatgaca agaagaacca aaagttcttc 3540

cctaatgctt tcatgaggct tcgggtcgtt atggatgtcg gaaaacccct cttgaaggaa 3600

cgagacgtta ttatgcatga cggtaagact attacttgtc agtataagta tgaaagatta 3660

cctgtcttct gctttgtttg tggattgatt ggacacgttg aaaaaaaatg tgcacttcga 3720

tttcaatact cagagatcga cttccctttt ctctaggagt attcgatcaa ggcattaaca 3780

tggaaggaag ctcaagctct aaaggcttca caatggaacc tgaaaaattt caacaagcct 3840

aaactgaaat cgaagtcaaa tcacccaacc gggagctcta aatcagcaaa cactcctcct 3900

ccacagtatc caatcatcgt gcacgatgct ccaggtattg caagccaggt attgcaagct 3960

aggagtagga tagagacctt aaacgtcgtt ggtgtgaaga gtcatcttca gacctaatgg 4020

agatagatgt agacggcggc acgaagactc tgaaacacca gaaaggctag tccaggataa 4080

ggatctgcta tcccaactga cctctcgtta gtcccaaggc ctctcaacta gagcaggagg 4140

aaggatggtc acaagactag gataatgatg tttccaatat gaacctgaat gtccatagct 4200

aattttttta gtcttgcttc tgcacttttt gtttattatg ttctggtgac tatgttattt 4260

acccttgtcc gtatgcttga gggtacccta gtagattggt tggttggttt ccatgtacca 4320

gaaggcttac cctattagtt gaaagttgaa actttgttcc ctactcaatt cctagttgtg 4380

taaatgtatg tatatgtaat gtgtataaaa cgtagtactt aaatgactag gagtggttct 4440

tgagaccgat gagagatggg agcagaacta aagatgatga cataattaag aacgaatttg 4500

aaaggctctt aggtttgaat cctattcgag aatgtttttg tcaaagatag tggcgatttt 4560

gaaccaaaga aaacatttaa aaaatcagta tccggttacg ttcatgcaaa tagaaagtgg 4620

tctaggatct gattgtaatt ttagacttaa agagtctctt aagattcaat cctggctgtg 4680

tacaaaacta caaataatat attttagact atttggcctt aactaaactt ccactcatta 4740

tttactgagg ttagagaata gacttgcgaa taaacacatt cccgagaaat actcatgatc 4800

ccataattag tcagagggta tgccaatcag atctaagaac acacattccc tcaaatttta 4860

atgcacatgt aatcatagtt tagcacaatt caaaaataat gtagtattaa agacagaaat 4920

ttgtagactt ttttttggcg ttaaaagaag actaagttta tacgtacatt ttattttaag 4980

tggaaaaccg aaattttcca tcgaaatata tgaatttagt atatatattt ctgcaatgta 5040

ctattttgct attttggcaa ctttcagtgg actactactt tattacaatg tgtatggatg 5100

catgagtttg agtatacaca tgtctaaatg catgctttgt aaaacgtaac ggaccacaaa 5160

agaggatcca tacaaataca tctcatagct tcctccatta ttttccgaca caaacagagc 5220

attttacaac aattaccaac aacaacaaac aacaaacaac attacaatta catttacaat 5280

taccatacca tggcctctat cgctatccct gctgctcttg ctggaactct tggatacgtt 5340

acctacaatg tggctaaccc tgatatccca gcttctgaga aagttcctgc ttacttcatg 5400

caggttgagt actggggacc tactatcgga actattggat acctcctctt catctacttc 5460

ggaaagcgta tcatgcagaa cagatctcaa cctttcggac tcaagaacgc tatgctcgtt 5520

tacaacttct accagacctt cttcaacagc tactgcatct accttttcgt tacttctcat 5580

agggctcagg gacttaaggt ttggggaaac atccctgata tgactgctaa ctcttgggga 5640

atctctcagg ttatctggct tcactacaac aacaagtacg ttgagcttct cgacaccttc 5700

ttcatggtga tgaggaagaa gttcgaccag ctttctttcc ttcacatcta ccaccacact 5760

cttctcatct ggtcatggtt cgttgttatg aagcttgagc ctgttggaga ttgctacttc 5820

ggatcttctg ttaacacctt cgtgcacgtg atcatgtact cttactacgg acttgctgct 5880

cttggagtta actgtttctg gaagaagtac atcacccaga tccagatgct tcagttctgt 5940

atctgtgctt ctcactctat ctacaccgct tacgttcaga ataccgcttt ctggcttcct 6000

taccttcaac tctgggttat ggtgaacatg ttcgttctct tcgccaactt ctaccgtaag 6060

aggtacaagt ctaagggtgc taagaagcag tgataaggcg cgcggcgcgc cgggccgccg 6120

ccatgtgaca gatcgaagga agaaagtgta ataagacgac tctcactact cgatcgctag 6180

tgattgtcat tgttatatat aataatgtta tctttcacaa cttatcgtaa tgcatgtgaa 6240

actataacac attaatccta cttgtcatat gataacactc tccccattta aaactcttgt 6300

caatttaaag atataagatt ctttaaatga ttaaaaaaaa tatattataa attcaatcac 6360

tcctactaat aaattattaa ttattattta ttgattaaaa aaatacttat actaatttag 6420

tctgaataga ataattagat tctagtctca tcccctttta aaccaactta gtaaacgttt 6480

ttttttttaa ttttatgaag ttaagttttt accttgtttt taaaaagaat cgttcataag 6540

atgccatgcc agaacattag ctacacgtta cacatagcat gcagccgcgg agaattgttt 6600

ttcttcgcca cttgtcactc ccttcaaaca cctaagagct tctctctcac agcacacaca 6660

tacaatcaca tgcgtgcatg cattattaca cgtgatcgcc atgcaaatct cctttatagc 6720

ctataaatta actcatccgc ttcactcttt actcaaacca aaactcatcg atacaaacaa 6780

gattaaaaac atacacgagg atcttttaca acaattacca acaacaacaa acaacaaaca 6840

acattacaat tacatttaca attaccatac catgcctcca agggactctt actcttatgc 6900

tgctcctcct tctgctcaac ttcacgaagt tgatactcct caagagcacg acaagaaaga 6960

gcttgttatc ggagataggg cttacgatgt taccaacttc gttaagagac accctggtgg 7020

aaagatcatt gcttaccaag ttggaactga tgctaccgat gcttacaagc agttccatgt 7080

tagatctgct aaggctgaca agatgcttaa gtctcttcct tctcgtcctg ttcacaaggg 7140

atactctcca agaagggctg atcttatcgc tgatttccaa gagttcacca agcaacttga 7200

ggctgaggga atgttcgagc cttctcttcc tcatgttgct tacagacttg ctgaggttat 7260

cgctatgcat gttgctggtg ctgctcttat ctggcatgga tacactttcg ctggaatcgc 7320

tatgcttgga gttgttcagg gaagatgtgg atggcttatg catgagggtg gacattactc 7380

tctcactgga aacattgctt tcgacagagc tatccaagtt gcttgttacg gacttggatg 7440

tggaatgtct ggtgcttggt ggcgtaacca gcataacaag caccatgcta ctcctcaaaa 7500

gcttcagcac gatgttgatc ttgataccct tcctctcgtt gctttccatg agagaatcgc 7560

tgctaaggtt aagtctcctg ctatgaaggc ttggctttct atgcaagcta agcttttcgc 7620

tcctgttacc actcttcttg ttgctcttgg atggcagctt taccttcatc ctagacacat 7680

gctcaggact aagcactacg atgagcttgc tatgctcgga atcagatacg gacttgttgg 7740

ataccttgct gctaactacg gtgctggata cgttctcgct tgttaccttc tttacgttca 7800

gcttggagct atgtacatct tctgcaactt cgctgtttct catactcacc tccctgttgt 7860

tgagcctaac gagcatgcta cttgggttga gtacgctgct aaccacacta ctaactgttc 7920

tccatcttgg tggtgtgatt ggtggatgtc ttaccttaac taccagatcg agcaccacct 7980

ttacccttct atgcctcaat tcagacaccc taagatcgct cctagagtta agcagctttt 8040

cgagaagcac ggacttcact acgatgttag aggatacttc gaggctatgg ctgatacttt 8100

cgctaacctt gataacgttg cccatgctcc tgagaagaaa atgcagtaat gagatcgttc 8160

aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 8220

catataattt ctgttgaatt acgttaagca cgtaataatt aacatgtaat gcatgacgtt 8280

atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 8340

aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 8400

agatcggtcg attaaaaatc ccaattatat ttggtctaat ttagtttggt attgagtaaa 8460

acaaattcga accaaaccaa aatataaata tatagttttt atatatatgc ctttaagact 8520

ttttatagaa ttttctttaa aaaatatcta gaaatatttg cgactcttct ggcatgtaat 8580

atttcgttaa atatgaagtg ctccattttt attaacttta aataattggt tgtacgatca 8640

ctttcttatc aagtgttact aaaatgcgtc aatctctttg ttcttccata ttcatatgtc 8700

aaaatctatc aaaattctta tatatctttt tcgaatttga agtgaaattt cgataattta 8760

aaattaaata gaacatatca ttatttaggt atcatattga tttttatact taattactaa 8820

atttggttaa ctttgaaagt gtacatcaac gaaaaattag tcaaacgact aaaataaata 8880

aatatcatgt gttattaaga aaattctcct ataagaatat tttaatagat catatgtttg 8940

taaaaaaaat taatttttac taacacatat atttacttat caaaaatttg acaaagtaag 9000

attaaaataa tattcatcta acaaaaaaaa aaccagaaaa tgctgaaaac ccggcaaaac 9060

cgaaccaatc caaaccgata tagttggttt ggtttgattt tgatataaac cgaaccaact 9120

cggtccattt gcacccctaa tcataatagc tttaatattt caagatatta ttaagttaac 9180

gttgtcaata tcctggaaat tttgcaaaat gaatcaagcc tatatggctg taatatgaat 9240

ttaaaagcag ctcgatgtgg tggtaatatg taatttactt gattctaaaa aaatatccca 9300

agtattaata atttctgcta ggaagaaggt tagctacgat ttacagcaaa gccagaatac 9360

aaagaaccat aaagtgattg aagctcgaaa tatacgaagg aacaaatatt tttaaaaaaa 9420

tacgcaatga cttggaacaa aagaaagtga tatatttttt gttcttaaac aagcatcccc 9480

tctaaagaat ggcagttttc ctttgcatgt aactattatg ctcccttcgt tacaaaaatt 9540

ttggactact attgggaact tcttctgaaa atagtgatag aacccacacg agcatgtgct 9600

ttccatttaa ttttaaaaac caagaaacat acatacataa cattccatca gcctctctct 9660

ctttttatta cggttaatga cttaaaacac atcttattat cccatcctta acacctagca 9720

gtgtctttat acgatctcat cgatcaccac ttcaaaacca tgcagactgc tgctgcccct 9780

ggagctggca tcggctaggc tgggtgccgc actgtcccgg aaggtcccta gcgacttgtt 9840

tagattgatg ggaccacctc tcaacttcct gctgctgtcc ctgctgctgg atgtcctgcc 9900

tcatctggcc gattgcacgc tccagtcccc tgcatgtgca ctcgctcctc aattgcttaa 9960

gatcatcgca gcagctatcg aagtgctggc tctgttgccc tcctccacgg ccttggttgt 10020

agtagtagct gccgccgccc ttctggactt tttcccacag gaaccgccga ataattcgat 10080

agaaccacac gagcatgtgc tttcatttat tttaaaaacc aagaaacata cataacattt 10140

catcagcctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctt 10200

tattacagct gttacactaa cttaaaacac attcatctca ttattattat tattatccat 10260

ccttaacacc tagcagtgtc tttgtacgat ctcataatcg atcacccctt catcaggtat 10320

ccttaggctt cactccaacg ttgttgcagt tacggaacat gtacacacca tcatggttct 10380

caacgaactg gcaagatctc caagttttcc aaaggctaac ccacatgttc tcatcggtgt 10440

gtctgtagtg ctctcccata actttcttga tgcactcggt agcttctcta gcatggtaga 10500

atgggatcct tgaaacgtag tgatggagca catgagtctc gatgatgtca tggaagatga 10560

ttccgaggat tccgaactct ctatcgatag tagcagcagc acccttagcg aaagtccact 10620

cttgagcatc gtaatgaggc atagaagaat cggtgtgctg aaggaaggta acgaaaacaa 10680

gccagtggtt aacaaggatc caaggacaga accatgtgat gaaagtaggc cagaatccga 10740

aaaccttgta agcggtgtaa acagaagtga gggtagcaag gattccaaga tcagaaagaa 10800

cgatgtacca gtagtccttc ttatcgaaaa cagggctaga aggccagtag tgagacttga 10860

agaacttaga aacaccaggg taaggttgtc cagtagcgtt agtagcaagg taaagagaaa 10920

gtcctccaag ctgttggaac aagagagcga aaacagagta gataggagtt tcctcagcga 10980

tatcgtgaag gctggtaact tggtgcttct ctttgaattc ctcggcggtg taaggaacga 11040

aaaccatatc tctggtcatg tgtccagtag ccttatggtg cttagcatga gagaacttcc 11100

agctgaagta aggaaccata acaagagagt ggagaaccca tccaacggta tcgttaaccc 11160

atccgtagtt agagaaagca gaatgtccac actcatgtcc aaggatccag attccgaatc 11220

cgaaacaaga gatagagaac acgtaagcag accaagcagc gaatctaagg aattcgttag 11280

ggagaagagg gatgtaggta agtccaacgt aagcgatagc agagatagcc acgatatctc 11340

tcaccacgta agacatagac ttcacgagag atctctcgta acagtgctta gggatagcgt 11400

caaggatatc cttgatggtg taatctggca ccttgaaaac gtttccgaag gtatcgatag 11460

cggtcttttg ctgcttgaaa gatgcaacgt ttccagaacg cctaacggtc ttagtagatc 11520

cctcaaggat ctcagatcca gacacggtaa ccttagacat ggtatggtaa ttgtaaatgt 11580

aattgtaatg ttgtttgttg tttgttgttg ttggtaattg ttgtaaaatt tttggtggtg 11640

attggttctt taaggtgtga gagtgagttg tgagttgtgt ggtgggtttg gtgagattgg 11700

ggatggtggg tttatatagt ggagactgag gaatggggtc gtgagtgtta actttgcatg 11760

ggctacacgt gggttctttt gggcttacac gtagtattat tcatgcaaat gcagccaata 11820

catatacggt attttaataa tgtgtgggaa tacaatatgc cgagtatttt actaattttg 11880

gcaatgacaa gtgtacattt ggattatctt acttggcctc tcttgcttta atttggatta 11940

tttttattct cttaccttgg ccgttcatat tcacatccct aaaggcaaga cagaattgaa 12000

tggtggccaa aaattaaaac gatggatatg acctacatag tgtaggatca attaacgtcg 12060

aaggaaaata ctgattctct caagcatacg gacaagggta aataacatag tcaccagaac 12120

ataataaaca aaaagtgcag aagcaagact aaaaaaatta gctatggaca ttcaggttca 12180

tattggaaac atcattatcc tagtcttgtg accatccttc ctcctgctct agttgagagg 12240

ccttgggact aacgagaggt cagttgggat agcagatcct tatcctggac tagcctttct 12300

ggtgtttcag agtcttcgtg ccgccgtcta catctatctc cattaggtct gaagatgact 12360

cttcacacca acgacgttta aggtctctat cctactccta gcttgcaata cctggcttgc 12420

aatacctgga gcatcgtgca cgatgattgg atactgtgga ggaggagtgt ttgctgattt 12480

agagctcccg gttgggtgat ttgacttcga tttcagttta ggcttgttga aatttttcag 12540

gttccattgt gaagccttta gagcttgagc ttccttccat gttaatgcct tgatcgaata 12600

ctcctagaga aaagggaagt cgatctctga gtattgaaat cgaagtgcac attttttttc 12660

aacgtgtcca atcaatccac aaacaaagca gaagacaggt aatctttcat acttatactg 12720

acaagtaata gtcttaccgt catgcataat aacgtctcgt tccttcaaga ggggttttcc 12780

gacatccata acgacccgaa gcctcatgaa agcattaggg aagaactttt ggttcttctt 12840

gtcatggcct ttataggtgt cagccgagct cgccaattcc cgtccgactg gctccgcaaa 12900

atattcgaac ggcaagttat ggacttgcaa ccataactcc acggtattga gcaggaccta 12960

ttgtgaagac tcatctcatg gagcttcaga atgtggttgt cagcaaacca atgaccgaaa 13020

tccatcacat gacggacgtc cagtgggtga gcgaaacgaa acaggaagcg cctatctttc 13080

agagtcgtga gctccacacc ggattccggc aactacgtgt tgggcaggct tcgccgtatt 13140

agagatatgt tgaggcagac ccatctgtgc cactcgtaca attacgagag ttgttttttt 13200

tgtgattttc ctagtttctc gttgatggtg agctcatatt ctacatcgta tggtctctca 13260

acgtcgtttc ctgtcatctg atatcccgtc atttgcatcc acgtgcgccg cctcccgtgc 13320

caagtcccta ggtgtcatgc acgccaaatt ggtggtggtg cgggctgccc tgtgcttctt 13380

accgatgggt ggaggttgag tttgggggtc tccgcggcga tggtagtggg ttgacggttt 13440

ggtgtgggtt gacggcattg atcaatttac ttcttgcttc aaattctttg gcagaaaaca 13500

attcattaga ttagaactgg aaaccagagt gatgagacgg attaagtcag attccaacag 13560

agttacatct cttaagaaat aatgtaaccc ctttagactt tatatatttg caattaaaaa 13620

aataatttaa cttttagact ttatatatag ttttaataac taagtttaac cactctatta 13680

tttatatcga aactatttgt atgtctcccc tctaaataaa cttggtattg tgtttacaga 13740

acctataatc aaataatcaa tactcaactg aagtttgtgc agttaattga agggattaac 13800

ggccaaaatg cactagtatt atcaaccgaa tagattcaca ctagatggcc atttccatca 13860

atatcatcgc cgttcttctt ctgtccacat atcccctctg aaacttgaga gacacctgca 13920

cttcattgtc cttattacgt gttacaaaat gaaacccatg catccatgca aactgaagaa 13980

tggcgcaaga acccttcccc tccatttctt atgtggcgac catccatttc accatctccc 14040

gctataaaac acccccatca cttcacctag aacatcatca ctacttgctt atccatccaa 14100

aagataccca cttttacaac aattaccaac aacaacaaac aacaaacaac attacaatta 14160

catttacaat taccatacca tgccacctag cgctgctaag caaatgggag cttctactgg 14220

tgttcatgct ggtgttactg actcttctgc tttcaccaga aaggatgttg ctgatagacc 14280

tgatctcacc atcgttggag attctgttta cgatgctaag gctttcagat ctgagcatcc 14340

tggtggtgct catttcgttt ctttgttcgg aggaagagat gctactgagg ctttcatgga 14400

ataccataga agggcttggc ctaagtctag aatgtctaga ttccacgttg gatctcttgc 14460

ttctactgag gaacctgttg ctgctgatga gggatacctt caactttgtg ctaggatcgc 14520

taagatggtg ccttctgttt cttctggatt cgctcctgct tcttactggg ttaaggctgg 14580

acttatcctt ggatctgcta tcgctcttga ggcttacatg ctttacgctg gaaagagact 14640

tctcccttct atcgttcttg gatggctttt cgctcttatc ggtcttaaca tccagcatga 14700

tgctaaccat ggtgctttgt ctaagtctgc ttctgttaac cttgctcttg gactttgtca 14760

ggattggatc ggaggatcta tgatcctttg gcttcaagag catgttgtta tgcaccacct 14820

ccacactaac gatgttgata aggatcctga tcaaaaggct cacggtgctc ttagactcaa 14880

gcctactgat gcttggtcac ctatgcattg gcttcagcat ctttaccttt tgcctggtga 14940

gactatgtac gctttcaagc ttttgttcct cgacatctct gagcttgtta tgtggcgttg 15000

ggagggtgag cctatctcta agcttgctgg atacctcttt atgccttctt tgcttctcaa 15060

gcttaccttc tgggctagat tcgttgcttt gcctctttac cttgctcctt ctgttcatac 15120

tgctgtgtgt atcgctgcta ctgttatgac tggatctttc tacctcgctt tcttcttctt 15180

catctcccac aacttcgagg gtgttgcttc tgttggacct gatggatcta tcacttctat 15240

gactagaggt gctagcttcc ttaagagaca agctgagact tcttctaacg ttggaggacc 15300

tcttcttgct actcttaacg gtggactcaa ctaccaaatt gagcatcact tgttccctag 15360

agttcaccat ggattctacc ctagacttgc tcctcttgtt aaggctgagc ttgaggctag 15420

aggaatcgag tacaagcact accctactat ctggtctaac cttgcttcta ccctcagaca 15480

tatgtacgct cttggaagaa ggcctagatc taaggctgag taatgacaag cttatgtgac 15540

gtgaaataat aacggtaaaa tatatgtaat aataataata ataaagccac aaagtgagaa 15600

tgaggggaag gggaaatgtg taatgagcca gtagccggtg gtgctaattt tgtatcgtat 15660

tgtcaataaa tcatgaattt tgtggttttt atgtgttttt ttaaatcatg aattttaaat 15720

tttataaaat aatctccaat cggaagaaca acattccata tccatgcatg gatgtttctt 15780

tacccaaatc tagttcttga gaggatgaag catcaccgaa cagttctgca actatccctc 15840

aaaagcttta aaatgaacaa caaggaacag agcaacgttc caaagatccc aaacgaaaca 15900

tattatctat actaatacta tattattaat tactactgcc cggaatcaca atccctgaat 15960

gattcctatt aactacaagc cttgttggcg gcggagaagt gatcggcgcg gcgagaagca 16020

gcggactcgg agacgaggcc ttggaagatc tgagtcgaac gggcagaatc agtattttcc 16080

ttcgacgtta attgatccta cactatgtag gtcatatcca tcgttttaat ttttggccac 16140

cattcaattc tgtcttgcct ttagggatgt gaatatgaac ggccaaggta agagaataaa 16200

aataatccaa attaaagcaa gagaggccaa gtaagataat ccaaatgtac acttgtcatt 16260

gccaaaatta gtaaaatact cggcatattg tattcccaca cattattaaa ataccgtata 16320

tgtattggct gcatttgcat gaataatact acgtgtaagc ccaaaagaac ccacgtgtag 16380

cccatgcaaa gttaacactc acgaccccat tcctcagtct ccactatata aacccaccat 16440

ccccaatctc accaaaccca ccacacaact cacaactcac tctcacacct taaagaacca 16500

atcaccacca aaaattttac aacaattacc aacaacaaca aacaacaaac aacattacaa 16560

ttacatttac aattaccata ccatgagcgc tgttaccgtt actggatctg atcctaagaa 16620

cagaggatct tctagcaaca ccgagcaaga ggttccaaaa gttgctatcg ataccaacgg 16680

aaacgtgttc tctgttcctg atttcaccat caaggacatc cttggagcta tccctcatga 16740

gtgttacgag agaagattgg ctacctctct ctactacgtg ttcagagata tcttctgcat 16800

gcttaccacc ggatacctta cccataagat cctttaccct ctcctcatct cttacacctc 16860

taacagcatc atcaagttca ctttctgggc cctttacact tacgttcaag gacttttcgg 16920

aaccggaatc tgggttctcg ctcatgagtg tggacatcaa gctttctctg attacggaat 16980

cgtgaacgat ttcgttggat ggacccttca ctcttacctt atggttcctt acttcagctg 17040

gaagtactct catggaaagc accataaggc tactggacac atgaccagag atatggtttt 17100

cgttcctgcc accaaagagg aattcaagaa gtctaggaac ttcttcggta acctcgctga 17160

gtactctgag gattctccac ttagaaccct ttacgagctt cttgttcaac aacttggagg 17220

atggatcgct tacctcttcg ttaacgttac aggacaacct taccctgatg ttccttcttg 17280

gaaatggaac cacttctggc ttacctctcc acttttcgag caaagagatg ctctctacat 17340

cttcctttct gatcttggaa tcctcaccca gggaatcgtt cttactcttt ggtacaagaa 17400

attcggagga tggtcccttt tcatcaactg gttcgttcct tacatctggg ttaaccactg 17460

gctcgttttc atcacattcc ttcagcacac tgatcctact atgcctcatt acaacgctga 17520

ggaatggact ttcgctaagg gtgctgctgc tactatcgat agaaagttcg gattcatcgg 17580

acctcacatc ttccatgata tcatcgagac tcatgtgctt caccactact gttctaggat 17640

cccattctac aacgctagac ctgcttctga ggctatcaag aaagttatgg gaaagcacta 17700

caggtctagc gacgagaaca tgtggaagtc actttggaag tctttcaggt cttgccaata 17760

cgttgacggt gataacggtg ttctcatgtt ccgtaacatc aacaactgcg gagttggagc 17820

tgctgagaag taatgaaggg gtgatcgatt atgagatcgt acaaagacac tgctaggtgt 17880

taaggatgga taataataat aataatgaga tgaatgtgtt ttaagttagt gtaacagctg 17940

taataaagag agagagagag agagagagag agagagagag agagagagag agagagaggc 18000

tgatgaaatg ttatgtatgt ttcttggttt ttaaaataaa tgaaagcaca tgctcgtgtg 18060

gttctatcga attattcggc ggttcctgtg ggaaaaagtc cagaagggcc gccgcagcta 18120

ctactacaac caaggccgtg gaggagggca acagagccag cacttcgata gctgctgcga 18180

tgatcttaag caattgagga gcgagtgcac atgcagggga ctggagcgtg caatcggcca 18240

gatgaggcag gacatccagc agcagggaca gcagcaggaa gttgagaggt ggtcccatca 18300

atctaaacaa gtcgctaggg accttccggg acagtgcggc acccagccta gccgatgcca 18360

gctccagggg cagcagcagt ctgcatggtt ttgaagtggt gatcgatgag atcgtataaa 18420

gacactgcta ggtgttaagg atgggataat aagatgtgtt ttaagtcatt aaccgtaata 18480

aaaagagaga gaggctgatg gaatgttatg tatgtatgtt tcttggtttt taaaattaaa 18540

tggaaagcac atgctcgtgt gggttctatc tcgattaaaa atcccaatta tatttggtct 18600

aatttagttt ggtattgagt aaaacaaatt cgaaccaaac caaaatataa atatatagtt 18660

tttatatata tgcctttaag actttttata gaattttctt taaaaaatat ctagaaatat 18720

ttgcgactct tctggcatgt aatatttcgt taaatatgaa gtgctccatt tttattaact 18780

ttaaataatt ggttgtacga tcactttctt atcaagtgtt actaaaatgc gtcaatctct 18840

ttgttcttcc atattcatat gtcaaaatct atcaaaattc ttatatatct ttttcgaatt 18900

tgaagtgaaa tttcgataat ttaaaattaa atagaacata tcattattta ggtatcatat 18960

tgatttttat acttaattac taaatttggt taactttgaa agtgtacatc aacgaaaaat 19020

tagtcaaacg actaaaataa ataaatatca tgtgttatta agaaaattct cctataagaa 19080

tattttaata gatcatatgt ttgtaaaaaa aattaatttt tactaacaca tatatttact 19140

tatcaaaaat ttgacaaagt aagattaaaa taatattcat ctaacaaaaa aaaaaccaga 19200

aaatgctgaa aacccggcaa aaccgaacca atccaaaccg atatagttgg tttggtttga 19260

ttttgatata aaccgaacca actcggtcca tttgcacccc taatcataat agctttaata 19320

tttcaagata ttattaagtt aacgttgtca atatcctgga aattttgcaa aatgaatcaa 19380

gcctatatgg ctgtaatatg aatttaaaag cagctcgatg tggtggtaat atgtaattta 19440

cttgattcta aaaaaatatc ccaagtatta ataatttctg ctaggaagaa ggttagctac 19500

gatttacagc aaagccagaa tacaaagaac cataaagtga ttgaagctcg aaatatacga 19560

aggaacaaat atttttaaaa aaatacgcaa tgacttggaa caaaagaaag tgatatattt 19620

tttgttctta aacaagcatc ccctctaaag aatggcagtt ttcctttgca tgtaactatt 19680

atgctccctt cgttacaaaa attttggact actattggga acttcttctg aaaatagtcc 19740

tgcaggctag tagattggtt ggttggtttc catgtaccag aaggcttacc ctattagttg 19800

aaagttgaaa ctttgttccc tactcaattc ctagttgtgt aaatgtatgt atatgtaatg 19860

tgtataaaac gtagtactta aatgactagg agtggttctt gagaccgatg agagatggga 19920

gcagaactaa agatgatgac ataattaaga acgaatttga aaggctctta ggtttgaatc 19980

ctattcgaga atgtttttgt caaagatagt ggcgattttg aaccaaagaa aacatttaaa 20040

aaatcagtat ccggttacgt tcatgcaaat agaaagtggt ctaggatctg attgtaattt 20100

tagacttaaa gagtctctta agattcaatc ctggctgtgt acaaaactac aaataatata 20160

ttttagacta tttggcctta actaaacttc cactcattat ttactgaggt tagagaatag 20220

acttgcgaat aaacacattc ccgagaaata ctcatgatcc cataattagt cagagggtat 20280

gccaatcaga tctaagaaca cacattccct caaattttaa tgcacatgta atcatagttt 20340

agcacaattc aaaaataatg tagtattaaa gacagaaatt tgtagacttt tttttggcgt 20400

taaaagaaga ctaagtttat acgtacattt tattttaagt ggaaaaccga aattttccat 20460

cgaaatatat gaatttagta tatatatttc tgcaatgtac tattttgcta ttttggcaac 20520

tttcagtgga ctactacttt attacaatgt gtatggatgc atgagtttga gtatacacat 20580

gtctaaatgc atgctttgta aaacgtaacg gaccacaaaa gaggatccat acaaatacat 20640

ctcatagctt cctccattat tttccgacac aaacagagca ttttacaaca attaccaaca 20700

acaacaaaca acaaacaaca ttacaattac atttacaatt accataccat ggaatttgct 20760

caacctctcg ttgctatggc tcaagagcag tacgctgcta tcgatgctgt tgttgctcct 20820

gctatcttct ctgctaccga ctctattgga tggggactca agcctatctc ttctgctact 20880

aaggatctcc ctctcgttga atctcctacc cctcttatcc tttctctcct cgcttacttc 20940

gctatcgttg gttctggact cgtttaccgt aaagtgttcc ctagaaccgt taagggacag 21000

gatcctttcc ttctcaaggc tcttatgctc gctcacaacg ttttccttat cggactcagc 21060

ctttacatgt gcctcaagct cgtttacgag gcttacgtga acaagtactc cttctgggga 21120

aacgcttaca accctgctca aaccgagatg gctaaggtga tctggatctt ctacgtgtcc 21180

aagatctacg agttcatgga caccttcatc atgcttctca agggaaacgt taaccaggtt 21240

tccttcctcc atgtttacca ccacggatct atctctggaa tctggtggat gatcacttat 21300

gctgctccag gtggagatgc ttacttctct gctgctctca actcttgggt tcatgtgtgc 21360

atgtacacct actacttcat ggctgctgtt cttcctaagg acgaaaagac caagagaaag 21420

tacctttggt ggggaagata ccttacccag atgcaaatgt tccagttctt catgaacctt 21480

ctccaggctg tttacctcct ctactcttct tctccttacc ctaagttcat tgctcaactc 21540

ctcgttgttt acatggttac cctcctcatg cttttcggaa acttctacta catgaagcac 21600

cacgcttcta agtgataagg gccgccgcca tgtgacagat cgaaggaaga aagtgtaata 21660

agacgactct cactactcga tcgctagtga ttgtcattgt tatatataat aatgttatct 21720

ttcacaactt atcgtaatgc atgtgaaact ataacacatt aatcctactt gtcatatgat 21780

aacactctcc ccatttaaaa ctcttgtcaa tttaaagata taagattctt taaatgatta 21840

aaaaaaatat attataaatt caatcactcc tactaataaa ttattaatta ttatttattg 21900

attaaaaaaa tacttatact aatttagtct gaatagaata attagattct agcctgcagg 21960

gcggccgcgg atcccatgga gtcaaagatt caaatagagg acctaacaga actcgccgta 22020

aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc 22080

gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca 22140

gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga 22200

ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc 22260

tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc tgccgacagt 22320

ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 22380

acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa 22440

tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagaga 22500

acacggggga ctgaattaaa tatgagccct gagaggcgtc ctgttgaaat cagacctgct 22560

actgctgctg atatggctgc tgtttgtgat atcgtgaacc actacatcga gacttctacc 22620

gttaacttca gaactgagcc tcaaactcct caagagtgga tcgatgatct tgagagactc 22680

caagatagat acccttggct tgttgctgag gttgagggtg ttgttgctgg aatcgcttac 22740

gctggacctt ggaaggctag aaacgcttac gattggactg ttgagtctac cgtttacgtt 22800

tcacacagac atcagagact tggacttgga tctacccttt acactcacct tctcaagtct 22860

atggaagctc agggattcaa gtctgttgtt gctgttatcg gactccctaa cgatccttct 22920

gttagacttc atgaggctct tggatacact gctagaggaa ctcttagagc tgctggatac 22980

aagcacggtg gatggcatga tgttggattc tggcaaagag atttcgagct tcctgctcct 23040

cctagacctg ttagaccagt tactcagatc tgaatttgcg tgatcgttca aacatttggc 23100

aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc 23160

tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat 23220

gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat 23280

agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcactagt 23340

gatgtacggt taaaaccacc ccagtacatt aaaaacgtcc gcaatgtgtt attaagttgt 23400

ctaagcgtca atttgtttac accacaatat atcctgccac cagccagcca acagctcccc 23460

gaccggcagc tcggcacaaa atcaccactc gatacaggca gcccatcagt cc 23512

<210> SEQ ID NO: 3

<211> LENGTH: 25787

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pGA7- mod_C nucleotide sequence

<400> SEQENCE: 3

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgccccgatc tagtaacata gatgacaccg cgcgcgataa 240

tttatcctag tttgcgcgct atattttgtt ttctatcgcg tattaaatgt ataattgcgg 300

gactctaatc ataaaaaccc atctcataaa taacgtcatg cattacatgt taattattac 360

gtgcttaacg taattcaaca gaaattatat gataatcatc gcaagaccgg caacaggatt 420

caatcttaag aaactttatt gccaaatgtt tgaacgatct gcccggaagc ggccaactcg 480

aaaatttaat taatcatcag tgagccttct cagcctttcc gttaacgtag tagtgctgtc 540

caactttgtc gaggttgctg aaagtagcct tccaagcacc gtagtaagag agcaccttgt 600

agttgagtcc ccacttctta gcgaaaggga cgaatcttct tgacacctca ggctgtctga 660

attgaggcat atcagggaag agatggtgga taacctggca gttaaggtat cccataagcc 720

agttaacgta tccacgagaa ggatcgatgt caacggtgtg atcaacagcg tagttaaccc 780

agctaaggtg cttgtcagat ggaacaacag ggaggtgagt gtgagaagta gagaagtgag 840

cgaagaggta catgtaagcg atccagtttc cgaaagtgaa ccaccagtaa gcaacaggcc 900

aagagtatcc ggtagcaagc ttgataacag cggttctaac aacgtgagaa acgagcatcc 960

aagaagcttc ctcgtagttc ttcttcctga gcacctgtct aggatggaga acgtagatcc 1020

agaaagcctg aacgagaagt ccagaagtaa caggaacgaa ggtccaagct tgaagtctag 1080

cccaagctct agagaatccc ctaggtctat tatcctccac agcggtgttg aagaaagcca 1140

cagcaggagt ggtatcaaga tccatgtcgt gtctaacttt ctgaggggta gcatggtgct 1200

tgttatgcat ctggttccac atctctccgc tggtagaaag tccgaatccg caagtcatag 1260

cctgaagtct cttatccacg tacacagatc cggtaagaga gttgtgtcca ccctcatgtt 1320

gaacccatcc acatctagct ccgaagaaag caccgtacac aacgctagca atgatagggt 1380

atccagcgta cataagagcg gttccaagag cgaaagtagc aagaagctcc aaaagacggt 1440

aagcaacatg ggtgatagaa ggcttgaaga atccgtccct ctcaagttca gctctccacc 1500

tagcgaaatc ctcaagcata ggagcatcct cagactcaga tctcttgatc tcagcaggtc 1560

tagaaggcaa agctctaagc atcttccaag ccttgaggct acgcatgtga aattctttga 1620

aagcctcagt agcatcagca ccagtgttag caagcatgta gaagatcacg cttccaccag 1680

gatgtttgaa gttggtcacg tcgtactcaa catcctcaac cctaacccat ctagtctcga 1740

aggtagcagc aagttcatga ggctcaaggg tcttaagatc aacaggagcg gtagaagcat 1800

ccttagcatc aagagcctca gcagatgact tagacctggt gagaggagat ctaggagaag 1860

atcttccatc ggtcttagga ggacacatgg cgcgccgatt ttcgagatgg taattgtaaa 1920

tgtaattgta atgttgtttg ttgtttgttg ttgttggtaa ttgttgtaaa attcgagttg 1980

gccgcttccg gggatcctcg tgtatgtttt taatcttgtt tgtatcgatg agttttggtt 2040

tgagtaaaga gtgaagcgga tgagttaatt tataggctat aaaggagatt tgcatggcga 2100

tcacgtgtaa taatgcatgc acgcatgtga ttgtatgtgt gtgctgtgag agagaagctc 2160

ttaggtgttt gaagggagtg acaagtggcg aagaaaaaca attctccgcg gctgcatgct 2220

atgtgtaacg tgtagctaat gttctggcat ggcatcttat gaacgattct ttttaaaaac 2280

aaggtaaaaa cttaacttca taaaattaaa aaaaaaaacg tttactaagt tggtttaaaa 2340

ggggatgaga ggcgccccgc ggaaagcttg ctagccaatt ggggcccaac gttctcgagt 2400

ttttctagaa ggaaactgaa ggcgggaaac gacaatctgc tagtggatct cccagtcacg 2460

acgttgtaaa acgggcgccc cgcggaaagc ttgcggccgc ggtaccgccc gttcgactca 2520

gatcttccaa ggcctcgtct ccgagtccgc tgcttctcgc cgcgccgatc acttctccgc 2580

cgccaacaag gcttgtagtt aataggaatc attcagggat tgtgattccg ggcagtagta 2640

attaataata tagtattagt atagataata tgtttcgttt gggatctttg gaacgttgct 2700

ctgttccttg ttgttcattt taaagctttt gagggatagt tgcagaactg ttcggtgatg 2760

cttcatcctc tcaagaacta gatttgggta aagaaacatc catgcatgga tatggaatgt 2820

tgttcttccg attggagatt attttataaa atttaaaatt catgatttaa aaaaacacat 2880

aaaaaccaca aaattcatga tttattgaca atacgataca aaattagcac caccggctac 2940

tggctcatta cacatttccc cttcccctca ttctcacttt gtggctttat tattattatt 3000

attacatata ttttaccgtt attatttcac gtcacataag cttgttaatt aatcattagt 3060

gagccttctc agcctttccg ttaacgtagt agtgctgtcc caccttatca aggttagaga 3120

aagtagcctt ccaagcaccg tagtaagaga gcaccttgta gttgagtccc cacttcttag 3180

cgaaaggaac gaatcttctg ctaacctcag gctgtctgaa ttgaggcata tcagggaaga 3240

ggtggtggat aacctgacag ttaaggtatc ccataagcca gttcacgtat cctctagaag 3300

gatcgatatc aacggtgtga tcaacagcgt agttaaccca agaaaggtgc ttatcagatg 3360

gaacaacagg gaggtgagta tgagaagtag agaagtgagc gaaaaggtac atgtaagcga 3420

tccagtttcc gaaagtgaac caccagtaag caacaggcca agagtatcca gtagcaagct 3480

tgataacagc ggttctaaca acatgagaaa cgagcatcca agaagcctct tcgtagttct 3540

tcttacggag aacttgtcta gggtggagaa cgtagatcca gaaagcttga acaagaagtc 3600

cagaggtaac aggaacgaaa gtccaagctt gaagtctagc ccaagctcta gagaatcctc 3660

taggtctgtt atcctcaaca gcagtgttga agaaagccac agcaggagtg gtatcaagat 3720

ccatatcgtg tctaaccttt tgaggggtag catggtgctt gttatgcatc tggttccaca 3780

tctcaccaga agtagaaagt ccgaatccac aagtcatagc ctgaagtctc ttgtccacgt 3840

aaacagatcc ggtaagagag ttatgtccac cctcatgttg aacccatcca catctagctc 3900

cgaagaaagc accgtaaaca acagaagcaa tgatagggta tccagcgtac ataagagcag 3960

ttccaagagc gaatgtagca agaagctcga gaagtctgta agccacatgg gtgatagaag 4020

gcttgaagaa tccatctctc tcaagctcag cacgccatct agcgaaatcc tcaagcatag 4080

gagcatcctc agactcagat ctcttgatct cagcaggtct agaaggcaaa gctctaagca 4140

tcttccaagc cttgagagaa cgcatgtgga attctttgaa agcctcagta gcatcagcac 4200

cagtgttagc aagcatgtag aagatcacag atccaccagg gtgcttgaag ttagtcacat 4260

cgtactcaac gtcctcaact ctaacccatc tagtctcgaa agtagcagca agctcatgag 4320

gctcaagagt cttaagatca acaggagcag tagaagcatc cttagcatca agagcctcag 4380

cagaagattt agacctggta agtggagatc taggagaaga tcttccatca gtcttaggag 4440

ggcacatggt atggtaattg taaatgtaat tgtaatgttg tttgttgttt gttgttgttg 4500

gtaattgttg taaaattaat taagtgggta tcttttggat ggataagcaa gtagtgatga 4560

tgttctaggt gaagtgatgg gggtgtttta tagcgggaga tggtgaaatg gatggtcgcc 4620

acataagaaa tggaggggaa gggttcttgc gccattcttc agtttgcatg gatgcatggg 4680

tttcattttg taacacgtaa taaggacaat gaagtgcagg tgtctctcaa gtttcagagg 4740

ggatatgtgg acagaagaag aacggcgatg atattgatgg aaatggccat ctagtgtgaa 4800

tctattcggt tgataatact agtgcatttt ggccgttaat cccttcaatt aactgcacaa 4860

acttcagttg agtattgatt atttgattat aggttctgta aacacaatac caagtttatt 4920

tagaggggag acatacaaat agtttcgata taaataatag agtggttaaa cttagttatt 4980

aaaactatat ataaagtcta aaagttaaat tattttttta attgcaaata tataaagtct 5040

aaaggggtta cattatttct taagagatgt aactctgttg gaatctgact taatccgtct 5100

catcactctg gtttccagtt ctaatctaat gaattgtttt ctgccaaaga atttgaagca 5160

agaagtaaat tgatcaatgc cgtcaaccca caccaaaccg tcaacccact accatcgccg 5220

cggagacccc caaactcaac ctccacccat cggtaagaag cacagggcag cccgcaccac 5280

caccaatttg gcgtgcatga cacctaggga cttggcacgg gaggcggcgc acgtggatgc 5340

aaatgacggg atatcagatg acaggaaacg acgttgagag accatacgat gtagaatatg 5400

agctcaccat caacgagaaa ctaggaaaat cacaaaaaaa acaactctcg taattgtacg 5460

agtggcacag atgggtctgc ctcaacatat ctctaatacg gcgaagcctg cccaacacgt 5520

agttgccgga atccggtgtg gagctcacga ctctgaaaga taggcgcttc ctgtttcgtt 5580

tcgctcaccc actggacgtc cgtcatgtga tggatttcgg tcattggttt gctgacaacc 5640

acattctgaa gctccatgag atgagtcttc acaataggtc ctgctcaata ccgtggagtt 5700

atggttgcaa gtccataact tgccgttcga atattttgcg gagccagtcg gacgggaatt 5760

ggcgagctcg gctgacacct ataaaggcca tgacaagaag aaccaaaagt tcttccctaa 5820

tgctttcatg aggcttcggg tcgttatgga tgtcggaaaa cccctcttga aggaacgaga 5880

cgttattatg catgacggta agactattac ttgtcagtat aagtatgaaa gattacctgt 5940

cttctgcttt gtttgtggat tgattggaca cgttgaaaaa aaatgtgcac ttcgatttca 6000

atactcagag atcgacttcc cttttctcta ggagtattcg atcaaggcat taacatggaa 6060

ggaagctcaa gctctaaagg cttcacaatg gaacctgaaa aatttcaaca agcctaaact 6120

gaaatcgaag tcaaatcacc caaccgggag ctctaaatca gcaaacactc ctcctccaca 6180

gtatccaatc atcgtgcacg atgctccagg tattgcaagc caggtattgc aagctaggag 6240

taggatagag accttaaacg tcgttggtgt gaagagtcat cttcagacct aatggagata 6300

gatgtagacg gcggcacgaa gactctgaaa caccagaaag gctagtccag gataaggatc 6360

tgctatccca actgacctct cgttagtccc aaggcctctc aactagagca ggaggaagga 6420

tggtcacaag actaggataa tgatgtttcc aatatgaacc tgaatgtcca tagctaattt 6480

ttttagtctt gcttctgcac tttttgttta ttatgttctg gtgactatgt tatttaccct 6540

tgtccgtatg cttgagggta ccctagtaga ttggttggtt ggtttccatg taccagaagg 6600

cttaccctat tagttgaaag ttgaaacttt gttccctact caattcctag ttgtgtaaat 6660

gtatgtatat gtaatgtgta taaaacgtag tacttaaatg actaggagtg gttcttgaga 6720

ccgatgagag atgggagcag aactaaagat gatgacataa ttaagaacga atttgaaagg 6780

ctcttaggtt tgaatcctat tcgagaatgt ttttgtcaaa gatagtggcg attttgaacc 6840

aaagaaaaca tttaaaaaat cagtatccgg ttacgttcat gcaaatagaa agtggtctag 6900

gatctgattg taattttaga cttaaagagt ctcttaagat tcaatcctgg ctgtgtacaa 6960

aactacaaat aatatatttt agactatttg gccttaacta aacttccact cattatttac 7020

tgaggttaga gaatagactt gcgaataaac acattcccga gaaatactca tgatcccata 7080

attagtcaga gggtatgcca atcagatcta agaacacaca ttccctcaaa ttttaatgca 7140

catgtaatca tagtttagca caattcaaaa ataatgtagt attaaagaca gaaatttgta 7200

gacttttttt tggcgttaaa agaagactaa gtttatacgt acattttatt ttaagtggaa 7260

aaccgaaatt ttccatcgaa atatatgaat ttagtatata tatttctgca atgtactatt 7320

ttgctatttt ggcaactttc agtggactac tactttatta caatgtgtat ggatgcatga 7380

gtttgagtat acacatgtct aaatgcatgc tttgtaaaac gtaacggacc acaaaagagg 7440

atccatacaa atacatctca tagcttcctc cattattttc cgacacaaac agagcatttt 7500

acaacaatta ccaacaacaa caaacaacaa acaacattac aattacattt acaattacca 7560

taccatggcc tctatcgcta tccctgctgc tcttgctgga actcttggat acgttaccta 7620

caatgtggct aaccctgata tcccagcttc tgagaaagtt cctgcttact tcatgcaggt 7680

tgagtactgg ggacctacta tcggaactat tggatacctc ctcttcatct acttcggaaa 7740

gcgtatcatg cagaacagat ctcaaccttt cggactcaag aacgctatgc tcgtttacaa 7800

cttctaccag accttcttca acagctactg catctacctt ttcgttactt ctcatagggc 7860

tcagggactt aaggtttggg gaaacatccc tgatatgact gctaactctt ggggaatctc 7920

tcaggttatc tggcttcact acaacaacaa gtacgttgag cttctcgaca ccttcttcat 7980

ggtgatgagg aagaagttcg accagctttc tttccttcac atctaccacc acactcttct 8040

catctggtca tggttcgttg ttatgaagct tgagcctgtt ggagattgct acttcggatc 8100

ttctgttaac accttcgtgc acgtgatcat gtactcttac tacggacttg ctgctcttgg 8160

agttaactgt ttctggaaga agtacatcac ccagatccag atgcttcagt tctgtatctg 8220

tgcttctcac tctatctaca ccgcttacgt tcagaatacc gctttctggc ttccttacct 8280

tcaactctgg gttatggtga acatgttcgt tctcttcgcc aacttctacc gtaagaggta 8340

caagtctaag ggtgctaaga agcagtgata aggcgcgcgg cgcgccgggc cgccgccatg 8400

tgacagatcg aaggaagaaa gtgtaataag acgactctca ctactcgatc gctagtgatt 8460

gtcattgtta tatataataa tgttatcttt cacaacttat cgtaatgcat gtgaaactat 8520

aacacattaa tcctacttgt catatgataa cactctcccc atttaaaact cttgtcaatt 8580

taaagatata agattcttta aatgattaaa aaaaatatat tataaattca atcactccta 8640

ctaataaatt attaattatt atttattgat taaaaaaata cttatactaa tttagtctga 8700

atagaataat tagattctag tctcatcccc ttttaaacca acttagtaaa cgtttttttt 8760

tttaatttta tgaagttaag tttttacctt gtttttaaaa agaatcgttc ataagatgcc 8820

atgccagaac attagctaca cgttacacat agcatgcagc cgcggagaat tgtttttctt 8880

cgccacttgt cactcccttc aaacacctaa gagcttctct ctcacagcac acacatacaa 8940

tcacatgcgt gcatgcatta ttacacgtga tcgccatgca aatctccttt atagcctata 9000

aattaactca tccgcttcac tctttactca aaccaaaact catcgataca aacaagatta 9060

aaaacataca cgaggatctt ttacaacaat taccaacaac aacaaacaac aaacaacatt 9120

acaattacat ttacaattac cataccatgc ctccaaggga ctcttactct tatgctgctc 9180

ctccttctgc tcaacttcac gaagttgata ctcctcaaga gcacgacaag aaagagcttg 9240

ttatcggaga tagggcttac gatgttacca acttcgttaa gagacaccct ggtggaaaga 9300

tcattgctta ccaagttgga actgatgcta ccgatgctta caagcagttc catgttagat 9360

ctgctaaggc tgacaagatg cttaagtctc ttccttctcg tcctgttcac aagggatact 9420

ctccaagaag ggctgatctt atcgctgatt tccaagagtt caccaagcaa cttgaggctg 9480

agggaatgtt cgagccttct cttcctcatg ttgcttacag acttgctgag gttatcgcta 9540

tgcatgttgc tggtgctgct cttatctggc atggatacac tttcgctgga atcgctatgc 9600

ttggagttgt tcagggaaga tgtggatggc ttatgcatga gggtggacat tactctctca 9660

ctggaaacat tgctttcgac agagctatcc aagttgcttg ttacggactt ggatgtggaa 9720

tgtctggtgc ttggtggcgt aaccagcata acaagcacca tgctactcct caaaagcttc 9780

agcacgatgt tgatcttgat acccttcctc tcgttgcttt ccatgagaga atcgctgcta 9840

aggttaagtc tcctgctatg aaggcttggc tttctatgca agctaagctt ttcgctcctg 9900

ttaccactct tcttgttgct cttggatggc agctttacct tcatcctaga cacatgctca 9960

ggactaagca ctacgatgag cttgctatgc tcggaatcag atacggactt gttggatacc 10020

ttgctgctaa ctacggtgct ggatacgttc tcgcttgtta ccttctttac gttcagcttg 10080

gagctatgta catcttctgc aacttcgctg tttctcatac tcacctccct gttgttgagc 10140

ctaacgagca tgctacttgg gttgagtacg ctgctaacca cactactaac tgttctccat 10200

cttggtggtg tgattggtgg atgtcttacc ttaactacca gatcgagcac cacctttacc 10260

cttctatgcc tcaattcaga caccctaaga tcgctcctag agttaagcag cttttcgaga 10320

agcacggact tcactacgat gttagaggat acttcgaggc tatggctgat actttcgcta 10380

accttgataa cgttgcccat gctcctgaga agaaaatgca gtaatgagat cgttcaaaca 10440

tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat 10500

aatttctgtt gaattacgtt aagcacgtaa taattaacat gtaatgcatg acgttattta 10560

tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca 10620

aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc 10680

ggtcgattaa aaatcccaat tatatttggt ctaatttagt ttggtattga gtaaaacaaa 10740

ttcgaaccaa accaaaatat aaatatatag tttttatata tatgccttta agacttttta 10800

tagaattttc tttaaaaaat atctagaaat atttgcgact cttctggcat gtaatatttc 10860

gttaaatatg aagtgctcca tttttattaa ctttaaataa ttggttgtac gatcactttc 10920

ttatcaagtg ttactaaaat gcgtcaatct ctttgttctt ccatattcat atgtcaaaat 10980

ctatcaaaat tcttatatat ctttttcgaa tttgaagtga aatttcgata atttaaaatt 11040

aaatagaaca tatcattatt taggtatcat attgattttt atacttaatt actaaatttg 11100

gttaactttg aaagtgtaca tcaacgaaaa attagtcaaa cgactaaaat aaataaatat 11160

catgtgttat taagaaaatt ctcctataag aatattttaa tagatcatat gtttgtaaaa 11220

aaaattaatt tttactaaca catatattta cttatcaaaa atttgacaaa gtaagattaa 11280

aataatattc atctaacaaa aaaaaaacca gaaaatgctg aaaacccggc aaaaccgaac 11340

caatccaaac cgatatagtt ggtttggttt gattttgata taaaccgaac caactcggtc 11400

catttgcacc cctaatcata atagctttaa tatttcaaga tattattaag ttaacgttgt 11460

caatatcctg gaaattttgc aaaatgaatc aagcctatat ggctgtaata tgaatttaaa 11520

agcagctcga tgtggtggta atatgtaatt tacttgattc taaaaaaata tcccaagtat 11580

taataatttc tgctaggaag aaggttagct acgatttaca gcaaagccag aatacaaaga 11640

accataaagt gattgaagct cgaaatatac gaaggaacaa atatttttaa aaaaatacgc 11700

aatgacttgg aacaaaagaa agtgatatat tttttgttct taaacaagca tcccctctaa 11760

agaatggcag ttttcctttg catgtaacta ttatgctccc ttcgttacaa aaattttgga 11820

ctactattgg gaacttcttc tgaaaatagt gatagaaccc acacgagcat gtgctttcca 11880

tttaatttta aaaaccaaga aacatacata cataacattc catcagcctc tctctctttt 11940

tattacggtt aatgacttaa aacacatctt attatcccat ccttaacacc tagcagtgtc 12000

tttatacgat ctcatcgatc accacttcaa aaccatgcag actgctgctg cccctggagc 12060

tggcatcggc taggctgggt gccgcactgt cccggaaggt ccctagcgac ttgtttagat 12120

tgatgggacc acctctcaac ttcctgctgc tgtccctgct gctggatgtc ctgcctcatc 12180

tggccgattg cacgctccag tcccctgcat gtgcactcgc tcctcaattg cttaagatca 12240

tcgcagcagc tatcgaagtg ctggctctgt tgccctcctc cacggccttg gttgtagtag 12300

tagctgccgc cgcccttctg gactttttcc cacaggaacc gccgaataat tcgatagaac 12360

cacacgagca tgtgctttca tttattttaa aaaccaagaa acatacataa catttcatca 12420

gcctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctttatta 12480

cagctgttac actaacttaa aacacattca tctcattatt attattatta tccatcctta 12540

acacctagca gtgtctttgt acgatctcat aatcgatcac cccttcatca ggtatcctta 12600

ggcttcactc caacgttgtt gcagttacgg aacatgtaca caccatcatg gttctcaacg 12660

aactggcaag atctccaagt tttccaaagg ctaacccaca tgttctcatc ggtgtgtctg 12720

tagtgctctc ccataacttt cttgatgcac tcggtagctt ctctagcatg gtagaatggg 12780

atccttgaaa cgtagtgatg gagcacatga gtctcgatga tgtcatggaa gatgattccg 12840

aggattccga actctctatc gatagtagca gcagcaccct tagcgaaagt ccactcttga 12900

gcatcgtaat gaggcataga agaatcggtg tgctgaagga aggtaacgaa aacaagccag 12960

tggttaacaa ggatccaagg acagaaccat gtgatgaaag taggccagaa tccgaaaacc 13020

ttgtaagcgg tgtaaacaga agtgagggta gcaaggattc caagatcaga aagaacgatg 13080

taccagtagt ccttcttatc gaaaacaggg ctagaaggcc agtagtgaga cttgaagaac 13140

ttagaaacac cagggtaagg ttgtccagta gcgttagtag caaggtaaag agaaagtcct 13200

ccaagctgtt ggaacaagag agcgaaaaca gagtagatag gagtttcctc agcgatatcg 13260

tgaaggctgg taacttggtg cttctctttg aattcctcgg cggtgtaagg aacgaaaacc 13320

atatctctgg tcatgtgtcc agtagcctta tggtgcttag catgagagaa cttccagctg 13380

aagtaaggaa ccataacaag agagtggaga acccatccaa cggtatcgtt aacccatccg 13440

tagttagaga aagcagaatg tccacactca tgtccaagga tccagattcc gaatccgaaa 13500

caagagatag agaacacgta agcagaccaa gcagcgaatc taaggaattc gttagggaga 13560

agagggatgt aggtaagtcc aacgtaagcg atagcagaga tagccacgat atctctcacc 13620

acgtaagaca tagacttcac gagagatctc tcgtaacagt gcttagggat agcgtcaagg 13680

atatccttga tggtgtaatc tggcaccttg aaaacgtttc cgaaggtatc gatagcggtc 13740

ttttgctgct tgaaagatgc aacgtttcca gaacgcctaa cggtcttagt agatccctca 13800

aggatctcag atccagacac ggtaacctta gacatggtat ggtaattgta aatgtaattg 13860

taatgttgtt tgttgtttgt tgttgttggt aattgttgta aaatttttgg tggtgattgg 13920

ttctttaagg tgtgagagtg agttgtgagt tgtgtggtgg gtttggtgag attggggatg 13980

gtgggtttat atagtggaga ctgaggaatg gggtcgtgag tgttaacttt gcatgggcta 14040

cacgtgggtt cttttgggct tacacgtagt attattcatg caaatgcagc caatacatat 14100

acggtatttt aataatgtgt gggaatacaa tatgccgagt attttactaa ttttggcaat 14160

gacaagtgta catttggatt atcttacttg gcctctcttg ctttaatttg gattattttt 14220

attctcttac cttggccgtt catattcaca tccctaaagg caagacagaa ttgaatggtg 14280

gccaaaaatt aaaacgatgg atatgaccta catagtgtag gatcaattaa cgtcgaagga 14340

aaatactgat tctctcaagc atacggacaa gggtaaataa catagtcacc agaacataat 14400

aaacaaaaag tgcagaagca agactaaaaa aattagctat ggacattcag gttcatattg 14460

gaaacatcat tatcctagtc ttgtgaccat ccttcctcct gctctagttg agaggccttg 14520

ggactaacga gaggtcagtt gggatagcag atccttatcc tggactagcc tttctggtgt 14580

ttcagagtct tcgtgccgcc gtctacatct atctccatta ggtctgaaga tgactcttca 14640

caccaacgac gtttaaggtc tctatcctac tcctagcttg caatacctgg cttgcaatac 14700

ctggagcatc gtgcacgatg attggatact gtggaggagg agtgtttgct gatttagagc 14760

tcccggttgg gtgatttgac ttcgatttca gtttaggctt gttgaaattt ttcaggttcc 14820

attgtgaagc ctttagagct tgagcttcct tccatgttaa tgccttgatc gaatactcct 14880

agagaaaagg gaagtcgatc tctgagtatt gaaatcgaag tgcacatttt ttttcaacgt 14940

gtccaatcaa tccacaaaca aagcagaaga caggtaatct ttcatactta tactgacaag 15000

taatagtctt accgtcatgc ataataacgt ctcgttcctt caagaggggt tttccgacat 15060

ccataacgac ccgaagcctc atgaaagcat tagggaagaa cttttggttc ttcttgtcat 15120

ggcctttata ggtgtcagcc gagctcgcca attcccgtcc gactggctcc gcaaaatatt 15180

cgaacggcaa gttatggact tgcaaccata actccacggt attgagcagg acctattgtg 15240

aagactcatc tcatggagct tcagaatgtg gttgtcagca aaccaatgac cgaaatccat 15300

cacatgacgg acgtccagtg ggtgagcgaa acgaaacagg aagcgcctat ctttcagagt 15360

cgtgagctcc acaccggatt ccggcaacta cgtgttgggc aggcttcgcc gtattagaga 15420

tatgttgagg cagacccatc tgtgccactc gtacaattac gagagttgtt ttttttgtga 15480

ttttcctagt ttctcgttga tggtgagctc atattctaca tcgtatggtc tctcaacgtc 15540

gtttcctgtc atctgatatc ccgtcatttg catccacgtg cgccgcctcc cgtgccaagt 15600

ccctaggtgt catgcacgcc aaattggtgg tggtgcgggc tgccctgtgc ttcttaccga 15660

tgggtggagg ttgagtttgg gggtctccgc ggcgatggta gtgggttgac ggtttggtgt 15720

gggttgacgg cattgatcaa tttacttctt gcttcaaatt ctttggcaga aaacaattca 15780

ttagattaga actggaaacc agagtgatga gacggattaa gtcagattcc aacagagtta 15840

catctcttaa gaaataatgt aaccccttta gactttatat atttgcaatt aaaaaaataa 15900

tttaactttt agactttata tatagtttta ataactaagt ttaaccactc tattatttat 15960

atcgaaacta tttgtatgtc tcccctctaa ataaacttgg tattgtgttt acagaaccta 16020

taatcaaata atcaatactc aactgaagtt tgtgcagtta attgaaggga ttaacggcca 16080

aaatgcacta gtattatcaa ccgaatagat tcacactaga tggccatttc catcaatatc 16140

atcgccgttc ttcttctgtc cacatatccc ctctgaaact tgagagacac ctgcacttca 16200

ttgtccttat tacgtgttac aaaatgaaac ccatgcatcc atgcaaactg aagaatggcg 16260

caagaaccct tcccctccat ttcttatgtg gcgaccatcc atttcaccat ctcccgctat 16320

aaaacacccc catcacttca cctagaacat catcactact tgcttatcca tccaaaagat 16380

acccactttt acaacaatta ccaacaacaa caaacaacaa acaacattac aattacattt 16440

acaattacca taccatgcca cctagcgctg ctaagcaaat gggagcttct actggtgttc 16500

atgctggtgt tactgactct tctgctttca ccagaaagga tgttgctgat agacctgatc 16560

tcaccatcgt tggagattct gtttacgatg ctaaggcttt cagatctgag catcctggtg 16620

gtgctcattt cgtttctttg ttcggaggaa gagatgctac tgaggctttc atggaatacc 16680

atagaagggc ttggcctaag tctagaatgt ctagattcca cgttggatct cttgcttcta 16740

ctgaggaacc tgttgctgct gatgagggat accttcaact ttgtgctagg atcgctaaga 16800

tggtgccttc tgtttcttct ggattcgctc ctgcttctta ctgggttaag gctggactta 16860

tccttggatc tgctatcgct cttgaggctt acatgcttta cgctggaaag agacttctcc 16920

cttctatcgt tcttggatgg cttttcgctc ttatcggtct taacatccag catgatgcta 16980

accatggtgc tttgtctaag tctgcttctg ttaaccttgc tcttggactt tgtcaggatt 17040

ggatcggagg atctatgatc ctttggcttc aagagcatgt tgttatgcac cacctccaca 17100

ctaacgatgt tgataaggat cctgatcaaa aggctcacgg tgctcttaga ctcaagccta 17160

ctgatgcttg gtcacctatg cattggcttc agcatcttta ccttttgcct ggtgagacta 17220

tgtacgcttt caagcttttg ttcctcgaca tctctgagct tgttatgtgg cgttgggagg 17280

gtgagcctat ctctaagctt gctggatacc tctttatgcc ttctttgctt ctcaagctta 17340

ccttctgggc tagattcgtt gctttgcctc tttaccttgc tccttctgtt catactgctg 17400

tgtgtatcgc tgctactgtt atgactggat ctttctacct cgctttcttc ttcttcatct 17460

cccacaactt cgagggtgtt gcttctgttg gacctgatgg atctatcact tctatgacta 17520

gaggtgctag cttccttaag agacaagctg agacttcttc taacgttgga ggacctcttc 17580

ttgctactct taacggtgga ctcaactacc aaattgagca tcacttgttc cctagagttc 17640

accatggatt ctaccctaga cttgctcctc ttgttaaggc tgagcttgag gctagaggaa 17700

tcgagtacaa gcactaccct actatctggt ctaaccttgc ttctaccctc agacatatgt 17760

acgctcttgg aagaaggcct agatctaagg ctgagtaatg acaagcttat gtgacgtgaa 17820

ataataacgg taaaatatat gtaataataa taataataaa gccacaaagt gagaatgagg 17880

ggaaggggaa atgtgtaatg agccagtagc cggtggtgct aattttgtat cgtattgtca 17940

ataaatcatg aattttgtgg tttttatgtg tttttttaaa tcatgaattt taaattttat 18000

aaaataatct ccaatcggaa gaacaacatt ccatatccat gcatggatgt ttctttaccc 18060

aaatctagtt cttgagagga tgaagcatca ccgaacagtt ctgcaactat ccctcaaaag 18120

ctttaaaatg aacaacaagg aacagagcaa cgttccaaag atcccaaacg aaacatatta 18180

tctatactaa tactatatta ttaattacta ctgcccggaa tcacaatccc tgaatgattc 18240

ctattaacta caagccttgt tggcggcgga gaagtgatcg gcgcggcgag aagcagcgga 18300

ctcggagacg aggccttgga agatctgagt cgaacgggca gaatcagtat tttccttcga 18360

cgttaattga tcctacacta tgtaggtcat atccatcgtt ttaatttttg gccaccattc 18420

aattctgtct tgcctttagg gatgtgaata tgaacggcca aggtaagaga ataaaaataa 18480

tccaaattaa agcaagagag gccaagtaag ataatccaaa tgtacacttg tcattgccaa 18540

aattagtaaa atactcggca tattgtattc ccacacatta ttaaaatacc gtatatgtat 18600

tggctgcatt tgcatgaata atactacgtg taagcccaaa agaacccacg tgtagcccat 18660

gcaaagttaa cactcacgac cccattcctc agtctccact atataaaccc accatcccca 18720

atctcaccaa acccaccaca caactcacaa ctcactctca caccttaaag aaccaatcac 18780

caccaaaaat tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattaca 18840

tttacaatta ccataccatg agcgctgtta ccgttactgg atctgatcct aagaacagag 18900

gatcttctag caacaccgag caagaggttc caaaagttgc tatcgatacc aacggaaacg 18960

tgttctctgt tcctgatttc accatcaagg acatccttgg agctatccct catgagtgtt 19020

acgagagaag attggctacc tctctctact acgtgttcag agatatcttc tgcatgctta 19080

ccaccggata ccttacccat aagatccttt accctctcct catctcttac acctctaaca 19140

gcatcatcaa gttcactttc tgggcccttt acacttacgt tcaaggactt ttcggaaccg 19200

gaatctgggt tctcgctcat gagtgtggac atcaagcttt ctctgattac ggaatcgtga 19260

acgatttcgt tggatggacc cttcactctt accttatggt tccttacttc agctggaagt 19320

actctcatgg aaagcaccat aaggctactg gacacatgac cagagatatg gttttcgttc 19380

ctgccaccaa agaggaattc aagaagtcta ggaacttctt cggtaacctc gctgagtact 19440

ctgaggattc tccacttaga accctttacg agcttcttgt tcaacaactt ggaggatgga 19500

tcgcttacct cttcgttaac gttacaggac aaccttaccc tgatgttcct tcttggaaat 19560

ggaaccactt ctggcttacc tctccacttt tcgagcaaag agatgctctc tacatcttcc 19620

tttctgatct tggaatcctc acccagggaa tcgttcttac tctttggtac aagaaattcg 19680

gaggatggtc ccttttcatc aactggttcg ttccttacat ctgggttaac cactggctcg 19740

ttttcatcac attccttcag cacactgatc ctactatgcc tcattacaac gctgaggaat 19800

ggactttcgc taagggtgct gctgctacta tcgatagaaa gttcggattc atcggacctc 19860

acatcttcca tgatatcatc gagactcatg tgcttcacca ctactgttct aggatcccat 19920

tctacaacgc tagacctgct tctgaggcta tcaagaaagt tatgggaaag cactacaggt 19980

ctagcgacga gaacatgtgg aagtcacttt ggaagtcttt caggtcttgc caatacgttg 20040

acggtgataa cggtgttctc atgttccgta acatcaacaa ctgcggagtt ggagctgctg 20100

agaagtaatg aaggggtgat cgattatgag atcgtacaaa gacactgcta ggtgttaagg 20160

atggataata ataataataa tgagatgaat gtgttttaag ttagtgtaac agctgtaata 20220

aagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gaggctgatg 20280

aaatgttatg tatgtttctt ggtttttaaa ataaatgaaa gcacatgctc gtgtggttct 20340

atcgaattat tcggcggttc ctgtgggaaa aagtccagaa gggccgccgc agctactact 20400

acaaccaagg ccgtggagga gggcaacaga gccagcactt cgatagctgc tgcgatgatc 20460

ttaagcaatt gaggagcgag tgcacatgca ggggactgga gcgtgcaatc ggccagatga 20520

ggcaggacat ccagcagcag ggacagcagc aggaagttga gaggtggtcc catcaatcta 20580

aacaagtcgc tagggacctt ccgggacagt gcggcaccca gcctagccga tgccagctcc 20640

aggggcagca gcagtctgca tggttttgaa gtggtgatcg atgagatcgt ataaagacac 20700

tgctaggtgt taaggatggg ataataagat gtgttttaag tcattaaccg taataaaaag 20760

agagagaggc tgatggaatg ttatgtatgt atgtttcttg gtttttaaaa ttaaatggaa 20820

agcacatgct cgtgtgggtt ctatctcgat taaaaatccc aattatattt ggtctaattt 20880

agtttggtat tgagtaaaac aaattcgaac caaaccaaaa tataaatata tagtttttat 20940

atatatgcct ttaagacttt ttatagaatt ttctttaaaa aatatctaga aatatttgcg 21000

actcttctgg catgtaatat ttcgttaaat atgaagtgct ccatttttat taactttaaa 21060

taattggttg tacgatcact ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt 21120

cttccatatt catatgtcaa aatctatcaa aattcttata tatctttttc gaatttgaag 21180

tgaaatttcg ataatttaaa attaaataga acatatcatt atttaggtat catattgatt 21240

tttatactta attactaaat ttggttaact ttgaaagtgt acatcaacga aaaattagtc 21300

aaacgactaa aataaataaa tatcatgtgt tattaagaaa attctcctat aagaatattt 21360

taatagatca tatgtttgta aaaaaaatta atttttacta acacatatat ttacttatca 21420

aaaatttgac aaagtaagat taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg 21480

ctgaaaaccc ggcaaaaccg aaccaatcca aaccgatata gttggtttgg tttgattttg 21540

atataaaccg aaccaactcg gtccatttgc acccctaatc ataatagctt taatatttca 21600

agatattatt aagttaacgt tgtcaatatc ctggaaattt tgcaaaatga atcaagccta 21660

tatggctgta atatgaattt aaaagcagct cgatgtggtg gtaatatgta atttacttga 21720

ttctaaaaaa atatcccaag tattaataat ttctgctagg aagaaggtta gctacgattt 21780

acagcaaagc cagaatacaa agaaccataa agtgattgaa gctcgaaata tacgaaggaa 21840

caaatatttt taaaaaaata cgcaatgact tggaacaaaa gaaagtgata tattttttgt 21900

tcttaaacaa gcatcccctc taaagaatgg cagttttcct ttgcatgtaa ctattatgct 21960

cccttcgtta caaaaatttt ggactactat tgggaacttc ttctgaaaat agtcctgcag 22020

gctagtagat tggttggttg gtttccatgt accagaaggc ttaccctatt agttgaaagt 22080

tgaaactttg ttccctactc aattcctagt tgtgtaaatg tatgtatatg taatgtgtat 22140

aaaacgtagt acttaaatga ctaggagtgg ttcttgagac cgatgagaga tgggagcaga 22200

actaaagatg atgacataat taagaacgaa tttgaaaggc tcttaggttt gaatcctatt 22260

cgagaatgtt tttgtcaaag atagtggcga ttttgaacca aagaaaacat ttaaaaaatc 22320

agtatccggt tacgttcatg caaatagaaa gtggtctagg atctgattgt aattttagac 22380

ttaaagagtc tcttaagatt caatcctggc tgtgtacaaa actacaaata atatatttta 22440

gactatttgg ccttaactaa acttccactc attatttact gaggttagag aatagacttg 22500

cgaataaaca cattcccgag aaatactcat gatcccataa ttagtcagag ggtatgccaa 22560

tcagatctaa gaacacacat tccctcaaat tttaatgcac atgtaatcat agtttagcac 22620

aattcaaaaa taatgtagta ttaaagacag aaatttgtag actttttttt ggcgttaaaa 22680

gaagactaag tttatacgta cattttattt taagtggaaa accgaaattt tccatcgaaa 22740

tatatgaatt tagtatatat atttctgcaa tgtactattt tgctattttg gcaactttca 22800

gtggactact actttattac aatgtgtatg gatgcatgag tttgagtata cacatgtcta 22860

aatgcatgct ttgtaaaacg taacggacca caaaagagga tccatacaaa tacatctcat 22920

agcttcctcc attattttcc gacacaaaca gagcatttta caacaattac caacaacaac 22980

aaacaacaaa caacattaca attacattta caattaccat accatggaat ttgctcaacc 23040

tctcgttgct atggctcaag agcagtacgc tgctatcgat gctgttgttg ctcctgctat 23100

cttctctgct accgactcta ttggatgggg actcaagcct atctcttctg ctactaagga 23160

tctccctctc gttgaatctc ctacccctct tatcctttct ctcctcgctt acttcgctat 23220

cgttggttct ggactcgttt accgtaaagt gttccctaga accgttaagg gacaggatcc 23280

tttccttctc aaggctctta tgctcgctca caacgttttc cttatcggac tcagccttta 23340

catgtgcctc aagctcgttt acgaggctta cgtgaacaag tactccttct ggggaaacgc 23400

ttacaaccct gctcaaaccg agatggctaa ggtgatctgg atcttctacg tgtccaagat 23460

ctacgagttc atggacacct tcatcatgct tctcaaggga aacgttaacc aggtttcctt 23520

cctccatgtt taccaccacg gatctatctc tggaatctgg tggatgatca cttatgctgc 23580

tccaggtgga gatgcttact tctctgctgc tctcaactct tgggttcatg tgtgcatgta 23640

cacctactac ttcatggctg ctgttcttcc taaggacgaa aagaccaaga gaaagtacct 23700

ttggtgggga agatacctta cccagatgca aatgttccag ttcttcatga accttctcca 23760

ggctgtttac ctcctctact cttcttctcc ttaccctaag ttcattgctc aactcctcgt 23820

tgtttacatg gttaccctcc tcatgctttt cggaaacttc tactacatga agcaccacgc 23880

ttctaagtga taagggccgc cgccatgtga cagatcgaag gaagaaagtg taataagacg 23940

actctcacta ctcgatcgct agtgattgtc attgttatat ataataatgt tatctttcac 24000

aacttatcgt aatgcatgtg aaactataac acattaatcc tacttgtcat atgataacac 24060

tctccccatt taaaactctt gtcaatttaa agatataaga ttctttaaat gattaaaaaa 24120

aatatattat aaattcaatc actcctacta ataaattatt aattattatt tattgattaa 24180

aaaaatactt atactaattt agtctgaata gaataattag attctagcct gcagggcggc 24240

cgcggatccc atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac 24300

tggcgaacag ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa 24360

catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga 24420

ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca 24480

ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa 24540

atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc 24600

caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 24660

ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca 24720

ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agagaacacg 24780

ggggactgaa ttaaatatga gccctgagag gcgtcctgtt gaaatcagac ctgctactgc 24840

tgctgatatg gctgctgttt gtgatatcgt gaaccactac atcgagactt ctaccgttaa 24900

cttcagaact gagcctcaaa ctcctcaaga gtggatcgat gatcttgaga gactccaaga 24960

tagataccct tggcttgttg ctgaggttga gggtgttgtt gctggaatcg cttacgctgg 25020

accttggaag gctagaaacg cttacgattg gactgttgag tctaccgttt acgtttcaca 25080

cagacatcag agacttggac ttggatctac cctttacact caccttctca agtctatgga 25140

agctcaggga ttcaagtctg ttgttgctgt tatcggactc cctaacgatc cttctgttag 25200

acttcatgag gctcttggat acactgctag aggaactctt agagctgctg gatacaagca 25260

cggtggatgg catgatgttg gattctggca aagagatttc gagcttcctg ctcctcctag 25320

acctgttaga ccagttactc agatctgaat ttgcgtgatc gttcaaacat ttggcaataa 25380

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 25440

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 25500

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 25560

gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatca ctagtgatgt 25620

acggttaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 25680

cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc tccccgaccg 25740

gcagctcggc acaaaatcac cactcgatac aggcagccca tcagtcc 25787

<210> SEQ ID NO: 4

<211> LENGTH: 22824

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pGA7- mod_D nucleotide sequence

<400> SEQENCE: 4

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgcctcgatt aaaaatccca attatatttg gtctaattta 240

gtttggtatt gagtaaaaca aattcgaacc aaaccaaaat ataaatatat agtttttata 300

tatatgcctt taagactttt tatagaattt tctttaaaaa atatctagaa atatttgcga 360

ctcttctggc atgtaatatt tcgttaaata tgaagtgctc catttttatt aactttaaat 420

aattggttgt acgatcactt tcttatcaag tgttactaaa atgcgtcaat ctctttgttc 480

ttccatattc atatgtcaaa atctatcaaa attcttatat atctttttcg aatttgaagt 540

gaaatttcga taatttaaaa ttaaatagaa catatcatta tttaggtatc atattgattt 600

ttatacttaa ttactaaatt tggttaactt tgaaagtgta catcaacgaa aaattagtca 660

aacgactaaa ataaataaat atcatgtgtt attaagaaaa ttctcctata agaatatttt 720

aatagatcat atgtttgtaa aaaaaattaa tttttactaa cacatatatt tacttatcaa 780

aaatttgaca aagtaagatt aaaataatat tcatctaaca aaaaaaaaac cagaaaatgc 840

tgaaaacccg gcaaaaccga accaatccaa accgatatag ttggtttggt ttgattttga 900

tataaaccga accaactcgg tccatttgca cccctaatca taatagcttt aatatttcaa 960

gatattatta agttaacgtt gtcaatatcc tggaaatttt gcaaaatgaa tcaagcctat 1020

atggctgtaa tatgaattta aaagcagctc gatgtggtgg taatatgtaa tttacttgat 1080

tctaaaaaaa tatcccaagt attaataatt tctgctagga agaaggttag ctacgattta 1140

cagcaaagcc agaatacaaa gaaccataaa gtgattgaag ctcgaaatat acgaaggaac 1200

aaatattttt aaaaaaatac gcaatgactt ggaacaaaag aaagtgatat attttttgtt 1260

cttaaacaag catcccctct aaagaatggc agttttcctt tgcatgtaac tattatgctc 1320

ccttcgttac aaaaattttg gactactatt gggaacttct tctgaaaata gtggcgcccc 1380

gcggaaagct tgctagccaa ttggggccca acgttctcga gtttttctag aaggaaactg 1440

aaggcgggaa acgacaatct gctagtggat ctcccagtca cgacgttgta aaacgggcgc 1500

cccgcggaaa gcttgcggcc gcccgatcta gtaacataga tgacaccgcg cgcgataatt 1560

tatcctagtt tgcgcgctat attttgtttt ctatcgcgta ttaaatgtat aattgcggga 1620

ctctaatcat aaaaacccat ctcataaata acgtcatgca ttacatgtta attattacgt 1680

gcttaacgta attcaacaga aattatatga taatcatcgc aagaccggca acaggattca 1740

atcttaagaa actttattgc caaatgtttg aacgatcggc gcgcctcatt agtgagcctt 1800

ctcagccttt ccgttaacgt agtagtgctg tcccacctta tcaaggttag agaaagtagc 1860

cttccaagca ccgtagtaag agagcacctt gtagttgagt ccccacttct tagcgaaagg 1920

aacgaatctt ctgctaacct caggctgtct gaattgaggc atatcaggga agaggtggtg 1980

gataacctga cagttaaggt atcccataag ccagttcacg tatcctctag aaggatcgat 2040

atcaacggtg tgatcaacag cgtagttaac ccaagaaagg tgcttatcag atggaacaac 2100

agggaggtga gtatgagaag tagagaagtg agcgaaaagg tacatgtaag cgatccagtt 2160

tccgaaagtg aaccaccagt aagcaacagg ccaagagtat ccagtagcaa gcttgataac 2220

agcggttcta acaacatgag aaacgagcat ccaagaagcc tcttcgtagt tcttcttacg 2280

gagaacttgt ctagggtgga gaacgtagat ccagaaagct tgaacaagaa gtccagaggt 2340

aacaggaacg aaagtccaag cttgaagtct agcccaagct ctagagaatc ctctaggtct 2400

gttatcctca acagcagtgt tgaagaaagc cacagcagga gtggtatcaa gatccatatc 2460

gtgtctaacc ttttgagggg tagcatggtg cttgttatgc atctggttcc acatctcacc 2520

agaagtagaa agtccgaatc cacaagtcat agcctgaagt ctcttgtcca cgtaaacaga 2580

tccggtaaga gagttatgtc caccctcatg ttgaacccat ccacatctag ctccgaagaa 2640

agcaccgtaa acaacagaag caatgatagg gtatccagcg tacataagag cagttccaag 2700

agcgaatgta gcaagaagct cgagaagtct gtaagccaca tgggtgatag aaggcttgaa 2760

gaatccatct ctctcaagct cagcacgcca tctagcgaaa tcctcaagca taggagcatc 2820

ctcagactca gatctcttga tctcagcagg tctagaaggc aaagctctaa gcatcttcca 2880

agccttgaga gaacgcatgt ggaattcttt gaaagcctca gtagcatcag caccagtgtt 2940

agcaagcatg tagaagatca cagatccacc agggtgcttg aagttagtca catcgtactc 3000

aacgtcctca actctaaccc atctagtctc gaaagtagca gcaagctcat gaggctcaag 3060

agtcttaaga tcaacaggag cagtagaagc atccttagca tcaagagcct cagcagaaga 3120

tttagacctg gtaagtggag atctaggaga agatcttcca tcagtcttag gagggcacat 3180

ggtatggtaa ttgtaaatgt aattgtaatg ttgtttgttg tttgttgttg ttggtaattg 3240

ttgtaaaaga tcctcgtgta tgtttttaat cttgtttgta tcgatgagtt ttggtttgag 3300

taaagagtga agcggatgag ttaatttata ggctataaag gagatttgca tggcgatcac 3360

gtgtaataat gcatgcacgc atgtgattgt atgtgtgtgc tgtgagagag aagctcttag 3420

gtgtttgaag ggagtgacaa gtggcgaaga aaaacaattc tccgcggctg catgctatgt 3480

gtaacgtgta gctaatgttc tggcatggca tcttatgaac gattcttttt aaaaacaagg 3540

taaaaactta acttcataaa attaaaaaaa aaaacgttta ctaagttggt ttaaaagggg 3600

atgagactag tagattggtt ggttggtttc catgtaccag aaggcttacc ctattagttg 3660

aaagttgaaa ctttgttccc tactcaattc ctagttgtgt aaatgtatgt atatgtaatg 3720

tgtataaaac gtagtactta aatgactagg agtggttctt gagaccgatg agagatggga 3780

gcagaactaa agatgatgac ataattaaga acgaatttga aaggctctta ggtttgaatc 3840

ctattcgaga atgtttttgt caaagatagt ggcgattttg aaccaaagaa aacatttaaa 3900

aaatcagtat ccggttacgt tcatgcaaat agaaagtggt ctaggatctg attgtaattt 3960

tagacttaaa gagtctctta agattcaatc ctggctgtgt acaaaactac aaataatata 4020

ttttagacta tttggcctta actaaacttc cactcattat ttactgaggt tagagaatag 4080

acttgcgaat aaacacattc ccgagaaata ctcatgatcc cataattagt cagagggtat 4140

gccaatcaga tctaagaaca cacattccct caaattttaa tgcacatgta atcatagttt 4200

agcacaattc aaaaataatg tagtattaaa gacagaaatt tgtagacttt tttttggcgt 4260

taaaagaaga ctaagtttat acgtacattt tattttaagt ggaaaaccga aattttccat 4320

cgaaatatat gaatttagta tatatatttc tgcaatgtac tattttgcta ttttggcaac 4380

tttcagtgga ctactacttt attacaatgt gtatggatgc atgagtttga gtatacacat 4440

gtctaaatgc atgctttgta aaacgtaacg gaccacaaaa gaggatccat acaaatacat 4500

ctcatagctt cctccattat tttccgacac aaacagagca ttttacaaca attaccaaca 4560

acaacaaaca acaaacaaca ttacaattac atttacaatt accataccat ggaattcgcc 4620

cagcctcttg ttgctatggc tcaagagcaa tacgctgcta tcgatgctgt tgttgctcct 4680

gctatcttct ctgctactga ttctatcgga tggggactta agcctatctc ttctgctact 4740

aaggacttgc ctcttgttga gtctcctaca cctctcatcc tttctttgct tgcttacttc 4800

gctatcgttg gatctggact cgtttacaga aaggttttcc ctagaaccgt gaagggacaa 4860

gatccattcc ttttgaaggc tcttatgctt gctcacaacg tgttccttat cggactttct 4920

ctttacatgt gcctcaagct tgtgtacgag gcttacgtta acaagtactc tttctgggga 4980

aacgcttaca accctgctca aactgagatg gctaaggtta tctggatctt ctacgtgagc 5040

aagatctacg agttcatgga taccttcatc atgctcctca agggaaatgt taaccaggtt 5100

agcttccttc acgtttacca tcacggatct atctctggaa tctggtggat gattacttac 5160

gctgctcctg gtggtgatgc ttacttctct gctgctctta actcttgggt tcacgtgtgt 5220

atgtacacct actattttat ggctgccgtg cttcctaagg acgagaaaac taagagaaag 5280

tacctctggt ggggaagata ccttactcaa atgcagatgt tccagttctt catgaacctt 5340

ctccaggctg tttaccttct ctactcttca tctccttacc ctaagtttat cgctcagctc 5400

ctcgtggtgt acatggttac tcttctcatg cttttcggaa acttctacta catgaagcac 5460

cacgctagca agtgatgagg cgcgccgggc cgccgccatg tgacagatcg aaggaagaaa 5520

gtgtaataag acgactctca ctactcgatc gctagtgatt gtcattgtta tatataataa 5580

tgttatcttt cacaacttat cgtaatgcat gtgaaactat aacacattaa tcctacttgt 5640

catatgataa cactctcccc atttaaaact cttgtcaatt taaagatata agattcttta 5700

aatgattaaa aaaaatatat tataaattca atcactccta ctaataaatt attaattatt 5760

atttattgat taaaaaaata cttatactaa tttagtctga atagaataat tagattctag 5820

tctcatcccc ttttaaacca acttagtaaa cgtttttttt tttaatttta tgaagttaag 5880

tttttacctt gtttttaaaa agaatcgttc ataagatgcc atgccagaac attagctaca 5940

cgttacacat agcatgcagc cgcggagaat tgtttttctt cgccacttgt cactcccttc 6000

aaacacctaa gagcttctct ctcacagcac acacatacaa tcacatgcgt gcatgcatta 6060

ttacacgtga tcgccatgca aatctccttt atagcctata aattaactca tccgcttcac 6120

tctttactca aaccaaaact catcgataca aacaagatta aaaacataca cgaggatctt 6180

ttacaacaat taccaacaac aacaaacaac aaacaacatt acaattacat ttacaattac 6240

cataccatgc ctccaaggga ctcttactct tatgctgctc ctccttctgc tcaacttcac 6300

gaagttgata ctcctcaaga gcacgacaag aaagagcttg ttatcggaga tagggcttac 6360

gatgttacca acttcgttaa gagacaccct ggtggaaaga tcattgctta ccaagttgga 6420

actgatgcta ccgatgctta caagcagttc catgttagat ctgctaaggc tgacaagatg 6480

cttaagtctc ttccttctcg tcctgttcac aagggatact ctccaagaag ggctgatctt 6540

atcgctgatt tccaagagtt caccaagcaa cttgaggctg agggaatgtt cgagccttct 6600

cttcctcatg ttgcttacag acttgctgag gttatcgcta tgcatgttgc tggtgctgct 6660

cttatctggc atggatacac tttcgctgga atcgctatgc ttggagttgt tcagggaaga 6720

tgtggatggc ttatgcatga gggtggacat tactctctca ctggaaacat tgctttcgac 6780

agagctatcc aagttgcttg ttacggactt ggatgtggaa tgtctggtgc ttggtggcgt 6840

aaccagcata acaagcacca tgctactcct caaaagcttc agcacgatgt tgatcttgat 6900

acccttcctc tcgttgcttt ccatgagaga atcgctgcta aggttaagtc tcctgctatg 6960

aaggcttggc tttctatgca agctaagctt ttcgctcctg ttaccactct tcttgttgct 7020

cttggatggc agctttacct tcatcctaga cacatgctca ggactaagca ctacgatgag 7080

cttgctatgc tcggaatcag atacggactt gttggatacc ttgctgctaa ctacggtgct 7140

ggatacgttc tcgcttgtta ccttctttac gttcagcttg gagctatgta catcttctgc 7200

aacttcgctg tttctcatac tcacctccct gttgttgagc ctaacgagca tgctacttgg 7260

gttgagtacg ctgctaacca cactactaac tgttctccat cttggtggtg tgattggtgg 7320

atgtcttacc ttaactacca gatcgagcac cacctttacc cttctatgcc tcaattcaga 7380

caccctaaga tcgctcctag agttaagcag cttttcgaga agcacggact tcactacgat 7440

gttagaggat acttcgaggc tatggctgat actttcgcta accttgataa cgttgcccat 7500

gctcctgaga agaaaatgca gtaatgagat cgttcaaaca tttggcaata aagtttctta 7560

agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 7620

aagcacgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 7680

agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 7740

gataaattat cgcgcgcggt gtcatctatg ttactagatc ggtcgattaa aaatcccaat 7800

tatatttggt ctaatttagt ttggtattga gtaaaacaaa ttcgaaccaa accaaaatat 7860

aaatatatag tttttatata tatgccttta agacttttta tagaattttc tttaaaaaat 7920

atctagaaat atttgcgact cttctggcat gtaatatttc gttaaatatg aagtgctcca 7980

tttttattaa ctttaaataa ttggttgtac gatcactttc ttatcaagtg ttactaaaat 8040

gcgtcaatct ctttgttctt ccatattcat atgtcaaaat ctatcaaaat tcttatatat 8100

ctttttcgaa tttgaagtga aatttcgata atttaaaatt aaatagaaca tatcattatt 8160

taggtatcat attgattttt atacttaatt actaaatttg gttaactttg aaagtgtaca 8220

tcaacgaaaa attagtcaaa cgactaaaat aaataaatat catgtgttat taagaaaatt 8280

ctcctataag aatattttaa tagatcatat gtttgtaaaa aaaattaatt tttactaaca 8340

catatattta cttatcaaaa atttgacaaa gtaagattaa aataatattc atctaacaaa 8400

aaaaaaacca gaaaatgctg aaaacccggc aaaaccgaac caatccaaac cgatatagtt 8460

ggtttggttt gattttgata taaaccgaac caactcggtc catttgcacc cctaatcata 8520

atagctttaa tatttcaaga tattattaag ttaacgttgt caatatcctg gaaattttgc 8580

aaaatgaatc aagcctatat ggctgtaata tgaatttaaa agcagctcga tgtggtggta 8640

atatgtaatt tacttgattc taaaaaaata tcccaagtat taataatttc tgctaggaag 8700

aaggttagct acgatttaca gcaaagccag aatacaaaga accataaagt gattgaagct 8760

cgaaatatac gaaggaacaa atatttttaa aaaaatacgc aatgacttgg aacaaaagaa 8820

agtgatatat tttttgttct taaacaagca tcccctctaa agaatggcag ttttcctttg 8880

catgtaacta ttatgctccc ttcgttacaa aaattttgga ctactattgg gaacttcttc 8940

tgaaaatagt gatagaaccc acacgagcat gtgctttcca tttaatttta aaaaccaaga 9000

aacatacata cataacattc catcagcctc tctctctttt tattacggtt aatgacttaa 9060

aacacatctt attatcccat ccttaacacc tagcagtgtc tttatacgat ctcatcgatc 9120

accacttcaa aaccatgcag actgctgctg cccctggagc tggcatcggc taggctgggt 9180

gccgcactgt cccggaaggt ccctagcgac ttgtttagat tgatgggacc acctctcaac 9240

ttcctgctgc tgtccctgct gctggatgtc ctgcctcatc tggccgattg cacgctccag 9300

tcccctgcat gtgcactcgc tcctcaattg cttaagatca tcgcagcagc tatcgaagtg 9360

ctggctctgt tgccctcctc cacggccttg gttgtagtag tagctgccgc cgcccttctg 9420

gactttttcc cacaggaacc gccgaataat tcgatagaac cacacgagca tgtgctttca 9480

tttattttaa aaaccaagaa acatacataa catttcatca gcctctctct ctctctctct 9540

ctctctctct ctctctctct ctctctctct ctctttatta cagctgttac actaacttaa 9600

aacacattca tctcattatt attattatta tccatcctta acacctagca gtgtctttgt 9660

acgatctcat aatcgatcac cccttcatca ggtatcctta ggcttcactc caacgttgtt 9720

gcagttacgg aacatgtaca caccatcatg gttctcaacg aactggcaag atctccaagt 9780

tttccaaagg ctaacccaca tgttctcatc ggtgtgtctg tagtgctctc ccataacttt 9840

cttgatgcac tcggtagctt ctctagcatg gtagaatggg atccttgaaa cgtagtgatg 9900

gagcacatga gtctcgatga tgtcatggaa gatgattccg aggattccga actctctatc 9960

gatagtagca gcagcaccct tagcgaaagt ccactcttga gcatcgtaat gaggcataga 10020

agaatcggtg tgctgaagga aggtaacgaa aacaagccag tggttaacaa ggatccaagg 10080

acagaaccat gtgatgaaag taggccagaa tccgaaaacc ttgtaagcgg tgtaaacaga 10140

agtgagggta gcaaggattc caagatcaga aagaacgatg taccagtagt ccttcttatc 10200

gaaaacaggg ctagaaggcc agtagtgaga cttgaagaac ttagaaacac cagggtaagg 10260

ttgtccagta gcgttagtag caaggtaaag agaaagtcct ccaagctgtt ggaacaagag 10320

agcgaaaaca gagtagatag gagtttcctc agcgatatcg tgaaggctgg taacttggtg 10380

cttctctttg aattcctcgg cggtgtaagg aacgaaaacc atatctctgg tcatgtgtcc 10440

agtagcctta tggtgcttag catgagagaa cttccagctg aagtaaggaa ccataacaag 10500

agagtggaga acccatccaa cggtatcgtt aacccatccg tagttagaga aagcagaatg 10560

tccacactca tgtccaagga tccagattcc gaatccgaaa caagagatag agaacacgta 10620

agcagaccaa gcagcgaatc taaggaattc gttagggaga agagggatgt aggtaagtcc 10680

aacgtaagcg atagcagaga tagccacgat atctctcacc acgtaagaca tagacttcac 10740

gagagatctc tcgtaacagt gcttagggat agcgtcaagg atatccttga tggtgtaatc 10800

tggcaccttg aaaacgtttc cgaaggtatc gatagcggtc ttttgctgct tgaaagatgc 10860

aacgtttcca gaacgcctaa cggtcttagt agatccctca aggatctcag atccagacac 10920

ggtaacctta gacatggtat ggtaattgta aatgtaattg taatgttgtt tgttgtttgt 10980

tgttgttggt aattgttgta aaatttttgg tggtgattgg ttctttaagg tgtgagagtg 11040

agttgtgagt tgtgtggtgg gtttggtgag attggggatg gtgggtttat atagtggaga 11100

ctgaggaatg gggtcgtgag tgttaacttt gcatgggcta cacgtgggtt cttttgggct 11160

tacacgtagt attattcatg caaatgcagc caatacatat acggtatttt aataatgtgt 11220

gggaatacaa tatgccgagt attttactaa ttttggcaat gacaagtgta catttggatt 11280

atcttacttg gcctctcttg ctttaatttg gattattttt attctcttac cttggccgtt 11340

catattcaca tccctaaagg caagacagaa ttgaatggtg gccaaaaatt aaaacgatgg 11400

atatgaccta catagtgtag gatcaattaa cgtcgaagga aaatactgat tctctcaagc 11460

atacggacaa gggtaaataa catagtcacc agaacataat aaacaaaaag tgcagaagca 11520

agactaaaaa aattagctat ggacattcag gttcatattg gaaacatcat tatcctagtc 11580

ttgtgaccat ccttcctcct gctctagttg agaggccttg ggactaacga gaggtcagtt 11640

gggatagcag atccttatcc tggactagcc tttctggtgt ttcagagtct tcgtgccgcc 11700

gtctacatct atctccatta ggtctgaaga tgactcttca caccaacgac gtttaaggtc 11760

tctatcctac tcctagcttg caatacctgg cttgcaatac ctggagcatc gtgcacgatg 11820

attggatact gtggaggagg agtgtttgct gatttagagc tcccggttgg gtgatttgac 11880

ttcgatttca gtttaggctt gttgaaattt ttcaggttcc attgtgaagc ctttagagct 11940

tgagcttcct tccatgttaa tgccttgatc gaatactcct agagaaaagg gaagtcgatc 12000

tctgagtatt gaaatcgaag tgcacatttt ttttcaacgt gtccaatcaa tccacaaaca 12060

aagcagaaga caggtaatct ttcatactta tactgacaag taatagtctt accgtcatgc 12120

ataataacgt ctcgttcctt caagaggggt tttccgacat ccataacgac ccgaagcctc 12180

atgaaagcat tagggaagaa cttttggttc ttcttgtcat ggcctttata ggtgtcagcc 12240

gagctcgcca attcccgtcc gactggctcc gcaaaatatt cgaacggcaa gttatggact 12300

tgcaaccata actccacggt attgagcagg acctattgtg aagactcatc tcatggagct 12360

tcagaatgtg gttgtcagca aaccaatgac cgaaatccat cacatgacgg acgtccagtg 12420

ggtgagcgaa acgaaacagg aagcgcctat ctttcagagt cgtgagctcc acaccggatt 12480

ccggcaacta cgtgttgggc aggcttcgcc gtattagaga tatgttgagg cagacccatc 12540

tgtgccactc gtacaattac gagagttgtt ttttttgtga ttttcctagt ttctcgttga 12600

tggtgagctc atattctaca tcgtatggtc tctcaacgtc gtttcctgtc atctgatatc 12660

ccgtcatttg catccacgtg cgccgcctcc cgtgccaagt ccctaggtgt catgcacgcc 12720

aaattggtgg tggtgcgggc tgccctgtgc ttcttaccga tgggtggagg ttgagtttgg 12780

gggtctccgc ggcgatggta gtgggttgac ggtttggtgt gggttgacgg cattgatcaa 12840

tttacttctt gcttcaaatt ctttggcaga aaacaattca ttagattaga actggaaacc 12900

agagtgatga gacggattaa gtcagattcc aacagagtta catctcttaa gaaataatgt 12960

aaccccttta gactttatat atttgcaatt aaaaaaataa tttaactttt agactttata 13020

tatagtttta ataactaagt ttaaccactc tattatttat atcgaaacta tttgtatgtc 13080

tcccctctaa ataaacttgg tattgtgttt acagaaccta taatcaaata atcaatactc 13140

aactgaagtt tgtgcagtta attgaaggga ttaacggcca aaatgcacta gtattatcaa 13200

ccgaatagat tcacactaga tggccatttc catcaatatc atcgccgttc ttcttctgtc 13260

cacatatccc ctctgaaact tgagagacac ctgcacttca ttgtccttat tacgtgttac 13320

aaaatgaaac ccatgcatcc atgcaaactg aagaatggcg caagaaccct tcccctccat 13380

ttcttatgtg gcgaccatcc atttcaccat ctcccgctat aaaacacccc catcacttca 13440

cctagaacat catcactact tgcttatcca tccaaaagat acccactttt acaacaatta 13500

ccaacaacaa caaacaacaa acaacattac aattacattt acaattacca taccatgcca 13560

cctagcgctg ctaagcaaat gggagcttct actggtgttc atgctggtgt tactgactct 13620

tctgctttca ccagaaagga tgttgctgat agacctgatc tcaccatcgt tggagattct 13680

gtttacgatg ctaaggcttt cagatctgag catcctggtg gtgctcattt cgtttctttg 13740

ttcggaggaa gagatgctac tgaggctttc atggaatacc atagaagggc ttggcctaag 13800

tctagaatgt ctagattcca cgttggatct cttgcttcta ctgaggaacc tgttgctgct 13860

gatgagggat accttcaact ttgtgctagg atcgctaaga tggtgccttc tgtttcttct 13920

ggattcgctc ctgcttctta ctgggttaag gctggactta tccttggatc tgctatcgct 13980

cttgaggctt acatgcttta cgctggaaag agacttctcc cttctatcgt tcttggatgg 14040

cttttcgctc ttatcggtct taacatccag catgatgcta accatggtgc tttgtctaag 14100

tctgcttctg ttaaccttgc tcttggactt tgtcaggatt ggatcggagg atctatgatc 14160

ctttggcttc aagagcatgt tgttatgcac cacctccaca ctaacgatgt tgataaggat 14220

cctgatcaaa aggctcacgg tgctcttaga ctcaagccta ctgatgcttg gtcacctatg 14280

cattggcttc agcatcttta ccttttgcct ggtgagacta tgtacgcttt caagcttttg 14340

ttcctcgaca tctctgagct tgttatgtgg cgttgggagg gtgagcctat ctctaagctt 14400

gctggatacc tctttatgcc ttctttgctt ctcaagctta ccttctgggc tagattcgtt 14460

gctttgcctc tttaccttgc tccttctgtt catactgctg tgtgtatcgc tgctactgtt 14520

atgactggat ctttctacct cgctttcttc ttcttcatct cccacaactt cgagggtgtt 14580

gcttctgttg gacctgatgg atctatcact tctatgacta gaggtgctag cttccttaag 14640

agacaagctg agacttcttc taacgttgga ggacctcttc ttgctactct taacggtgga 14700

ctcaactacc aaattgagca tcacttgttc cctagagttc accatggatt ctaccctaga 14760

cttgctcctc ttgttaaggc tgagcttgag gctagaggaa tcgagtacaa gcactaccct 14820

actatctggt ctaaccttgc ttctaccctc agacatatgt acgctcttgg aagaaggcct 14880

agatctaagg ctgagtaatg acaagcttat gtgacgtgaa ataataacgg taaaatatat 14940

gtaataataa taataataaa gccacaaagt gagaatgagg ggaaggggaa atgtgtaatg 15000

agccagtagc cggtggtgct aattttgtat cgtattgtca ataaatcatg aattttgtgg 15060

tttttatgtg tttttttaaa tcatgaattt taaattttat aaaataatct ccaatcggaa 15120

gaacaacatt ccatatccat gcatggatgt ttctttaccc aaatctagtt cttgagagga 15180

tgaagcatca ccgaacagtt ctgcaactat ccctcaaaag ctttaaaatg aacaacaagg 15240

aacagagcaa cgttccaaag atcccaaacg aaacatatta tctatactaa tactatatta 15300

ttaattacta ctgcccggaa tcacaatccc tgaatgattc ctattaacta caagccttgt 15360

tggcggcgga gaagtgatcg gcgcggcgag aagcagcgga ctcggagacg aggccttgga 15420

agatctgagt cgaacgggca gaatcagtat tttccttcga cgttaattga tcctacacta 15480

tgtaggtcat atccatcgtt ttaatttttg gccaccattc aattctgtct tgcctttagg 15540

gatgtgaata tgaacggcca aggtaagaga ataaaaataa tccaaattaa agcaagagag 15600

gccaagtaag ataatccaaa tgtacacttg tcattgccaa aattagtaaa atactcggca 15660

tattgtattc ccacacatta ttaaaatacc gtatatgtat tggctgcatt tgcatgaata 15720

atactacgtg taagcccaaa agaacccacg tgtagcccat gcaaagttaa cactcacgac 15780

cccattcctc agtctccact atataaaccc accatcccca atctcaccaa acccaccaca 15840

caactcacaa ctcactctca caccttaaag aaccaatcac caccaaaaat tttacaacaa 15900

ttaccaacaa caacaaacaa caaacaacat tacaattaca tttacaatta ccataccatg 15960

agcgctgtta ccgttactgg atctgatcct aagaacagag gatcttctag caacaccgag 16020

caagaggttc caaaagttgc tatcgatacc aacggaaacg tgttctctgt tcctgatttc 16080

accatcaagg acatccttgg agctatccct catgagtgtt acgagagaag attggctacc 16140

tctctctact acgtgttcag agatatcttc tgcatgctta ccaccggata ccttacccat 16200

aagatccttt accctctcct catctcttac acctctaaca gcatcatcaa gttcactttc 16260

tgggcccttt acacttacgt tcaaggactt ttcggaaccg gaatctgggt tctcgctcat 16320

gagtgtggac atcaagcttt ctctgattac ggaatcgtga acgatttcgt tggatggacc 16380

cttcactctt accttatggt tccttacttc agctggaagt actctcatgg aaagcaccat 16440

aaggctactg gacacatgac cagagatatg gttttcgttc ctgccaccaa agaggaattc 16500

aagaagtcta ggaacttctt cggtaacctc gctgagtact ctgaggattc tccacttaga 16560

accctttacg agcttcttgt tcaacaactt ggaggatgga tcgcttacct cttcgttaac 16620

gttacaggac aaccttaccc tgatgttcct tcttggaaat ggaaccactt ctggcttacc 16680

tctccacttt tcgagcaaag agatgctctc tacatcttcc tttctgatct tggaatcctc 16740

acccagggaa tcgttcttac tctttggtac aagaaattcg gaggatggtc ccttttcatc 16800

aactggttcg ttccttacat ctgggttaac cactggctcg ttttcatcac attccttcag 16860

cacactgatc ctactatgcc tcattacaac gctgaggaat ggactttcgc taagggtgct 16920

gctgctacta tcgatagaaa gttcggattc atcggacctc acatcttcca tgatatcatc 16980

gagactcatg tgcttcacca ctactgttct aggatcccat tctacaacgc tagacctgct 17040

tctgaggcta tcaagaaagt tatgggaaag cactacaggt ctagcgacga gaacatgtgg 17100

aagtcacttt ggaagtcttt caggtcttgc caatacgttg acggtgataa cggtgttctc 17160

atgttccgta acatcaacaa ctgcggagtt ggagctgctg agaagtaatg aaggggtgat 17220

cgattatgag atcgtacaaa gacactgcta ggtgttaagg atggataata ataataataa 17280

tgagatgaat gtgttttaag ttagtgtaac agctgtaata aagagagaga gagagagaga 17340

gagagagaga gagagagaga gagagagaga gaggctgatg aaatgttatg tatgtttctt 17400

ggtttttaaa ataaatgaaa gcacatgctc gtgtggttct atcgaattat tcggcggttc 17460

ctgtgggaaa aagtccagaa gggccgccgc agctactact acaaccaagg ccgtggagga 17520

gggcaacaga gccagcactt cgatagctgc tgcgatgatc ttaagcaatt gaggagcgag 17580

tgcacatgca ggggactgga gcgtgcaatc ggccagatga ggcaggacat ccagcagcag 17640

ggacagcagc aggaagttga gaggtggtcc catcaatcta aacaagtcgc tagggacctt 17700

ccgggacagt gcggcaccca gcctagccga tgccagctcc aggggcagca gcagtctgca 17760

tggttttgaa gtggtgatcg atgagatcgt ataaagacac tgctaggtgt taaggatggg 17820

ataataagat gtgttttaag tcattaaccg taataaaaag agagagaggc tgatggaatg 17880

ttatgtatgt atgtttcttg gtttttaaaa ttaaatggaa agcacatgct cgtgtgggtt 17940

ctatctcgat taaaaatccc aattatattt ggtctaattt agtttggtat tgagtaaaac 18000

aaattcgaac caaaccaaaa tataaatata tagtttttat atatatgcct ttaagacttt 18060

ttatagaatt ttctttaaaa aatatctaga aatatttgcg actcttctgg catgtaatat 18120

ttcgttaaat atgaagtgct ccatttttat taactttaaa taattggttg tacgatcact 18180

ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt cttccatatt catatgtcaa 18240

aatctatcaa aattcttata tatctttttc gaatttgaag tgaaatttcg ataatttaaa 18300

attaaataga acatatcatt atttaggtat catattgatt tttatactta attactaaat 18360

ttggttaact ttgaaagtgt acatcaacga aaaattagtc aaacgactaa aataaataaa 18420

tatcatgtgt tattaagaaa attctcctat aagaatattt taatagatca tatgtttgta 18480

aaaaaaatta atttttacta acacatatat ttacttatca aaaatttgac aaagtaagat 18540

taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc ggcaaaaccg 18600

aaccaatcca aaccgatata gttggtttgg tttgattttg atataaaccg aaccaactcg 18660

gtccatttgc acccctaatc ataatagctt taatatttca agatattatt aagttaacgt 18720

tgtcaatatc ctggaaattt tgcaaaatga atcaagccta tatggctgta atatgaattt 18780

aaaagcagct cgatgtggtg gtaatatgta atttacttga ttctaaaaaa atatcccaag 18840

tattaataat ttctgctagg aagaaggtta gctacgattt acagcaaagc cagaatacaa 18900

agaaccataa agtgattgaa gctcgaaata tacgaaggaa caaatatttt taaaaaaata 18960

cgcaatgact tggaacaaaa gaaagtgata tattttttgt tcttaaacaa gcatcccctc 19020

taaagaatgg cagttttcct ttgcatgtaa ctattatgct cccttcgtta caaaaatttt 19080

ggactactat tgggaacttc ttctgaaaat agtcctgcag gctagtagat tggttggttg 19140

gtttccatgt accagaaggc ttaccctatt agttgaaagt tgaaactttg ttccctactc 19200

aattcctagt tgtgtaaatg tatgtatatg taatgtgtat aaaacgtagt acttaaatga 19260

ctaggagtgg ttcttgagac cgatgagaga tgggagcaga actaaagatg atgacataat 19320

taagaacgaa tttgaaaggc tcttaggttt gaatcctatt cgagaatgtt tttgtcaaag 19380

atagtggcga ttttgaacca aagaaaacat ttaaaaaatc agtatccggt tacgttcatg 19440

caaatagaaa gtggtctagg atctgattgt aattttagac ttaaagagtc tcttaagatt 19500

caatcctggc tgtgtacaaa actacaaata atatatttta gactatttgg ccttaactaa 19560

acttccactc attatttact gaggttagag aatagacttg cgaataaaca cattcccgag 19620

aaatactcat gatcccataa ttagtcagag ggtatgccaa tcagatctaa gaacacacat 19680

tccctcaaat tttaatgcac atgtaatcat agtttagcac aattcaaaaa taatgtagta 19740

ttaaagacag aaatttgtag actttttttt ggcgttaaaa gaagactaag tttatacgta 19800

cattttattt taagtggaaa accgaaattt tccatcgaaa tatatgaatt tagtatatat 19860

atttctgcaa tgtactattt tgctattttg gcaactttca gtggactact actttattac 19920

aatgtgtatg gatgcatgag tttgagtata cacatgtcta aatgcatgct ttgtaaaacg 19980

taacggacca caaaagagga tccatacaaa tacatctcat agcttcctcc attattttcc 20040

gacacaaaca gagcatttta caacaattac caacaacaac aaacaacaaa caacattaca 20100

attacattta caattaccat accatggcct ctatcgctat ccctgctgct cttgctggaa 20160

ctcttggata cgttacctac aatgtggcta accctgatat cccagcttct gagaaagttc 20220

ctgcttactt catgcaggtt gagtactggg gacctactat cggaactatt ggatacctcc 20280

tcttcatcta cttcggaaag cgtatcatgc agaacagatc tcaacctttc ggactcaaga 20340

acgctatgct cgtttacaac ttctaccaga ccttcttcaa cagctactgc atctaccttt 20400

tcgttacttc tcatagggct cagggactta aggtttgggg aaacatccct gatatgactg 20460

ctaactcttg gggaatctct caggttatct ggcttcacta caacaacaag tacgttgagc 20520

ttctcgacac cttcttcatg gtgatgagga agaagttcga ccagctttct ttccttcaca 20580

tctaccacca cactcttctc atctggtcat ggttcgttgt tatgaagctt gagcctgttg 20640

gagattgcta cttcggatct tctgttaaca ccttcgtgca cgtgatcatg tactcttact 20700

acggacttgc tgctcttgga gttaactgtt tctggaagaa gtacatcacc cagatccaga 20760

tgcttcagtt ctgtatctgt gcttctcact ctatctacac cgcttacgtt cagaataccg 20820

ctttctggct tccttacctt caactctggg ttatggtgaa catgttcgtt ctcttcgcca 20880

acttctaccg taagaggtac aagtctaagg gtgctaagaa gcagtgataa gggccgccgc 20940

catgtgacag atcgaaggaa gaaagtgtaa taagacgact ctcactactc gatcgctagt 21000

gattgtcatt gttatatata ataatgttat ctttcacaac ttatcgtaat gcatgtgaaa 21060

ctataacaca ttaatcctac ttgtcatatg ataacactct ccccatttaa aactcttgtc 21120

aatttaaaga tataagattc tttaaatgat taaaaaaaat atattataaa ttcaatcact 21180

cctactaata aattattaat tattatttat tgattaaaaa aatacttata ctaatttagt 21240

ctgaatagaa taattagatt ctagcctgca gggcggccgc ggatcccatg gagtcaaaga 21300

ttcaaataga ggacctaaca gaactcgccg taaagactgg cgaacagttc atacagagtc 21360

tcttacgact caatgacaag aagaaaatct tcgtcaacat ggtggagcac gacacacttg 21420

tctactccaa aaatatcaaa gatacagtct cagaagacca aagggcaatt gagacttttc 21480

aacaaagggt aatatccgga aacctcctcg gattccattg cccagctatc tgtcacttta 21540

ttgtgaagat agtggaaaag gaaggtggct cctacaaatg ccatcattgc gataaaggaa 21600

aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa agatggaccc ccacccacga 21660

ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg 21720

atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa gacccttcct 21780

ctatataagg aagttcattt catttggaga gaacacgggg gactgaatta aatatgagcc 21840

ctgagaggcg tcctgttgaa atcagacctg ctactgctgc tgatatggct gctgtttgtg 21900

atatcgtgaa ccactacatc gagacttcta ccgttaactt cagaactgag cctcaaactc 21960

ctcaagagtg gatcgatgat cttgagagac tccaagatag atacccttgg cttgttgctg 22020

aggttgaggg tgttgttgct ggaatcgctt acgctggacc ttggaaggct agaaacgctt 22080

acgattggac tgttgagtct accgtttacg tttcacacag acatcagaga cttggacttg 22140

gatctaccct ttacactcac cttctcaagt ctatggaagc tcagggattc aagtctgttg 22200

ttgctgttat cggactccct aacgatcctt ctgttagact tcatgaggct cttggataca 22260

ctgctagagg aactcttaga gctgctggat acaagcacgg tggatggcat gatgttggat 22320

tctggcaaag agatttcgag cttcctgctc ctcctagacc tgttagacca gttactcaga 22380

tctgaatttg cgtgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 22440

tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 22500

taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 22560

atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 22620

cgcggtgtca tctatgttac tagatcacta gtgatgtacg gttaaaacca ccccagtaca 22680

ttaaaaacgt ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt acaccacaat 22740

atatcctgcc accagccagc caacagctcc ccgaccggca gctcggcaca aaatcaccac 22800

tcgatacagg cagcccatca gtcc 22824

<210> SEQ ID NO: 5

<211> LENGTH: 24809

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pga7- mod_e nucleotide sequence

<400> SEQENCE: 5

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgcctcgatt aaaaatccca attatatttg gtctaattta 240

gtttggtatt gagtaaaaca aattcgaacc aaaccaaaat ataaatatat agtttttata 300

tatatgcctt taagactttt tatagaattt tctttaaaaa atatctagaa atatttgcga 360

ctcttctggc atgtaatatt tcgttaaata tgaagtgctc catttttatt aactttaaat 420

aattggttgt acgatcactt tcttatcaag tgttactaaa atgcgtcaat ctctttgttc 480

ttccatattc atatgtcaaa atctatcaaa attcttatat atctttttcg aatttgaagt 540

gaaatttcga taatttaaaa ttaaatagaa catatcatta tttaggtatc atattgattt 600

ttatacttaa ttactaaatt tggttaactt tgaaagtgta catcaacgaa aaattagtca 660

aacgactaaa ataaataaat atcatgtgtt attaagaaaa ttctcctata agaatatttt 720

aatagatcat atgtttgtaa aaaaaattaa tttttactaa cacatatatt tacttatcaa 780

aaatttgaca aagtaagatt aaaataatat tcatctaaca aaaaaaaaac cagaaaatgc 840

tgaaaacccg gcaaaaccga accaatccaa accgatatag ttggtttggt ttgattttga 900

tataaaccga accaactcgg tccatttgca cccctaatca taatagcttt aatatttcaa 960

gatattatta agttaacgtt gtcaatatcc tggaaatttt gcaaaatgaa tcaagcctat 1020

atggctgtaa tatgaattta aaagcagctc gatgtggtgg taatatgtaa tttacttgat 1080

tctaaaaaaa tatcccaagt attaataatt tctgctagga agaaggttag ctacgattta 1140

cagcaaagcc agaatacaaa gaaccataaa gtgattgaag ctcgaaatat acgaaggaac 1200

aaatattttt aaaaaaatac gcaatgactt ggaacaaaag aaagtgatat attttttgtt 1260

cttaaacaag catcccctct aaagaatggc agttttcctt tgcatgtaac tattatgctc 1320

ccttcgttac aaaaattttg gactactatt gggaacttct tctgaaaata gtggcgcccc 1380

gcggaaagct tgctagccaa ttggggccca acgttctcga gtttttctag aaggaaactg 1440

aaggcgggaa acgacaatct gctagtggat ctcccagtca cgacgttgta aaacgggcgc 1500

cccgcggaaa gcttgcggcc gcggtaccgc ccgttcgact cagatcttcc aaggcctcgt 1560

ctccgagtcc gctgcttctc gccgcgccga tcacttctcc gccgccaaca aggcttgtag 1620

ttaataggaa tcattcaggg attgtgattc cgggcagtag taattaataa tatagtatta 1680

gtatagataa tatgtttcgt ttgggatctt tggaacgttg ctctgttcct tgttgttcat 1740

tttaaagctt ttgagggata gttgcagaac tgttcggtga tgcttcatcc tctcaagaac 1800

tagatttggg taaagaaaca tccatgcatg gatatggaat gttgttcttc cgattggaga 1860

ttattttata aaatttaaaa ttcatgattt aaaaaaacac ataaaaacca caaaattcat 1920

gatttattga caatacgata caaaattagc accaccggct actggctcat tacacatttc 1980

cccttcccct cattctcact ttgtggcttt attattatta ttattacata tattttaccg 2040

ttattatttc acgtcacata agcttgttaa ttaatcatta gtgagccttc tcagcctttc 2100

cgttaacgta gtagtgctgt cccaccttat caaggttaga gaaagtagcc ttccaagcac 2160

cgtagtaaga gagcaccttg tagttgagtc cccacttctt agcgaaagga acgaatcttc 2220

tgctaacctc aggctgtctg aattgaggca tatcagggaa gaggtggtgg ataacctgac 2280

agttaaggta tcccataagc cagttcacgt atcctctaga aggatcgata tcaacggtgt 2340

gatcaacagc gtagttaacc caagaaaggt gcttatcaga tggaacaaca gggaggtgag 2400

tatgagaagt agagaagtga gcgaaaaggt acatgtaagc gatccagttt ccgaaagtga 2460

accaccagta agcaacaggc caagagtatc cagtagcaag cttgataaca gcggttctaa 2520

caacatgaga aacgagcatc caagaagcct cttcgtagtt cttcttacgg agaacttgtc 2580

tagggtggag aacgtagatc cagaaagctt gaacaagaag tccagaggta acaggaacga 2640

aagtccaagc ttgaagtcta gcccaagctc tagagaatcc tctaggtctg ttatcctcaa 2700

cagcagtgtt gaagaaagcc acagcaggag tggtatcaag atccatatcg tgtctaacct 2760

tttgaggggt agcatggtgc ttgttatgca tctggttcca catctcacca gaagtagaaa 2820

gtccgaatcc acaagtcata gcctgaagtc tcttgtccac gtaaacagat ccggtaagag 2880

agttatgtcc accctcatgt tgaacccatc cacatctagc tccgaagaaa gcaccgtaaa 2940

caacagaagc aatgataggg tatccagcgt acataagagc agttccaaga gcgaatgtag 3000

caagaagctc gagaagtctg taagccacat gggtgataga aggcttgaag aatccatctc 3060

tctcaagctc agcacgccat ctagcgaaat cctcaagcat aggagcatcc tcagactcag 3120

atctcttgat ctcagcaggt ctagaaggca aagctctaag catcttccaa gccttgagag 3180

aacgcatgtg gaattctttg aaagcctcag tagcatcagc accagtgtta gcaagcatgt 3240

agaagatcac agatccacca gggtgcttga agttagtcac atcgtactca acgtcctcaa 3300

ctctaaccca tctagtctcg aaagtagcag caagctcatg aggctcaaga gtcttaagat 3360

caacaggagc agtagaagca tccttagcat caagagcctc agcagaagat ttagacctgg 3420

taagtggaga tctaggagaa gatcttccat cagtcttagg agggcacatg gtatggtaat 3480

tgtaaatgta attgtaatgt tgtttgttgt ttgttgttgt tggtaattgt tgtaaaatta 3540

attaagtggg tatcttttgg atggataagc aagtagtgat gatgttctag gtgaagtgat 3600

gggggtgttt tatagcggga gatggtgaaa tggatggtcg ccacataaga aatggagggg 3660

aagggttctt gcgccattct tcagtttgca tggatgcatg ggtttcattt tgtaacacgt 3720

aataaggaca atgaagtgca ggtgtctctc aagtttcaga ggggatatgt ggacagaaga 3780

agaacggcga tgatattgat ggaaatggcc atctagtgtg aatctattcg gttgataata 3840

ctagtgcatt ttggccgtta atcccttcaa ttaactgcac aaacttcagt tgagtattga 3900

ttatttgatt ataggttctg taaacacaat accaagttta tttagagggg agacatacaa 3960

atagtttcga tataaataat agagtggtta aacttagtta ttaaaactat atataaagtc 4020

taaaagttaa attatttttt taattgcaaa tatataaagt ctaaaggggt tacattattt 4080

cttaagagat gtaactctgt tggaatctga cttaatccgt ctcatcactc tggtttccag 4140

ttctaatcta atgaattgtt ttctgccaaa gaatttgaag caagaagtaa attgatcaat 4200

gccgtcaacc cacaccaaac cgtcaaccca ctaccatcgc cgcggagacc cccaaactca 4260

acctccaccc atcggtaaga agcacagggc agcccgcacc accaccaatt tggcgtgcat 4320

gacacctagg gacttggcac gggaggcggc gcacgtggat gcaaatgacg ggatatcaga 4380

tgacaggaaa cgacgttgag agaccatacg atgtagaata tgagctcacc atcaacgaga 4440

aactaggaaa atcacaaaaa aaacaactct cgtaattgta cgagtggcac agatgggtct 4500

gcctcaacat atctctaata cggcgaagcc tgcccaacac gtagttgccg gaatccggtg 4560

tggagctcac gactctgaaa gataggcgct tcctgtttcg tttcgctcac ccactggacg 4620

tccgtcatgt gatggatttc ggtcattggt ttgctgacaa ccacattctg aagctccatg 4680

agatgagtct tcacaatagg tcctgctcaa taccgtggag ttatggttgc aagtccataa 4740

cttgccgttc gaatattttg cggagccagt cggacgggaa ttggcgagct cggctgacac 4800

ctataaaggc catgacaaga agaaccaaaa gttcttccct aatgctttca tgaggcttcg 4860

ggtcgttatg gatgtcggaa aacccctctt gaaggaacga gacgttatta tgcatgacgg 4920

taagactatt acttgtcagt ataagtatga aagattacct gtcttctgct ttgtttgtgg 4980

attgattgga cacgttgaaa aaaaatgtgc acttcgattt caatactcag agatcgactt 5040

cccttttctc taggagtatt cgatcaaggc attaacatgg aaggaagctc aagctctaaa 5100

ggcttcacaa tggaacctga aaaatttcaa caagcctaaa ctgaaatcga agtcaaatca 5160

cccaaccggg agctctaaat cagcaaacac tcctcctcca cagtatccaa tcatcgtgca 5220

cgatgctcca ggtattgcaa gccaggtatt gcaagctagg agtaggatag agaccttaaa 5280

cgtcgttggt gtgaagagtc atcttcagac ctaatggaga tagatgtaga cggcggcacg 5340

aagactctga aacaccagaa aggctagtcc aggataagga tctgctatcc caactgacct 5400

ctcgttagtc ccaaggcctc tcaactagag caggaggaag gatggtcaca agactaggat 5460

aatgatgttt ccaatatgaa cctgaatgtc catagctaat ttttttagtc ttgcttctgc 5520

actttttgtt tattatgttc tggtgactat gttatttacc cttgtccgta tgcttgaggg 5580

taccctagta gattggttgg ttggtttcca tgtaccagaa ggcttaccct attagttgaa 5640

agttgaaact ttgttcccta ctcaattcct agttgtgtaa atgtatgtat atgtaatgtg 5700

tataaaacgt agtacttaaa tgactaggag tggttcttga gaccgatgag agatgggagc 5760

agaactaaag atgatgacat aattaagaac gaatttgaaa ggctcttagg tttgaatcct 5820

attcgagaat gtttttgtca aagatagtgg cgattttgaa ccaaagaaaa catttaaaaa 5880

atcagtatcc ggttacgttc atgcaaatag aaagtggtct aggatctgat tgtaatttta 5940

gacttaaaga gtctcttaag attcaatcct ggctgtgtac aaaactacaa ataatatatt 6000

ttagactatt tggccttaac taaacttcca ctcattattt actgaggtta gagaatagac 6060

ttgcgaataa acacattccc gagaaatact catgatccca taattagtca gagggtatgc 6120

caatcagatc taagaacaca cattccctca aattttaatg cacatgtaat catagtttag 6180

cacaattcaa aaataatgta gtattaaaga cagaaatttg tagacttttt tttggcgtta 6240

aaagaagact aagtttatac gtacatttta ttttaagtgg aaaaccgaaa ttttccatcg 6300

aaatatatga atttagtata tatatttctg caatgtacta ttttgctatt ttggcaactt 6360

tcagtggact actactttat tacaatgtgt atggatgcat gagtttgagt atacacatgt 6420

ctaaatgcat gctttgtaaa acgtaacgga ccacaaaaga ggatccatac aaatacatct 6480

catagcttcc tccattattt tccgacacaa acagagcatt ttacaacaat taccaacaac 6540

aacaaacaac aaacaacatt acaattacat ttacaattac cataccatgg cctctatcgc 6600

tatccctgct gctcttgctg gaactcttgg atacgttacc tacaatgtgg ctaaccctga 6660

tatcccagct tctgagaaag ttcctgctta cttcatgcag gttgagtact ggggacctac 6720

tatcggaact attggatacc tcctcttcat ctacttcgga aagcgtatca tgcagaacag 6780

atctcaacct ttcggactca agaacgctat gctcgtttac aacttctacc agaccttctt 6840

caacagctac tgcatctacc ttttcgttac ttctcatagg gctcagggac ttaaggtttg 6900

gggaaacatc cctgatatga ctgctaactc ttggggaatc tctcaggtta tctggcttca 6960

ctacaacaac aagtacgttg agcttctcga caccttcttc atggtgatga ggaagaagtt 7020

cgaccagctt tctttccttc acatctacca ccacactctt ctcatctggt catggttcgt 7080

tgttatgaag cttgagcctg ttggagattg ctacttcgga tcttctgtta acaccttcgt 7140

gcacgtgatc atgtactctt actacggact tgctgctctt ggagttaact gtttctggaa 7200

gaagtacatc acccagatcc agatgcttca gttctgtatc tgtgcttctc actctatcta 7260

caccgcttac gttcagaata ccgctttctg gcttccttac cttcaactct gggttatggt 7320

gaacatgttc gttctcttcg ccaacttcta ccgtaagagg tacaagtcta agggtgctaa 7380

gaagcagtga taaggcgcgc ggcgcgccgg gccgccgcca tgtgacagat cgaaggaaga 7440

aagtgtaata agacgactct cactactcga tcgctagtga ttgtcattgt tatatataat 7500

aatgttatct ttcacaactt atcgtaatgc atgtgaaact ataacacatt aatcctactt 7560

gtcatatgat aacactctcc ccatttaaaa ctcttgtcaa tttaaagata taagattctt 7620

taaatgatta aaaaaaatat attataaatt caatcactcc tactaataaa ttattaatta 7680

ttatttattg attaaaaaaa tacttatact aatttagtct gaatagaata attagattct 7740

agtctcatcc ccttttaaac caacttagta aacgtttttt tttttaattt tatgaagtta 7800

agtttttacc ttgtttttaa aaagaatcgt tcataagatg ccatgccaga acattagcta 7860

cacgttacac atagcatgca gccgcggaga attgtttttc ttcgccactt gtcactccct 7920

tcaaacacct aagagcttct ctctcacagc acacacatac aatcacatgc gtgcatgcat 7980

tattacacgt gatcgccatg caaatctcct ttatagccta taaattaact catccgcttc 8040

actctttact caaaccaaaa ctcatcgata caaacaagat taaaaacata cacgaggatc 8100

ttttacaaca attaccaaca acaacaaaca acaaacaaca ttacaattac atttacaatt 8160

accataccat gcctccaagg gactcttact cttatgctgc tcctccttct gctcaacttc 8220

acgaagttga tactcctcaa gagcacgaca agaaagagct tgttatcgga gatagggctt 8280

acgatgttac caacttcgtt aagagacacc ctggtggaaa gatcattgct taccaagttg 8340

gaactgatgc taccgatgct tacaagcagt tccatgttag atctgctaag gctgacaaga 8400

tgcttaagtc tcttccttct cgtcctgttc acaagggata ctctccaaga agggctgatc 8460

ttatcgctga tttccaagag ttcaccaagc aacttgaggc tgagggaatg ttcgagcctt 8520

ctcttcctca tgttgcttac agacttgctg aggttatcgc tatgcatgtt gctggtgctg 8580

ctcttatctg gcatggatac actttcgctg gaatcgctat gcttggagtt gttcagggaa 8640

gatgtggatg gcttatgcat gagggtggac attactctct cactggaaac attgctttcg 8700

acagagctat ccaagttgct tgttacggac ttggatgtgg aatgtctggt gcttggtggc 8760

gtaaccagca taacaagcac catgctactc ctcaaaagct tcagcacgat gttgatcttg 8820

atacccttcc tctcgttgct ttccatgaga gaatcgctgc taaggttaag tctcctgcta 8880

tgaaggcttg gctttctatg caagctaagc ttttcgctcc tgttaccact cttcttgttg 8940

ctcttggatg gcagctttac cttcatccta gacacatgct caggactaag cactacgatg 9000

agcttgctat gctcggaatc agatacggac ttgttggata ccttgctgct aactacggtg 9060

ctggatacgt tctcgcttgt taccttcttt acgttcagct tggagctatg tacatcttct 9120

gcaacttcgc tgtttctcat actcacctcc ctgttgttga gcctaacgag catgctactt 9180

gggttgagta cgctgctaac cacactacta actgttctcc atcttggtgg tgtgattggt 9240

ggatgtctta ccttaactac cagatcgagc accaccttta cccttctatg cctcaattca 9300

gacaccctaa gatcgctcct agagttaagc agcttttcga gaagcacgga cttcactacg 9360

atgttagagg atacttcgag gctatggctg atactttcgc taaccttgat aacgttgccc 9420

atgctcctga gaagaaaatg cagtaatgag atcgttcaaa catttggcaa taaagtttct 9480

taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg 9540

ttaagcacgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga 9600

ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact 9660

aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcggtcgatt aaaaatccca 9720

attatatttg gtctaattta gtttggtatt gagtaaaaca aattcgaacc aaaccaaaat 9780

ataaatatat agtttttata tatatgcctt taagactttt tatagaattt tctttaaaaa 9840

atatctagaa atatttgcga ctcttctggc atgtaatatt tcgttaaata tgaagtgctc 9900

catttttatt aactttaaat aattggttgt acgatcactt tcttatcaag tgttactaaa 9960

atgcgtcaat ctctttgttc ttccatattc atatgtcaaa atctatcaaa attcttatat 10020

atctttttcg aatttgaagt gaaatttcga taatttaaaa ttaaatagaa catatcatta 10080

tttaggtatc atattgattt ttatacttaa ttactaaatt tggttaactt tgaaagtgta 10140

catcaacgaa aaattagtca aacgactaaa ataaataaat atcatgtgtt attaagaaaa 10200

ttctcctata agaatatttt aatagatcat atgtttgtaa aaaaaattaa tttttactaa 10260

cacatatatt tacttatcaa aaatttgaca aagtaagatt aaaataatat tcatctaaca 10320

aaaaaaaaac cagaaaatgc tgaaaacccg gcaaaaccga accaatccaa accgatatag 10380

ttggtttggt ttgattttga tataaaccga accaactcgg tccatttgca cccctaatca 10440

taatagcttt aatatttcaa gatattatta agttaacgtt gtcaatatcc tggaaatttt 10500

gcaaaatgaa tcaagcctat atggctgtaa tatgaattta aaagcagctc gatgtggtgg 10560

taatatgtaa tttacttgat tctaaaaaaa tatcccaagt attaataatt tctgctagga 10620

agaaggttag ctacgattta cagcaaagcc agaatacaaa gaaccataaa gtgattgaag 10680

ctcgaaatat acgaaggaac aaatattttt aaaaaaatac gcaatgactt ggaacaaaag 10740

aaagtgatat attttttgtt cttaaacaag catcccctct aaagaatggc agttttcctt 10800

tgcatgtaac tattatgctc ccttcgttac aaaaattttg gactactatt gggaacttct 10860

tctgaaaata gtgatagaac ccacacgagc atgtgctttc catttaattt taaaaaccaa 10920

gaaacataca tacataacat tccatcagcc tctctctctt tttattacgg ttaatgactt 10980

aaaacacatc ttattatccc atccttaaca cctagcagtg tctttatacg atctcatcga 11040

tcaccacttc aaaaccatgc agactgctgc tgcccctgga gctggcatcg gctaggctgg 11100

gtgccgcact gtcccggaag gtccctagcg acttgtttag attgatggga ccacctctca 11160

acttcctgct gctgtccctg ctgctggatg tcctgcctca tctggccgat tgcacgctcc 11220

agtcccctgc atgtgcactc gctcctcaat tgcttaagat catcgcagca gctatcgaag 11280

tgctggctct gttgccctcc tccacggcct tggttgtagt agtagctgcc gccgcccttc 11340

tggacttttt cccacaggaa ccgccgaata attcgataga accacacgag catgtgcttt 11400

catttatttt aaaaaccaag aaacatacat aacatttcat cagcctctct ctctctctct 11460

ctctctctct ctctctctct ctctctctct ctctctttat tacagctgtt acactaactt 11520

aaaacacatt catctcatta ttattattat tatccatcct taacacctag cagtgtcttt 11580

gtacgatctc ataatcgatc accccttcat caggtatcct taggcttcac tccaacgttg 11640

ttgcagttac ggaacatgta cacaccatca tggttctcaa cgaactggca agatctccaa 11700

gttttccaaa ggctaaccca catgttctca tcggtgtgtc tgtagtgctc tcccataact 11760

ttcttgatgc actcggtagc ttctctagca tggtagaatg ggatccttga aacgtagtga 11820

tggagcacat gagtctcgat gatgtcatgg aagatgattc cgaggattcc gaactctcta 11880

tcgatagtag cagcagcacc cttagcgaaa gtccactctt gagcatcgta atgaggcata 11940

gaagaatcgg tgtgctgaag gaaggtaacg aaaacaagcc agtggttaac aaggatccaa 12000

ggacagaacc atgtgatgaa agtaggccag aatccgaaaa ccttgtaagc ggtgtaaaca 12060

gaagtgaggg tagcaaggat tccaagatca gaaagaacga tgtaccagta gtccttctta 12120

tcgaaaacag ggctagaagg ccagtagtga gacttgaaga acttagaaac accagggtaa 12180

ggttgtccag tagcgttagt agcaaggtaa agagaaagtc ctccaagctg ttggaacaag 12240

agagcgaaaa cagagtagat aggagtttcc tcagcgatat cgtgaaggct ggtaacttgg 12300

tgcttctctt tgaattcctc ggcggtgtaa ggaacgaaaa ccatatctct ggtcatgtgt 12360

ccagtagcct tatggtgctt agcatgagag aacttccagc tgaagtaagg aaccataaca 12420

agagagtgga gaacccatcc aacggtatcg ttaacccatc cgtagttaga gaaagcagaa 12480

tgtccacact catgtccaag gatccagatt ccgaatccga aacaagagat agagaacacg 12540

taagcagacc aagcagcgaa tctaaggaat tcgttaggga gaagagggat gtaggtaagt 12600

ccaacgtaag cgatagcaga gatagccacg atatctctca ccacgtaaga catagacttc 12660

acgagagatc tctcgtaaca gtgcttaggg atagcgtcaa ggatatcctt gatggtgtaa 12720

tctggcacct tgaaaacgtt tccgaaggta tcgatagcgg tcttttgctg cttgaaagat 12780

gcaacgtttc cagaacgcct aacggtctta gtagatccct caaggatctc agatccagac 12840

acggtaacct tagacatggt atggtaattg taaatgtaat tgtaatgttg tttgttgttt 12900

gttgttgttg gtaattgttg taaaattttt ggtggtgatt ggttctttaa ggtgtgagag 12960

tgagttgtga gttgtgtggt gggtttggtg agattgggga tggtgggttt atatagtgga 13020

gactgaggaa tggggtcgtg agtgttaact ttgcatgggc tacacgtggg ttcttttggg 13080

cttacacgta gtattattca tgcaaatgca gccaatacat atacggtatt ttaataatgt 13140

gtgggaatac aatatgccga gtattttact aattttggca atgacaagtg tacatttgga 13200

ttatcttact tggcctctct tgctttaatt tggattattt ttattctctt accttggccg 13260

ttcatattca catccctaaa ggcaagacag aattgaatgg tggccaaaaa ttaaaacgat 13320

ggatatgacc tacatagtgt aggatcaatt aacgtcgaag gaaaatactg attctctcaa 13380

gcatacggac aagggtaaat aacatagtca ccagaacata ataaacaaaa agtgcagaag 13440

caagactaaa aaaattagct atggacattc aggttcatat tggaaacatc attatcctag 13500

tcttgtgacc atccttcctc ctgctctagt tgagaggcct tgggactaac gagaggtcag 13560

ttgggatagc agatccttat cctggactag cctttctggt gtttcagagt cttcgtgccg 13620

ccgtctacat ctatctccat taggtctgaa gatgactctt cacaccaacg acgtttaagg 13680

tctctatcct actcctagct tgcaatacct ggcttgcaat acctggagca tcgtgcacga 13740

tgattggata ctgtggagga ggagtgtttg ctgatttaga gctcccggtt gggtgatttg 13800

acttcgattt cagtttaggc ttgttgaaat ttttcaggtt ccattgtgaa gcctttagag 13860

cttgagcttc cttccatgtt aatgccttga tcgaatactc ctagagaaaa gggaagtcga 13920

tctctgagta ttgaaatcga agtgcacatt ttttttcaac gtgtccaatc aatccacaaa 13980

caaagcagaa gacaggtaat ctttcatact tatactgaca agtaatagtc ttaccgtcat 14040

gcataataac gtctcgttcc ttcaagaggg gttttccgac atccataacg acccgaagcc 14100

tcatgaaagc attagggaag aacttttggt tcttcttgtc atggccttta taggtgtcag 14160

ccgagctcgc caattcccgt ccgactggct ccgcaaaata ttcgaacggc aagttatgga 14220

cttgcaacca taactccacg gtattgagca ggacctattg tgaagactca tctcatggag 14280

cttcagaatg tggttgtcag caaaccaatg accgaaatcc atcacatgac ggacgtccag 14340

tgggtgagcg aaacgaaaca ggaagcgcct atctttcaga gtcgtgagct ccacaccgga 14400

ttccggcaac tacgtgttgg gcaggcttcg ccgtattaga gatatgttga ggcagaccca 14460

tctgtgccac tcgtacaatt acgagagttg ttttttttgt gattttccta gtttctcgtt 14520

gatggtgagc tcatattcta catcgtatgg tctctcaacg tcgtttcctg tcatctgata 14580

tcccgtcatt tgcatccacg tgcgccgcct cccgtgccaa gtccctaggt gtcatgcacg 14640

ccaaattggt ggtggtgcgg gctgccctgt gcttcttacc gatgggtgga ggttgagttt 14700

gggggtctcc gcggcgatgg tagtgggttg acggtttggt gtgggttgac ggcattgatc 14760

aatttacttc ttgcttcaaa ttctttggca gaaaacaatt cattagatta gaactggaaa 14820

ccagagtgat gagacggatt aagtcagatt ccaacagagt tacatctctt aagaaataat 14880

gtaacccctt tagactttat atatttgcaa ttaaaaaaat aatttaactt ttagacttta 14940

tatatagttt taataactaa gtttaaccac tctattattt atatcgaaac tatttgtatg 15000

tctcccctct aaataaactt ggtattgtgt ttacagaacc tataatcaaa taatcaatac 15060

tcaactgaag tttgtgcagt taattgaagg gattaacggc caaaatgcac tagtattatc 15120

aaccgaatag attcacacta gatggccatt tccatcaata tcatcgccgt tcttcttctg 15180

tccacatatc ccctctgaaa cttgagagac acctgcactt cattgtcctt attacgtgtt 15240

acaaaatgaa acccatgcat ccatgcaaac tgaagaatgg cgcaagaacc cttcccctcc 15300

atttcttatg tggcgaccat ccatttcacc atctcccgct ataaaacacc cccatcactt 15360

cacctagaac atcatcacta cttgcttatc catccaaaag atacccactt ttacaacaat 15420

taccaacaac aacaaacaac aaacaacatt acaattacat ttacaattac cataccatgc 15480

cacctagcgc tgctaagcaa atgggagctt ctactggtgt tcatgctggt gttactgact 15540

cttctgcttt caccagaaag gatgttgctg atagacctga tctcaccatc gttggagatt 15600

ctgtttacga tgctaaggct ttcagatctg agcatcctgg tggtgctcat ttcgtttctt 15660

tgttcggagg aagagatgct actgaggctt tcatggaata ccatagaagg gcttggccta 15720

agtctagaat gtctagattc cacgttggat ctcttgcttc tactgaggaa cctgttgctg 15780

ctgatgaggg ataccttcaa ctttgtgcta ggatcgctaa gatggtgcct tctgtttctt 15840

ctggattcgc tcctgcttct tactgggtta aggctggact tatccttgga tctgctatcg 15900

ctcttgaggc ttacatgctt tacgctggaa agagacttct cccttctatc gttcttggat 15960

ggcttttcgc tcttatcggt cttaacatcc agcatgatgc taaccatggt gctttgtcta 16020

agtctgcttc tgttaacctt gctcttggac tttgtcagga ttggatcgga ggatctatga 16080

tcctttggct tcaagagcat gttgttatgc accacctcca cactaacgat gttgataagg 16140

atcctgatca aaaggctcac ggtgctctta gactcaagcc tactgatgct tggtcaccta 16200

tgcattggct tcagcatctt taccttttgc ctggtgagac tatgtacgct ttcaagcttt 16260

tgttcctcga catctctgag cttgttatgt ggcgttggga gggtgagcct atctctaagc 16320

ttgctggata cctctttatg ccttctttgc ttctcaagct taccttctgg gctagattcg 16380

ttgctttgcc tctttacctt gctccttctg ttcatactgc tgtgtgtatc gctgctactg 16440

ttatgactgg atctttctac ctcgctttct tcttcttcat ctcccacaac ttcgagggtg 16500

ttgcttctgt tggacctgat ggatctatca cttctatgac tagaggtgct agcttcctta 16560

agagacaagc tgagacttct tctaacgttg gaggacctct tcttgctact cttaacggtg 16620

gactcaacta ccaaattgag catcacttgt tccctagagt tcaccatgga ttctacccta 16680

gacttgctcc tcttgttaag gctgagcttg aggctagagg aatcgagtac aagcactacc 16740

ctactatctg gtctaacctt gcttctaccc tcagacatat gtacgctctt ggaagaaggc 16800

ctagatctaa ggctgagtaa tgacaagctt atgtgacgtg aaataataac ggtaaaatat 16860

atgtaataat aataataata aagccacaaa gtgagaatga ggggaagggg aaatgtgtaa 16920

tgagccagta gccggtggtg ctaattttgt atcgtattgt caataaatca tgaattttgt 16980

ggtttttatg tgttttttta aatcatgaat tttaaatttt ataaaataat ctccaatcgg 17040

aagaacaaca ttccatatcc atgcatggat gtttctttac ccaaatctag ttcttgagag 17100

gatgaagcat caccgaacag ttctgcaact atccctcaaa agctttaaaa tgaacaacaa 17160

ggaacagagc aacgttccaa agatcccaaa cgaaacatat tatctatact aatactatat 17220

tattaattac tactgcccgg aatcacaatc cctgaatgat tcctattaac tacaagcctt 17280

gttggcggcg gagaagtgat cggcgcggcg agaagcagcg gactcggaga cgaggccttg 17340

gaagatctga gtcgaacggg cagaatcagt attttccttc gacgttaatt gatcctacac 17400

tatgtaggtc atatccatcg ttttaatttt tggccaccat tcaattctgt cttgccttta 17460

gggatgtgaa tatgaacggc caaggtaaga gaataaaaat aatccaaatt aaagcaagag 17520

aggccaagta agataatcca aatgtacact tgtcattgcc aaaattagta aaatactcgg 17580

catattgtat tcccacacat tattaaaata ccgtatatgt attggctgca tttgcatgaa 17640

taatactacg tgtaagccca aaagaaccca cgtgtagccc atgcaaagtt aacactcacg 17700

accccattcc tcagtctcca ctatataaac ccaccatccc caatctcacc aaacccacca 17760

cacaactcac aactcactct cacaccttaa agaaccaatc accaccaaaa attttacaac 17820

aattaccaac aacaacaaac aacaaacaac attacaatta catttacaat taccatacca 17880

tgagcgctgt taccgttact ggatctgatc ctaagaacag aggatcttct agcaacaccg 17940

agcaagaggt tccaaaagtt gctatcgata ccaacggaaa cgtgttctct gttcctgatt 18000

tcaccatcaa ggacatcctt ggagctatcc ctcatgagtg ttacgagaga agattggcta 18060

cctctctcta ctacgtgttc agagatatct tctgcatgct taccaccgga taccttaccc 18120

ataagatcct ttaccctctc ctcatctctt acacctctaa cagcatcatc aagttcactt 18180

tctgggccct ttacacttac gttcaaggac ttttcggaac cggaatctgg gttctcgctc 18240

atgagtgtgg acatcaagct ttctctgatt acggaatcgt gaacgatttc gttggatgga 18300

cccttcactc ttaccttatg gttccttact tcagctggaa gtactctcat ggaaagcacc 18360

ataaggctac tggacacatg accagagata tggttttcgt tcctgccacc aaagaggaat 18420

tcaagaagtc taggaacttc ttcggtaacc tcgctgagta ctctgaggat tctccactta 18480

gaacccttta cgagcttctt gttcaacaac ttggaggatg gatcgcttac ctcttcgtta 18540

acgttacagg acaaccttac cctgatgttc cttcttggaa atggaaccac ttctggctta 18600

cctctccact tttcgagcaa agagatgctc tctacatctt cctttctgat cttggaatcc 18660

tcacccaggg aatcgttctt actctttggt acaagaaatt cggaggatgg tcccttttca 18720

tcaactggtt cgttccttac atctgggtta accactggct cgttttcatc acattccttc 18780

agcacactga tcctactatg cctcattaca acgctgagga atggactttc gctaagggtg 18840

ctgctgctac tatcgataga aagttcggat tcatcggacc tcacatcttc catgatatca 18900

tcgagactca tgtgcttcac cactactgtt ctaggatccc attctacaac gctagacctg 18960

cttctgaggc tatcaagaaa gttatgggaa agcactacag gtctagcgac gagaacatgt 19020

ggaagtcact ttggaagtct ttcaggtctt gccaatacgt tgacggtgat aacggtgttc 19080

tcatgttccg taacatcaac aactgcggag ttggagctgc tgagaagtaa tgaaggggtg 19140

atcgattatg agatcgtaca aagacactgc taggtgttaa ggatggataa taataataat 19200

aatgagatga atgtgtttta agttagtgta acagctgtaa taaagagaga gagagagaga 19260

gagagagaga gagagagaga gagagagaga gagaggctga tgaaatgtta tgtatgtttc 19320

ttggttttta aaataaatga aagcacatgc tcgtgtggtt ctatcgaatt attcggcggt 19380

tcctgtggga aaaagtccag aagggccgcc gcagctacta ctacaaccaa ggccgtggag 19440

gagggcaaca gagccagcac ttcgatagct gctgcgatga tcttaagcaa ttgaggagcg 19500

agtgcacatg caggggactg gagcgtgcaa tcggccagat gaggcaggac atccagcagc 19560

agggacagca gcaggaagtt gagaggtggt cccatcaatc taaacaagtc gctagggacc 19620

ttccgggaca gtgcggcacc cagcctagcc gatgccagct ccaggggcag cagcagtctg 19680

catggttttg aagtggtgat cgatgagatc gtataaagac actgctaggt gttaaggatg 19740

ggataataag atgtgtttta agtcattaac cgtaataaaa agagagagag gctgatggaa 19800

tgttatgtat gtatgtttct tggtttttaa aattaaatgg aaagcacatg ctcgtgtggg 19860

ttctatctcg attaaaaatc ccaattatat ttggtctaat ttagtttggt attgagtaaa 19920

acaaattcga accaaaccaa aatataaata tatagttttt atatatatgc ctttaagact 19980

ttttatagaa ttttctttaa aaaatatcta gaaatatttg cgactcttct ggcatgtaat 20040

atttcgttaa atatgaagtg ctccattttt attaacttta aataattggt tgtacgatca 20100

ctttcttatc aagtgttact aaaatgcgtc aatctctttg ttcttccata ttcatatgtc 20160

aaaatctatc aaaattctta tatatctttt tcgaatttga agtgaaattt cgataattta 20220

aaattaaata gaacatatca ttatttaggt atcatattga tttttatact taattactaa 20280

atttggttaa ctttgaaagt gtacatcaac gaaaaattag tcaaacgact aaaataaata 20340

aatatcatgt gttattaaga aaattctcct ataagaatat tttaatagat catatgtttg 20400

taaaaaaaat taatttttac taacacatat atttacttat caaaaatttg acaaagtaag 20460

attaaaataa tattcatcta acaaaaaaaa aaccagaaaa tgctgaaaac ccggcaaaac 20520

cgaaccaatc caaaccgata tagttggttt ggtttgattt tgatataaac cgaaccaact 20580

cggtccattt gcacccctaa tcataatagc tttaatattt caagatatta ttaagttaac 20640

gttgtcaata tcctggaaat tttgcaaaat gaatcaagcc tatatggctg taatatgaat 20700

ttaaaagcag ctcgatgtgg tggtaatatg taatttactt gattctaaaa aaatatccca 20760

agtattaata atttctgcta ggaagaaggt tagctacgat ttacagcaaa gccagaatac 20820

aaagaaccat aaagtgattg aagctcgaaa tatacgaagg aacaaatatt tttaaaaaaa 20880

tacgcaatga cttggaacaa aagaaagtga tatatttttt gttcttaaac aagcatcccc 20940

tctaaagaat ggcagttttc ctttgcatgt aactattatg ctcccttcgt tacaaaaatt 21000

ttggactact attgggaact tcttctgaaa atagtcctgc aggctagtag attggttggt 21060

tggtttccat gtaccagaag gcttacccta ttagttgaaa gttgaaactt tgttccctac 21120

tcaattccta gttgtgtaaa tgtatgtata tgtaatgtgt ataaaacgta gtacttaaat 21180

gactaggagt ggttcttgag accgatgaga gatgggagca gaactaaaga tgatgacata 21240

attaagaacg aatttgaaag gctcttaggt ttgaatccta ttcgagaatg tttttgtcaa 21300

agatagtggc gattttgaac caaagaaaac atttaaaaaa tcagtatccg gttacgttca 21360

tgcaaataga aagtggtcta ggatctgatt gtaattttag acttaaagag tctcttaaga 21420

ttcaatcctg gctgtgtaca aaactacaaa taatatattt tagactattt ggccttaact 21480

aaacttccac tcattattta ctgaggttag agaatagact tgcgaataaa cacattcccg 21540

agaaatactc atgatcccat aattagtcag agggtatgcc aatcagatct aagaacacac 21600

attccctcaa attttaatgc acatgtaatc atagtttagc acaattcaaa aataatgtag 21660

tattaaagac agaaatttgt agactttttt ttggcgttaa aagaagacta agtttatacg 21720

tacattttat tttaagtgga aaaccgaaat tttccatcga aatatatgaa tttagtatat 21780

atatttctgc aatgtactat tttgctattt tggcaacttt cagtggacta ctactttatt 21840

acaatgtgta tggatgcatg agtttgagta tacacatgtc taaatgcatg ctttgtaaaa 21900

cgtaacggac cacaaaagag gatccataca aatacatctc atagcttcct ccattatttt 21960

ccgacacaaa cagagcattt tacaacaatt accaacaaca acaaacaaca aacaacatta 22020

caattacatt tacaattacc ataccatgga atttgctcaa cctctcgttg ctatggctca 22080

agagcagtac gctgctatcg atgctgttgt tgctcctgct atcttctctg ctaccgactc 22140

tattggatgg ggactcaagc ctatctcttc tgctactaag gatctccctc tcgttgaatc 22200

tcctacccct cttatccttt ctctcctcgc ttacttcgct atcgttggtt ctggactcgt 22260

ttaccgtaaa gtgttcccta gaaccgttaa gggacaggat cctttccttc tcaaggctct 22320

tatgctcgct cacaacgttt tccttatcgg actcagcctt tacatgtgcc tcaagctcgt 22380

ttacgaggct tacgtgaaca agtactcctt ctggggaaac gcttacaacc ctgctcaaac 22440

cgagatggct aaggtgatct ggatcttcta cgtgtccaag atctacgagt tcatggacac 22500

cttcatcatg cttctcaagg gaaacgttaa ccaggtttcc ttcctccatg tttaccacca 22560

cggatctatc tctggaatct ggtggatgat cacttatgct gctccaggtg gagatgctta 22620

cttctctgct gctctcaact cttgggttca tgtgtgcatg tacacctact acttcatggc 22680

tgctgttctt cctaaggacg aaaagaccaa gagaaagtac ctttggtggg gaagatacct 22740

tacccagatg caaatgttcc agttcttcat gaaccttctc caggctgttt acctcctcta 22800

ctcttcttct ccttacccta agttcattgc tcaactcctc gttgtttaca tggttaccct 22860

cctcatgctt ttcggaaact tctactacat gaagcaccac gcttctaagt gataagggcc 22920

gccgccatgt gacagatcga aggaagaaag tgtaataaga cgactctcac tactcgatcg 22980

ctagtgattg tcattgttat atataataat gttatctttc acaacttatc gtaatgcatg 23040

tgaaactata acacattaat cctacttgtc atatgataac actctcccca tttaaaactc 23100

ttgtcaattt aaagatataa gattctttaa atgattaaaa aaaatatatt ataaattcaa 23160

tcactcctac taataaatta ttaattatta tttattgatt aaaaaaatac ttatactaat 23220

ttagtctgaa tagaataatt agattctagc ctgcagggcg gccgcggatc ccatggagtc 23280

aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac agttcataca 23340

gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg agcacgacac 23400

acttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg caattgagac 23460

ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag ctatctgtca 23520

ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc attgcgataa 23580

aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 23640

cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 23700

atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc 23760

ttcctctata taaggaagtt catttcattt ggagagaaca cgggggactg aattaaatat 23820

gagccctgag aggcgtcctg ttgaaatcag acctgctact gctgctgata tggctgctgt 23880

ttgtgatatc gtgaaccact acatcgagac ttctaccgtt aacttcagaa ctgagcctca 23940

aactcctcaa gagtggatcg atgatcttga gagactccaa gatagatacc cttggcttgt 24000

tgctgaggtt gagggtgttg ttgctggaat cgcttacgct ggaccttgga aggctagaaa 24060

cgcttacgat tggactgttg agtctaccgt ttacgtttca cacagacatc agagacttgg 24120

acttggatct accctttaca ctcaccttct caagtctatg gaagctcagg gattcaagtc 24180

tgttgttgct gttatcggac tccctaacga tccttctgtt agacttcatg aggctcttgg 24240

atacactgct agaggaactc ttagagctgc tggatacaag cacggtggat ggcatgatgt 24300

tggattctgg caaagagatt tcgagcttcc tgctcctcct agacctgtta gaccagttac 24360

tcagatctga atttgcgtga tcgttcaaac atttggcaat aaagtttctt aagattgaat 24420

cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta 24480

ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg 24540

caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 24600

tcgcgcgcgg tgtcatctat gttactagat cactagtgat gtacggttaa aaccacccca 24660

gtacattaaa aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc 24720

acaatatatc ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc 24780

accactcgat acaggcagcc catcagtcc 24809

<210> SEQ ID NO: 6

<211> LENGTH: 26543

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pGA7- mod_F nucleotide sequence

<400> SEQENCE: 6

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgggcggccg cctagaatct aattattcta ttcagactaa 240

attagtataa gtattttttt aatcaataaa tattaattaa taatttatta gtaggagtga 300

ttgaatttat aatatatttt ttttaatcat ttaaagaatc ttatatcttt aaattgacaa 360

gagttttaaa tggggagagt gttatcatat gacaagtagg attaatgtgt tatagtttca 420

catgcattac gataagttgt gaaagataac attattatat ataacaatga caatcactag 480

cgatcgagta gtgagagtcg tcttattaca ctttcttcct tcgatctgtc acatggcggc 540

ggcccgcgat cgcgataatt ctcagtgcgc cttctccgcc ttgccgttga cgtagtagtg 600

ctgcccgacc ttatccaagt tcgagaacgt cgccttccag gcgccgtaat aggacagcac 660

cttgtagttc agcccccact tcttcgcgaa cgggacgaac cgccggctca cctccggctg 720

gcgaaactgc ggcatgtccg ggaacaggtg atgaatgacc tggcagttca gatatcccat 780

caaccagttc acgtacccgc gcgacgggtc gatgtccacg gtgtgatcga ccgcgtagtt 840

cacccagctc aggtgcttat ccgagggcac gaccgggagg tgcgtgtggc tcgtggagaa 900

gtgcgcgaag aggtacatgt acgcgatcca gttgccgaag gtgaaccacc agtacgcgac 960

gggccacgag taccccgtcg cgagtttaat caccgcggtc ctgacgacgt gagagacgag 1020

catccacgac gcctcctcgt agttcttctt tcgcaacacc tgccgcgggt gcaggacgta 1080

gatccagaac gcctggacga gcagcccgga ggtcaccggg acgaacgtcc acgcctgaag 1140

ccgagcccac gcgcgggaga accccctcgg ccggttgtcc tccacggcgg tgttaaaaaa 1200

cgccaccgcg ggggtcgtgt ccaggtccat gtcgtgcctc actttctgcg gcgtcgcgtg 1260

gtgcttattg tgcatctggt tccacatctc cccgctcgtg gacagcccga acccgcacgt 1320

catcgcttgg aggcgcttgt cgacgtagac ggaccccgtg agcgagttgt gcccgccctc 1380

gtgctggacc caaccgcacc gagcgccgaa gaacgcgccg tacacgacgg acgcgatgat 1440

cgggtacccg gcgtacatga gggcggtgcc gagggcgaag gtcgcgagga gctcgagtaa 1500

ccgatacgcg acgtgcgtta tcgagggctt aaagaacccg tcgcgttcga gctccgcgcg 1560

ccaccgcgcg aaatcctcca acatcggcgc gtcctcgctc tcgctgcgtt tgatctccgc 1620

ggggcgcgac ggcagcgctc tgagcatctt ccacgcctta agcgatcgca tgtggaactc 1680

cttgaacgcc tccgtggcgt ccgcgcccgt gttcgcgagc atgtagaata tcacgctgcc 1740

tcccgggtgt ttgaagtttg tgacgtcgta ctcgacgtcc tccacgcgca cccatcgcgt 1800

ctcgaacgtc gccgcgagct cgtgcggctc gagcgttttg agatcgacgg gcgcggtcga 1860

cgcgtccttg gcgtcgagcg cctccgcgga ggatttgctg cgcgtcagcg gcgatcgcgg 1920

ggacgatcgg ccgtccgtct tcggcgggca catcgtcgcg cgcgcgactt aaaccgacga 1980

cggacggacg aacctgcaac ggcgaattat caattgacgc gttgctctgt ttgtgtcgga 2040

aaataatgga ggaagctatg agatgtattt gcatggatcc tcttttgtgg tccgttacgt 2100

tttgcaaagc atgcatttag acatgtgtat actcaaactc atgcatccat acacattgta 2160

ataaagtagt agtccactga aagttgccaa aatagcaaaa tagtacattg cagaaatata 2220

tatactaaat tcatatattt cgatggaaaa tttcggtttt ccacttaaaa taaaatgtac 2280

gtataaactt agtcttcctt taacgccaaa aaaaagtcta caaatttctg tctttaatac 2340

tacattattt ttgaattgtg ctaaactatg attacatgtg cattaaaatt tgagggaatg 2400

tgtgttctta gatctgattg gcataccctc tgactaatta tgggatcatg agtatttctc 2460

gggaatgtgt ttattcgcaa gtctattctc taacctcagt aaataatgag tggaagttta 2520

gttaaggcca aatagtctaa aatatattat ttgtagtttt gtacacagcc aggattgaat 2580

cttaagagac tctttaagtc taaaattaca atcagatcct agaccacttt ctatttgcat 2640

gaacgtaacc ggatactgat tttttaaatg ttttctttgg ttcaaaatcg ccactatctt 2700

tgacaaaaac attctcgaat aggattcaaa cctaagagcc tttcaaattc gttcttaatt 2760

atgtcatcat ctttagttct gctcccatct ctcatcggtc tcaagaacca ctcctagtca 2820

tttaagtact acgttttata cgcattacat atacatacat ttacacaact aggaattgag 2880

tagggaacaa agtttcaact ttcaactaat agggtaagcc ttctggtaca tggaaaccaa 2940

ccaaccaatc tactaggcgg ccgcccgtcg ggatcttctg caagcatctc tatttcctga 3000

aggtctaacc tcgaagattt aagatttaat tacgtttata attacaaaat tgattctagt 3060

atctttaatt taatgcttat acattattaa ttaatttagt actttcaatt tgttttcaga 3120

aattatttta ctatttttta taaaataaaa gggagaaaat ggctatttaa actgaaggcg 3180

ggaaacgaca atctgctagt ggatctccca gtcacgacgt tgtaaaacgg gcgccccgcg 3240

gaaagcttgc ggccgcggta ccgcccgttc gactcagatc ttccaaggcc tcgtctccga 3300

gtccgctgct tctcgccgcg ccgatcactt ctccgccgcc aacaaggctt gtagttaata 3360

ggaatcattc agggattgtg attccgggca gtagtaatta ataatatagt attagtatag 3420

ataatatgtt tcgtttggga tctttggaac gttgctctgt tccttgttgt tcattttaaa 3480

gcttttgagg gatagttgca gaactgttcg gtgatgcttc atcctctcaa gaactagatt 3540

tgggtaaaga aacatccatg catggatatg gaatgttgtt cttccgattg gagattattt 3600

tataaaattt aaaattcatg atttaaaaaa acacataaaa accacaaaat tcatgattta 3660

ttgacaatac gatacaaaat tagcaccacc ggctactggc tcattacaca tttccccttc 3720

ccctcattct cactttgtgg ctttattatt attattatta catatatttt accgttatta 3780

tttcacgtca cataagcttg ttaattaatc attagtgagc cttctcagcc tttccgttaa 3840

cgtagtagtg ctgtcccacc ttatcaaggt tagagaaagt agccttccaa gcaccgtagt 3900

aagagagcac cttgtagttg agtccccact tcttagcgaa aggaacgaat cttctgctaa 3960

cctcaggctg tctgaattga ggcatatcag ggaagaggtg gtggataacc tgacagttaa 4020

ggtatcccat aagccagttc acgtatcctc tagaaggatc gatatcaacg gtgtgatcaa 4080

cagcgtagtt aacccaagaa aggtgcttat cagatggaac aacagggagg tgagtatgag 4140

aagtagagaa gtgagcgaaa aggtacatgt aagcgatcca gtttccgaaa gtgaaccacc 4200

agtaagcaac aggccaagag tatccagtag caagcttgat aacagcggtt ctaacaacat 4260

gagaaacgag catccaagaa gcctcttcgt agttcttctt acggagaact tgtctagggt 4320

ggagaacgta gatccagaaa gcttgaacaa gaagtccaga ggtaacagga acgaaagtcc 4380

aagcttgaag tctagcccaa gctctagaga atcctctagg tctgttatcc tcaacagcag 4440

tgttgaagaa agccacagca ggagtggtat caagatccat atcgtgtcta accttttgag 4500

gggtagcatg gtgcttgtta tgcatctggt tccacatctc accagaagta gaaagtccga 4560

atccacaagt catagcctga agtctcttgt ccacgtaaac agatccggta agagagttat 4620

gtccaccctc atgttgaacc catccacatc tagctccgaa gaaagcaccg taaacaacag 4680

aagcaatgat agggtatcca gcgtacataa gagcagttcc aagagcgaat gtagcaagaa 4740

gctcgagaag tctgtaagcc acatgggtga tagaaggctt gaagaatcca tctctctcaa 4800

gctcagcacg ccatctagcg aaatcctcaa gcataggagc atcctcagac tcagatctct 4860

tgatctcagc aggtctagaa ggcaaagctc taagcatctt ccaagccttg agagaacgca 4920

tgtggaattc tttgaaagcc tcagtagcat cagcaccagt gttagcaagc atgtagaaga 4980

tcacagatcc accagggtgc ttgaagttag tcacatcgta ctcaacgtcc tcaactctaa 5040

cccatctagt ctcgaaagta gcagcaagct catgaggctc aagagtctta agatcaacag 5100

gagcagtaga agcatcctta gcatcaagag cctcagcaga agatttagac ctggtaagtg 5160

gagatctagg agaagatctt ccatcagtct taggagggca catggtatgg taattgtaaa 5220

tgtaattgta atgttgtttg ttgtttgttg ttgttggtaa ttgttgtaaa attaattaag 5280

tgggtatctt ttggatggat aagcaagtag tgatgatgtt ctaggtgaag tgatgggggt 5340

gttttatagc gggagatggt gaaatggatg gtcgccacat aagaaatgga ggggaagggt 5400

tcttgcgcca ttcttcagtt tgcatggatg catgggtttc attttgtaac acgtaataag 5460

gacaatgaag tgcaggtgtc tctcaagttt cagaggggat atgtggacag aagaagaacg 5520

gcgatgatat tgatggaaat ggccatctag tgtgaatcta ttcggttgat aatactagtg 5580

cattttggcc gttaatccct tcaattaact gcacaaactt cagttgagta ttgattattt 5640

gattataggt tctgtaaaca caataccaag tttatttaga ggggagacat acaaatagtt 5700

tcgatataaa taatagagtg gttaaactta gttattaaaa ctatatataa agtctaaaag 5760

ttaaattatt tttttaattg caaatatata aagtctaaag gggttacatt atttcttaag 5820

agatgtaact ctgttggaat ctgacttaat ccgtctcatc actctggttt ccagttctaa 5880

tctaatgaat tgttttctgc caaagaattt gaagcaagaa gtaaattgat caatgccgtc 5940

aacccacacc aaaccgtcaa cccactacca tcgccgcgga gacccccaaa ctcaacctcc 6000

acccatcggt aagaagcaca gggcagcccg caccaccacc aatttggcgt gcatgacacc 6060

tagggacttg gcacgggagg cggcgcacgt ggatgcaaat gacgggatat cagatgacag 6120

gaaacgacgt tgagagacca tacgatgtag aatatgagct caccatcaac gagaaactag 6180

gaaaatcaca aaaaaaacaa ctctcgtaat tgtacgagtg gcacagatgg gtctgcctca 6240

acatatctct aatacggcga agcctgccca acacgtagtt gccggaatcc ggtgtggagc 6300

tcacgactct gaaagatagg cgcttcctgt ttcgtttcgc tcacccactg gacgtccgtc 6360

atgtgatgga tttcggtcat tggtttgctg acaaccacat tctgaagctc catgagatga 6420

gtcttcacaa taggtcctgc tcaataccgt ggagttatgg ttgcaagtcc ataacttgcc 6480

gttcgaatat tttgcggagc cagtcggacg ggaattggcg agctcggctg acacctataa 6540

aggccatgac aagaagaacc aaaagttctt ccctaatgct ttcatgaggc ttcgggtcgt 6600

tatggatgtc ggaaaacccc tcttgaagga acgagacgtt attatgcatg acggtaagac 6660

tattacttgt cagtataagt atgaaagatt acctgtcttc tgctttgttt gtggattgat 6720

tggacacgtt gaaaaaaaat gtgcacttcg atttcaatac tcagagatcg acttcccttt 6780

tctctaggag tattcgatca aggcattaac atggaaggaa gctcaagctc taaaggcttc 6840

acaatggaac ctgaaaaatt tcaacaagcc taaactgaaa tcgaagtcaa atcacccaac 6900

cgggagctct aaatcagcaa acactcctcc tccacagtat ccaatcatcg tgcacgatgc 6960

tccaggtatt gcaagccagg tattgcaagc taggagtagg atagagacct taaacgtcgt 7020

tggtgtgaag agtcatcttc agacctaatg gagatagatg tagacggcgg cacgaagact 7080

ctgaaacacc agaaaggcta gtccaggata aggatctgct atcccaactg acctctcgtt 7140

agtcccaagg cctctcaact agagcaggag gaaggatggt cacaagacta ggataatgat 7200

gtttccaata tgaacctgaa tgtccatagc taattttttt agtcttgctt ctgcactttt 7260

tgtttattat gttctggtga ctatgttatt tacccttgtc cgtatgcttg agggtaccct 7320

agtagattgg ttggttggtt tccatgtacc agaaggctta ccctattagt tgaaagttga 7380

aactttgttc cctactcaat tcctagttgt gtaaatgtat gtatatgtaa tgtgtataaa 7440

acgtagtact taaatgacta ggagtggttc ttgagaccga tgagagatgg gagcagaact 7500

aaagatgatg acataattaa gaacgaattt gaaaggctct taggtttgaa tcctattcga 7560

gaatgttttt gtcaaagata gtggcgattt tgaaccaaag aaaacattta aaaaatcagt 7620

atccggttac gttcatgcaa atagaaagtg gtctaggatc tgattgtaat tttagactta 7680

aagagtctct taagattcaa tcctggctgt gtacaaaact acaaataata tattttagac 7740

tatttggcct taactaaact tccactcatt atttactgag gttagagaat agacttgcga 7800

ataaacacat tcccgagaaa tactcatgat cccataatta gtcagagggt atgccaatca 7860

gatctaagaa cacacattcc ctcaaatttt aatgcacatg taatcatagt ttagcacaat 7920

tcaaaaataa tgtagtatta aagacagaaa tttgtagact tttttttggc gttaaaagaa 7980

gactaagttt atacgtacat tttattttaa gtggaaaacc gaaattttcc atcgaaatat 8040

atgaatttag tatatatatt tctgcaatgt actattttgc tattttggca actttcagtg 8100

gactactact ttattacaat gtgtatggat gcatgagttt gagtatacac atgtctaaat 8160

gcatgctttg taaaacgtaa cggaccacaa aagaggatcc atacaaatac atctcatagc 8220

ttcctccatt attttccgac acaaacagag cattttacaa caattaccaa caacaacaaa 8280

caacaaacaa cattacaatt acatttacaa ttaccatacc atggcctcta tcgctatccc 8340

tgctgctctt gctggaactc ttggatacgt tacctacaat gtggctaacc ctgatatccc 8400

agcttctgag aaagttcctg cttacttcat gcaggttgag tactggggac ctactatcgg 8460

aactattgga tacctcctct tcatctactt cggaaagcgt atcatgcaga acagatctca 8520

acctttcgga ctcaagaacg ctatgctcgt ttacaacttc taccagacct tcttcaacag 8580

ctactgcatc taccttttcg ttacttctca tagggctcag ggacttaagg tttggggaaa 8640

catccctgat atgactgcta actcttgggg aatctctcag gttatctggc ttcactacaa 8700

caacaagtac gttgagcttc tcgacacctt cttcatggtg atgaggaaga agttcgacca 8760

gctttctttc cttcacatct accaccacac tcttctcatc tggtcatggt tcgttgttat 8820

gaagcttgag cctgttggag attgctactt cggatcttct gttaacacct tcgtgcacgt 8880

gatcatgtac tcttactacg gacttgctgc tcttggagtt aactgtttct ggaagaagta 8940

catcacccag atccagatgc ttcagttctg tatctgtgct tctcactcta tctacaccgc 9000

ttacgttcag aataccgctt tctggcttcc ttaccttcaa ctctgggtta tggtgaacat 9060

gttcgttctc ttcgccaact tctaccgtaa gaggtacaag tctaagggtg ctaagaagca 9120

gtgataaggc gcgcggcgcg ccgggccgcc gccatgtgac agatcgaagg aagaaagtgt 9180

aataagacga ctctcactac tcgatcgcta gtgattgtca ttgttatata taataatgtt 9240

atctttcaca acttatcgta atgcatgtga aactataaca cattaatcct acttgtcata 9300

tgataacact ctccccattt aaaactcttg tcaatttaaa gatataagat tctttaaatg 9360

attaaaaaaa atatattata aattcaatca ctcctactaa taaattatta attattattt 9420

attgattaaa aaaatactta tactaattta gtctgaatag aataattaga ttctagtctc 9480

atcccctttt aaaccaactt agtaaacgtt ttttttttta attttatgaa gttaagtttt 9540

taccttgttt ttaaaaagaa tcgttcataa gatgccatgc cagaacatta gctacacgtt 9600

acacatagca tgcagccgcg gagaattgtt tttcttcgcc acttgtcact cccttcaaac 9660

acctaagagc ttctctctca cagcacacac atacaatcac atgcgtgcat gcattattac 9720

acgtgatcgc catgcaaatc tcctttatag cctataaatt aactcatccg cttcactctt 9780

tactcaaacc aaaactcatc gatacaaaca agattaaaaa catacacgag gatcttttac 9840

aacaattacc aacaacaaca aacaacaaac aacattacaa ttacatttac aattaccata 9900

ccatgcctcc aagggactct tactcttatg ctgctcctcc ttctgctcaa cttcacgaag 9960

ttgatactcc tcaagagcac gacaagaaag agcttgttat cggagatagg gcttacgatg 10020

ttaccaactt cgttaagaga caccctggtg gaaagatcat tgcttaccaa gttggaactg 10080

atgctaccga tgcttacaag cagttccatg ttagatctgc taaggctgac aagatgctta 10140

agtctcttcc ttctcgtcct gttcacaagg gatactctcc aagaagggct gatcttatcg 10200

ctgatttcca agagttcacc aagcaacttg aggctgaggg aatgttcgag ccttctcttc 10260

ctcatgttgc ttacagactt gctgaggtta tcgctatgca tgttgctggt gctgctctta 10320

tctggcatgg atacactttc gctggaatcg ctatgcttgg agttgttcag ggaagatgtg 10380

gatggcttat gcatgagggt ggacattact ctctcactgg aaacattgct ttcgacagag 10440

ctatccaagt tgcttgttac ggacttggat gtggaatgtc tggtgcttgg tggcgtaacc 10500

agcataacaa gcaccatgct actcctcaaa agcttcagca cgatgttgat cttgataccc 10560

ttcctctcgt tgctttccat gagagaatcg ctgctaaggt taagtctcct gctatgaagg 10620

cttggctttc tatgcaagct aagcttttcg ctcctgttac cactcttctt gttgctcttg 10680

gatggcagct ttaccttcat cctagacaca tgctcaggac taagcactac gatgagcttg 10740

ctatgctcgg aatcagatac ggacttgttg gataccttgc tgctaactac ggtgctggat 10800

acgttctcgc ttgttacctt ctttacgttc agcttggagc tatgtacatc ttctgcaact 10860

tcgctgtttc tcatactcac ctccctgttg ttgagcctaa cgagcatgct acttgggttg 10920

agtacgctgc taaccacact actaactgtt ctccatcttg gtggtgtgat tggtggatgt 10980

cttaccttaa ctaccagatc gagcaccacc tttacccttc tatgcctcaa ttcagacacc 11040

ctaagatcgc tcctagagtt aagcagcttt tcgagaagca cggacttcac tacgatgtta 11100

gaggatactt cgaggctatg gctgatactt tcgctaacct tgataacgtt gcccatgctc 11160

ctgagaagaa aatgcagtaa tgagatcgtt caaacatttg gcaataaagt ttcttaagat 11220

tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc 11280

acgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag 11340

tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata 11400

aattatcgcg cgcggtgtca tctatgttac tagatcggtc gattaaaaat cccaattata 11460

tttggtctaa tttagtttgg tattgagtaa aacaaattcg aaccaaacca aaatataaat 11520

atatagtttt tatatatatg cctttaagac tttttataga attttcttta aaaaatatct 11580

agaaatattt gcgactcttc tggcatgtaa tatttcgtta aatatgaagt gctccatttt 11640

tattaacttt aaataattgg ttgtacgatc actttcttat caagtgttac taaaatgcgt 11700

caatctcttt gttcttccat attcatatgt caaaatctat caaaattctt atatatcttt 11760

ttcgaatttg aagtgaaatt tcgataattt aaaattaaat agaacatatc attatttagg 11820

tatcatattg atttttatac ttaattacta aatttggtta actttgaaag tgtacatcaa 11880

cgaaaaatta gtcaaacgac taaaataaat aaatatcatg tgttattaag aaaattctcc 11940

tataagaata ttttaataga tcatatgttt gtaaaaaaaa ttaattttta ctaacacata 12000

tatttactta tcaaaaattt gacaaagtaa gattaaaata atattcatct aacaaaaaaa 12060

aaaccagaaa atgctgaaaa cccggcaaaa ccgaaccaat ccaaaccgat atagttggtt 12120

tggtttgatt ttgatataaa ccgaaccaac tcggtccatt tgcaccccta atcataatag 12180

ctttaatatt tcaagatatt attaagttaa cgttgtcaat atcctggaaa ttttgcaaaa 12240

tgaatcaagc ctatatggct gtaatatgaa tttaaaagca gctcgatgtg gtggtaatat 12300

gtaatttact tgattctaaa aaaatatccc aagtattaat aatttctgct aggaagaagg 12360

ttagctacga tttacagcaa agccagaata caaagaacca taaagtgatt gaagctcgaa 12420

atatacgaag gaacaaatat ttttaaaaaa atacgcaatg acttggaaca aaagaaagtg 12480

atatattttt tgttcttaaa caagcatccc ctctaaagaa tggcagtttt cctttgcatg 12540

taactattat gctcccttcg ttacaaaaat tttggactac tattgggaac ttcttctgaa 12600

aatagtgata gaacccacac gagcatgtgc tttccattta attttaaaaa ccaagaaaca 12660

tacatacata acattccatc agcctctctc tctttttatt acggttaatg acttaaaaca 12720

catcttatta tcccatcctt aacacctagc agtgtcttta tacgatctca tcgatcacca 12780

cttcaaaacc atgcagactg ctgctgcccc tggagctggc atcggctagg ctgggtgccg 12840

cactgtcccg gaaggtccct agcgacttgt ttagattgat gggaccacct ctcaacttcc 12900

tgctgctgtc cctgctgctg gatgtcctgc ctcatctggc cgattgcacg ctccagtccc 12960

ctgcatgtgc actcgctcct caattgctta agatcatcgc agcagctatc gaagtgctgg 13020

ctctgttgcc ctcctccacg gccttggttg tagtagtagc tgccgccgcc cttctggact 13080

ttttcccaca ggaaccgccg aataattcga tagaaccaca cgagcatgtg ctttcattta 13140

ttttaaaaac caagaaacat acataacatt tcatcagcct ctctctctct ctctctctct 13200

ctctctctct ctctctctct ctctctctct ttattacagc tgttacacta acttaaaaca 13260

cattcatctc attattatta ttattatcca tccttaacac ctagcagtgt ctttgtacga 13320

tctcataatc gatcacccct tcatcaggta tccttaggct tcactccaac gttgttgcag 13380

ttacggaaca tgtacacacc atcatggttc tcaacgaact ggcaagatct ccaagttttc 13440

caaaggctaa cccacatgtt ctcatcggtg tgtctgtagt gctctcccat aactttcttg 13500

atgcactcgg tagcttctct agcatggtag aatgggatcc ttgaaacgta gtgatggagc 13560

acatgagtct cgatgatgtc atggaagatg attccgagga ttccgaactc tctatcgata 13620

gtagcagcag cacccttagc gaaagtccac tcttgagcat cgtaatgagg catagaagaa 13680

tcggtgtgct gaaggaaggt aacgaaaaca agccagtggt taacaaggat ccaaggacag 13740

aaccatgtga tgaaagtagg ccagaatccg aaaaccttgt aagcggtgta aacagaagtg 13800

agggtagcaa ggattccaag atcagaaaga acgatgtacc agtagtcctt cttatcgaaa 13860

acagggctag aaggccagta gtgagacttg aagaacttag aaacaccagg gtaaggttgt 13920

ccagtagcgt tagtagcaag gtaaagagaa agtcctccaa gctgttggaa caagagagcg 13980

aaaacagagt agataggagt ttcctcagcg atatcgtgaa ggctggtaac ttggtgcttc 14040

tctttgaatt cctcggcggt gtaaggaacg aaaaccatat ctctggtcat gtgtccagta 14100

gccttatggt gcttagcatg agagaacttc cagctgaagt aaggaaccat aacaagagag 14160

tggagaaccc atccaacggt atcgttaacc catccgtagt tagagaaagc agaatgtcca 14220

cactcatgtc caaggatcca gattccgaat ccgaaacaag agatagagaa cacgtaagca 14280

gaccaagcag cgaatctaag gaattcgtta gggagaagag ggatgtaggt aagtccaacg 14340

taagcgatag cagagatagc cacgatatct ctcaccacgt aagacataga cttcacgaga 14400

gatctctcgt aacagtgctt agggatagcg tcaaggatat ccttgatggt gtaatctggc 14460

accttgaaaa cgtttccgaa ggtatcgata gcggtctttt gctgcttgaa agatgcaacg 14520

tttccagaac gcctaacggt cttagtagat ccctcaagga tctcagatcc agacacggta 14580

accttagaca tggtatggta attgtaaatg taattgtaat gttgtttgtt gtttgttgtt 14640

gttggtaatt gttgtaaaat ttttggtggt gattggttct ttaaggtgtg agagtgagtt 14700

gtgagttgtg tggtgggttt ggtgagattg gggatggtgg gtttatatag tggagactga 14760

ggaatggggt cgtgagtgtt aactttgcat gggctacacg tgggttcttt tgggcttaca 14820

cgtagtatta ttcatgcaaa tgcagccaat acatatacgg tattttaata atgtgtggga 14880

atacaatatg ccgagtattt tactaatttt ggcaatgaca agtgtacatt tggattatct 14940

tacttggcct ctcttgcttt aatttggatt atttttattc tcttaccttg gccgttcata 15000

ttcacatccc taaaggcaag acagaattga atggtggcca aaaattaaaa cgatggatat 15060

gacctacata gtgtaggatc aattaacgtc gaaggaaaat actgattctc tcaagcatac 15120

ggacaagggt aaataacata gtcaccagaa cataataaac aaaaagtgca gaagcaagac 15180

taaaaaaatt agctatggac attcaggttc atattggaaa catcattatc ctagtcttgt 15240

gaccatcctt cctcctgctc tagttgagag gccttgggac taacgagagg tcagttggga 15300

tagcagatcc ttatcctgga ctagcctttc tggtgtttca gagtcttcgt gccgccgtct 15360

acatctatct ccattaggtc tgaagatgac tcttcacacc aacgacgttt aaggtctcta 15420

tcctactcct agcttgcaat acctggcttg caatacctgg agcatcgtgc acgatgattg 15480

gatactgtgg aggaggagtg tttgctgatt tagagctccc ggttgggtga tttgacttcg 15540

atttcagttt aggcttgttg aaatttttca ggttccattg tgaagccttt agagcttgag 15600

cttccttcca tgttaatgcc ttgatcgaat actcctagag aaaagggaag tcgatctctg 15660

agtattgaaa tcgaagtgca catttttttt caacgtgtcc aatcaatcca caaacaaagc 15720

agaagacagg taatctttca tacttatact gacaagtaat agtcttaccg tcatgcataa 15780

taacgtctcg ttccttcaag aggggttttc cgacatccat aacgacccga agcctcatga 15840

aagcattagg gaagaacttt tggttcttct tgtcatggcc tttataggtg tcagccgagc 15900

tcgccaattc ccgtccgact ggctccgcaa aatattcgaa cggcaagtta tggacttgca 15960

accataactc cacggtattg agcaggacct attgtgaaga ctcatctcat ggagcttcag 16020

aatgtggttg tcagcaaacc aatgaccgaa atccatcaca tgacggacgt ccagtgggtg 16080

agcgaaacga aacaggaagc gcctatcttt cagagtcgtg agctccacac cggattccgg 16140

caactacgtg ttgggcaggc ttcgccgtat tagagatatg ttgaggcaga cccatctgtg 16200

ccactcgtac aattacgaga gttgtttttt ttgtgatttt cctagtttct cgttgatggt 16260

gagctcatat tctacatcgt atggtctctc aacgtcgttt cctgtcatct gatatcccgt 16320

catttgcatc cacgtgcgcc gcctcccgtg ccaagtccct aggtgtcatg cacgccaaat 16380

tggtggtggt gcgggctgcc ctgtgcttct taccgatggg tggaggttga gtttgggggt 16440

ctccgcggcg atggtagtgg gttgacggtt tggtgtgggt tgacggcatt gatcaattta 16500

cttcttgctt caaattcttt ggcagaaaac aattcattag attagaactg gaaaccagag 16560

tgatgagacg gattaagtca gattccaaca gagttacatc tcttaagaaa taatgtaacc 16620

cctttagact ttatatattt gcaattaaaa aaataattta acttttagac tttatatata 16680

gttttaataa ctaagtttaa ccactctatt atttatatcg aaactatttg tatgtctccc 16740

ctctaaataa acttggtatt gtgtttacag aacctataat caaataatca atactcaact 16800

gaagtttgtg cagttaattg aagggattaa cggccaaaat gcactagtat tatcaaccga 16860

atagattcac actagatggc catttccatc aatatcatcg ccgttcttct tctgtccaca 16920

tatcccctct gaaacttgag agacacctgc acttcattgt ccttattacg tgttacaaaa 16980

tgaaacccat gcatccatgc aaactgaaga atggcgcaag aacccttccc ctccatttct 17040

tatgtggcga ccatccattt caccatctcc cgctataaaa cacccccatc acttcaccta 17100

gaacatcatc actacttgct tatccatcca aaagataccc acttttacaa caattaccaa 17160

caacaacaaa caacaaacaa cattacaatt acatttacaa ttaccatacc atgccaccta 17220

gcgctgctaa gcaaatggga gcttctactg gtgttcatgc tggtgttact gactcttctg 17280

ctttcaccag aaaggatgtt gctgatagac ctgatctcac catcgttgga gattctgttt 17340

acgatgctaa ggctttcaga tctgagcatc ctggtggtgc tcatttcgtt tctttgttcg 17400

gaggaagaga tgctactgag gctttcatgg aataccatag aagggcttgg cctaagtcta 17460

gaatgtctag attccacgtt ggatctcttg cttctactga ggaacctgtt gctgctgatg 17520

agggatacct tcaactttgt gctaggatcg ctaagatggt gccttctgtt tcttctggat 17580

tcgctcctgc ttcttactgg gttaaggctg gacttatcct tggatctgct atcgctcttg 17640

aggcttacat gctttacgct ggaaagagac ttctcccttc tatcgttctt ggatggcttt 17700

tcgctcttat cggtcttaac atccagcatg atgctaacca tggtgctttg tctaagtctg 17760

cttctgttaa ccttgctctt ggactttgtc aggattggat cggaggatct atgatccttt 17820

ggcttcaaga gcatgttgtt atgcaccacc tccacactaa cgatgttgat aaggatcctg 17880

atcaaaaggc tcacggtgct cttagactca agcctactga tgcttggtca cctatgcatt 17940

ggcttcagca tctttacctt ttgcctggtg agactatgta cgctttcaag cttttgttcc 18000

tcgacatctc tgagcttgtt atgtggcgtt gggagggtga gcctatctct aagcttgctg 18060

gatacctctt tatgccttct ttgcttctca agcttacctt ctgggctaga ttcgttgctt 18120

tgcctcttta ccttgctcct tctgttcata ctgctgtgtg tatcgctgct actgttatga 18180

ctggatcttt ctacctcgct ttcttcttct tcatctccca caacttcgag ggtgttgctt 18240

ctgttggacc tgatggatct atcacttcta tgactagagg tgctagcttc cttaagagac 18300

aagctgagac ttcttctaac gttggaggac ctcttcttgc tactcttaac ggtggactca 18360

actaccaaat tgagcatcac ttgttcccta gagttcacca tggattctac cctagacttg 18420

ctcctcttgt taaggctgag cttgaggcta gaggaatcga gtacaagcac taccctacta 18480

tctggtctaa ccttgcttct accctcagac atatgtacgc tcttggaaga aggcctagat 18540

ctaaggctga gtaatgacaa gcttatgtga cgtgaaataa taacggtaaa atatatgtaa 18600

taataataat aataaagcca caaagtgaga atgaggggaa ggggaaatgt gtaatgagcc 18660

agtagccggt ggtgctaatt ttgtatcgta ttgtcaataa atcatgaatt ttgtggtttt 18720

tatgtgtttt tttaaatcat gaattttaaa ttttataaaa taatctccaa tcggaagaac 18780

aacattccat atccatgcat ggatgtttct ttacccaaat ctagttcttg agaggatgaa 18840

gcatcaccga acagttctgc aactatccct caaaagcttt aaaatgaaca acaaggaaca 18900

gagcaacgtt ccaaagatcc caaacgaaac atattatcta tactaatact atattattaa 18960

ttactactgc ccggaatcac aatccctgaa tgattcctat taactacaag ccttgttggc 19020

ggcggagaag tgatcggcgc ggcgagaagc agcggactcg gagacgaggc cttggaagat 19080

ctgagtcgaa cgggcagaat cagtattttc cttcgacgtt aattgatcct acactatgta 19140

ggtcatatcc atcgttttaa tttttggcca ccattcaatt ctgtcttgcc tttagggatg 19200

tgaatatgaa cggccaaggt aagagaataa aaataatcca aattaaagca agagaggcca 19260

agtaagataa tccaaatgta cacttgtcat tgccaaaatt agtaaaatac tcggcatatt 19320

gtattcccac acattattaa aataccgtat atgtattggc tgcatttgca tgaataatac 19380

tacgtgtaag cccaaaagaa cccacgtgta gcccatgcaa agttaacact cacgacccca 19440

ttcctcagtc tccactatat aaacccacca tccccaatct caccaaaccc accacacaac 19500

tcacaactca ctctcacacc ttaaagaacc aatcaccacc aaaaatttta caacaattac 19560

caacaacaac aaacaacaaa caacattaca attacattta caattaccat accatgagcg 19620

ctgttaccgt tactggatct gatcctaaga acagaggatc ttctagcaac accgagcaag 19680

aggttccaaa agttgctatc gataccaacg gaaacgtgtt ctctgttcct gatttcacca 19740

tcaaggacat ccttggagct atccctcatg agtgttacga gagaagattg gctacctctc 19800

tctactacgt gttcagagat atcttctgca tgcttaccac cggatacctt acccataaga 19860

tcctttaccc tctcctcatc tcttacacct ctaacagcat catcaagttc actttctggg 19920

ccctttacac ttacgttcaa ggacttttcg gaaccggaat ctgggttctc gctcatgagt 19980

gtggacatca agctttctct gattacggaa tcgtgaacga tttcgttgga tggacccttc 20040

actcttacct tatggttcct tacttcagct ggaagtactc tcatggaaag caccataagg 20100

ctactggaca catgaccaga gatatggttt tcgttcctgc caccaaagag gaattcaaga 20160

agtctaggaa cttcttcggt aacctcgctg agtactctga ggattctcca cttagaaccc 20220

tttacgagct tcttgttcaa caacttggag gatggatcgc ttacctcttc gttaacgtta 20280

caggacaacc ttaccctgat gttccttctt ggaaatggaa ccacttctgg cttacctctc 20340

cacttttcga gcaaagagat gctctctaca tcttcctttc tgatcttgga atcctcaccc 20400

agggaatcgt tcttactctt tggtacaaga aattcggagg atggtccctt ttcatcaact 20460

ggttcgttcc ttacatctgg gttaaccact ggctcgtttt catcacattc cttcagcaca 20520

ctgatcctac tatgcctcat tacaacgctg aggaatggac tttcgctaag ggtgctgctg 20580

ctactatcga tagaaagttc ggattcatcg gacctcacat cttccatgat atcatcgaga 20640

ctcatgtgct tcaccactac tgttctagga tcccattcta caacgctaga cctgcttctg 20700

aggctatcaa gaaagttatg ggaaagcact acaggtctag cgacgagaac atgtggaagt 20760

cactttggaa gtctttcagg tcttgccaat acgttgacgg tgataacggt gttctcatgt 20820

tccgtaacat caacaactgc ggagttggag ctgctgagaa gtaatgaagg ggtgatcgat 20880

tatgagatcg tacaaagaca ctgctaggtg ttaaggatgg ataataataa taataatgag 20940

atgaatgtgt tttaagttag tgtaacagct gtaataaaga gagagagaga gagagagaga 21000

gagagagaga gagagagaga gagagagagg ctgatgaaat gttatgtatg tttcttggtt 21060

tttaaaataa atgaaagcac atgctcgtgt ggttctatcg aattattcgg cggttcctgt 21120

gggaaaaagt ccagaagggc cgccgcagct actactacaa ccaaggccgt ggaggagggc 21180

aacagagcca gcacttcgat agctgctgcg atgatcttaa gcaattgagg agcgagtgca 21240

catgcagggg actggagcgt gcaatcggcc agatgaggca ggacatccag cagcagggac 21300

agcagcagga agttgagagg tggtcccatc aatctaaaca agtcgctagg gaccttccgg 21360

gacagtgcgg cacccagcct agccgatgcc agctccaggg gcagcagcag tctgcatggt 21420

tttgaagtgg tgatcgatga gatcgtataa agacactgct aggtgttaag gatgggataa 21480

taagatgtgt tttaagtcat taaccgtaat aaaaagagag agaggctgat ggaatgttat 21540

gtatgtatgt ttcttggttt ttaaaattaa atggaaagca catgctcgtg tgggttctat 21600

ctcgattaaa aatcccaatt atatttggtc taatttagtt tggtattgag taaaacaaat 21660

tcgaaccaaa ccaaaatata aatatatagt ttttatatat atgcctttaa gactttttat 21720

agaattttct ttaaaaaata tctagaaata tttgcgactc ttctggcatg taatatttcg 21780

ttaaatatga agtgctccat ttttattaac tttaaataat tggttgtacg atcactttct 21840

tatcaagtgt tactaaaatg cgtcaatctc tttgttcttc catattcata tgtcaaaatc 21900

tatcaaaatt cttatatatc tttttcgaat ttgaagtgaa atttcgataa tttaaaatta 21960

aatagaacat atcattattt aggtatcata ttgattttta tacttaatta ctaaatttgg 22020

ttaactttga aagtgtacat caacgaaaaa ttagtcaaac gactaaaata aataaatatc 22080

atgtgttatt aagaaaattc tcctataaga atattttaat agatcatatg tttgtaaaaa 22140

aaattaattt ttactaacac atatatttac ttatcaaaaa tttgacaaag taagattaaa 22200

ataatattca tctaacaaaa aaaaaaccag aaaatgctga aaacccggca aaaccgaacc 22260

aatccaaacc gatatagttg gtttggtttg attttgatat aaaccgaacc aactcggtcc 22320

atttgcaccc ctaatcataa tagctttaat atttcaagat attattaagt taacgttgtc 22380

aatatcctgg aaattttgca aaatgaatca agcctatatg gctgtaatat gaatttaaaa 22440

gcagctcgat gtggtggtaa tatgtaattt acttgattct aaaaaaatat cccaagtatt 22500

aataatttct gctaggaaga aggttagcta cgatttacag caaagccaga atacaaagaa 22560

ccataaagtg attgaagctc gaaatatacg aaggaacaaa tatttttaaa aaaatacgca 22620

atgacttgga acaaaagaaa gtgatatatt ttttgttctt aaacaagcat cccctctaaa 22680

gaatggcagt tttcctttgc atgtaactat tatgctccct tcgttacaaa aattttggac 22740

tactattggg aacttcttct gaaaatagtc ctgcaggcta gtagattggt tggttggttt 22800

ccatgtacca gaaggcttac cctattagtt gaaagttgaa actttgttcc ctactcaatt 22860

cctagttgtg taaatgtatg tatatgtaat gtgtataaaa cgtagtactt aaatgactag 22920

gagtggttct tgagaccgat gagagatggg agcagaacta aagatgatga cataattaag 22980

aacgaatttg aaaggctctt aggtttgaat cctattcgag aatgtttttg tcaaagatag 23040

tggcgatttt gaaccaaaga aaacatttaa aaaatcagta tccggttacg ttcatgcaaa 23100

tagaaagtgg tctaggatct gattgtaatt ttagacttaa agagtctctt aagattcaat 23160

cctggctgtg tacaaaacta caaataatat attttagact atttggcctt aactaaactt 23220

ccactcatta tttactgagg ttagagaata gacttgcgaa taaacacatt cccgagaaat 23280

actcatgatc ccataattag tcagagggta tgccaatcag atctaagaac acacattccc 23340

tcaaatttta atgcacatgt aatcatagtt tagcacaatt caaaaataat gtagtattaa 23400

agacagaaat ttgtagactt ttttttggcg ttaaaagaag actaagttta tacgtacatt 23460

ttattttaag tggaaaaccg aaattttcca tcgaaatata tgaatttagt atatatattt 23520

ctgcaatgta ctattttgct attttggcaa ctttcagtgg actactactt tattacaatg 23580

tgtatggatg catgagtttg agtatacaca tgtctaaatg catgctttgt aaaacgtaac 23640

ggaccacaaa agaggatcca tacaaataca tctcatagct tcctccatta ttttccgaca 23700

caaacagagc attttacaac aattaccaac aacaacaaac aacaaacaac attacaatta 23760

catttacaat taccatacca tggaatttgc tcaacctctc gttgctatgg ctcaagagca 23820

gtacgctgct atcgatgctg ttgttgctcc tgctatcttc tctgctaccg actctattgg 23880

atggggactc aagcctatct cttctgctac taaggatctc cctctcgttg aatctcctac 23940

ccctcttatc ctttctctcc tcgcttactt cgctatcgtt ggttctggac tcgtttaccg 24000

taaagtgttc cctagaaccg ttaagggaca ggatcctttc cttctcaagg ctcttatgct 24060

cgctcacaac gttttcctta tcggactcag cctttacatg tgcctcaagc tcgtttacga 24120

ggcttacgtg aacaagtact ccttctgggg aaacgcttac aaccctgctc aaaccgagat 24180

ggctaaggtg atctggatct tctacgtgtc caagatctac gagttcatgg acaccttcat 24240

catgcttctc aagggaaacg ttaaccaggt ttccttcctc catgtttacc accacggatc 24300

tatctctgga atctggtgga tgatcactta tgctgctcca ggtggagatg cttacttctc 24360

tgctgctctc aactcttggg ttcatgtgtg catgtacacc tactacttca tggctgctgt 24420

tcttcctaag gacgaaaaga ccaagagaaa gtacctttgg tggggaagat accttaccca 24480

gatgcaaatg ttccagttct tcatgaacct tctccaggct gtttacctcc tctactcttc 24540

ttctccttac cctaagttca ttgctcaact cctcgttgtt tacatggtta ccctcctcat 24600

gcttttcgga aacttctact acatgaagca ccacgcttct aagtgataag ggccgccgcc 24660

atgtgacaga tcgaaggaag aaagtgtaat aagacgactc tcactactcg atcgctagtg 24720

attgtcattg ttatatataa taatgttatc tttcacaact tatcgtaatg catgtgaaac 24780

tataacacat taatcctact tgtcatatga taacactctc cccatttaaa actcttgtca 24840

atttaaagat ataagattct ttaaatgatt aaaaaaaata tattataaat tcaatcactc 24900

ctactaataa attattaatt attatttatt gattaaaaaa atacttatac taatttagtc 24960

tgaatagaat aattagattc tagcctgcag ggcggccgcg gatcccatgg agtcaaagat 25020

tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct 25080

cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt 25140

ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca 25200

acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat 25260

tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 25320

ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 25380

gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 25440

tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 25500

tatataagga agttcatttc atttggagag aacacggggg actgaattaa atatgagccc 25560

tgagaggcgt cctgttgaaa tcagacctgc tactgctgct gatatggctg ctgtttgtga 25620

tatcgtgaac cactacatcg agacttctac cgttaacttc agaactgagc ctcaaactcc 25680

tcaagagtgg atcgatgatc ttgagagact ccaagataga tacccttggc ttgttgctga 25740

ggttgagggt gttgttgctg gaatcgctta cgctggacct tggaaggcta gaaacgctta 25800

cgattggact gttgagtcta ccgtttacgt ttcacacaga catcagagac ttggacttgg 25860

atctaccctt tacactcacc ttctcaagtc tatggaagct cagggattca agtctgttgt 25920

tgctgttatc ggactcccta acgatccttc tgttagactt catgaggctc ttggatacac 25980

tgctagagga actcttagag ctgctggata caagcacggt ggatggcatg atgttggatt 26040

ctggcaaaga gatttcgagc ttcctgctcc tcctagacct gttagaccag ttactcagat 26100

ctgaatttgc gtgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 26160

gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 26220

aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 26280

tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 26340

gcggtgtcat ctatgttact agatcactag tgatgtacgg ttaaaaccac cccagtacat 26400

taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata 26460

tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact 26520

cgatacaggc agcccatcag tcc 26543

<210> SEQ ID NO: 7

<211> LENGTH: 23760

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pGA7- mod_G nucleotide sequence

<400> SEQENCE: 7

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgccccgcgg aaagcttgcg gccgcggtac cgcccgttcg 240

actcagatct tccaaggcct cgtctccgag tccgctgctt ctcgccgcgc cgatcacttc 300

tccgccgcca acaaggcttg tagttaatag gaatcattca gggattgtga ttccgggcag 360

tagtaattaa taatatagta ttagtataga taatatgttt cgtttgggat ctttggaacg 420

ttgctctgtt ccttgttgtt cattttaaag cttttgaggg atagttgcag aactgttcgg 480

tgatgcttca tcctctcaag aactagattt gggtaaagaa acatccatgc atggatatgg 540

aatgttgttc ttccgattgg agattatttt ataaaattta aaattcatga tttaaaaaaa 600

cacataaaaa ccacaaaatt catgatttat tgacaatacg atacaaaatt agcaccaccg 660

gctactggct cattacacat ttccccttcc cctcattctc actttgtggc tttattatta 720

ttattattac atatatttta ccgttattat ttcacgtcac ataagcttgt taattaatta 780

tcactgcttc ttagcaccct tagacttgta cctcttacgg tagaagttgg cgaagagaac 840

gaacatgttc accataaccc agagttgaag gtaaggaagc cagaaagcgg tattctgaac 900

gtaagcggtg tagatagagt gagaagcaca gatacagaac tgaagcatct ggatctgggt 960

gatgtacttc ttccagaaac agttaactcc aagagcagca agtccgtagt aagagtacat 1020

gatcacgtgc acgaaggtgt taacagaaga tccgaagtag caatctccaa caggctcaag 1080

cttcataaca acgaaccatg accagatgag aagagtgtgg tggtagatgt gaaggaaaga 1140

aagctggtcg aacttcttcc tcatcaccat gaagaaggtg tcgagaagct caacgtactt 1200

gttgttgtag tgaagccaga taacctgaga gattccccaa gagttagcag tcatatcagg 1260

gatgtttccc caaaccttaa gtccctgagc cctatgagaa gtaacgaaaa ggtagatgca 1320

gtagctgttg aagaaggtct ggtagaagtt gtaaacgagc atagcgttct tgagtccgaa 1380

aggttgagat ctgttctgca tgatacgctt tccgaagtag atgaagagga ggtatccaat 1440

agttccgata gtaggtcccc agtactcaac ctgcatgaag taagcaggaa ctttctcaga 1500

agctgggata tcagggttag ccacattgta ggtaacgtat ccaagagttc cagcaagagc 1560

agcagggata gcgatagagg ccatggtatg gtaattgtaa atgtaattgt aatgttgttt 1620

gttgtttgtt gttgttggta attgttgtaa aattaattaa gtgggtatct tttggatgga 1680

taagcaagta gtgatgatgt tctaggtgaa gtgatggggg tgttttatag cgggagatgg 1740

tgaaatggat ggtcgccaca taagaaatgg aggggaaggg ttcttgcgcc attcttcagt 1800

ttgcatggat gcatgggttt cattttgtaa cacgtaataa ggacaatgaa gtgcaggtgt 1860

ctctcaagtt tcagagggga tatgtggaca gaagaagaac ggcgatgata ttgatggaaa 1920

tggccatcta gtgtgaatct attcggttga taatactagt gcattttggc cgttaatccc 1980

ttcaattaac tgcacaaact tcagttgagt attgattatt tgattatagg ttctgtaaac 2040

acaataccaa gtttatttag aggggagaca tacaaatagt ttcgatataa ataatagagt 2100

ggttaaactt agttattaaa actatatata aagtctaaaa gttaaattat ttttttaatt 2160

gcaaatatat aaagtctaaa ggggttacat tatttcttaa gagatgtaac tctgttggaa 2220

tctgacttaa tccgtctcat cactctggtt tccagttcta atctaatgaa ttgttttctg 2280

ccaaagaatt tgaagcaaga agtaaattga tcaatgccgt caacccacac caaaccgtca 2340

acccactacc atcgccgcgg agacccccaa actcaacctc cacccatcgg taagaagcac 2400

agggcagccc gcaccaccac caatttggcg tgcatgacac ctagggactt ggcacgggag 2460

gcggcgcacg tggatgcaaa tgacgggata tcagatgaca ggaaacgacg ttgagagacc 2520

atacgatgta gaatatgagc tcaccatcaa cgagaaacta ggaaaatcac aaaaaaaaca 2580

actctcgtaa ttgtacgagt ggcacagatg ggtctgcctc aacatatctc taatacggcg 2640

aagcctgccc aacacgtagt tgccggaatc cggtgtggag ctcacgactc tgaaagatag 2700

gcgcttcctg tttcgtttcg ctcacccact ggacgtccgt catgtgatgg atttcggtca 2760

ttggtttgct gacaaccaca ttctgaagct ccatgagatg agtcttcaca ataggtcctg 2820

ctcaataccg tggagttatg gttgcaagtc cataacttgc cgttcgaata ttttgcggag 2880

ccagtcggac gggaattggc gagctcggct gacacctata aaggccatga caagaagaac 2940

caaaagttct tccctaatgc tttcatgagg cttcgggtcg ttatggatgt cggaaaaccc 3000

ctcttgaagg aacgagacgt tattatgcat gacggtaaga ctattacttg tcagtataag 3060

tatgaaagat tacctgtctt ctgctttgtt tgtggattga ttggacacgt tgaaaaaaaa 3120

tgtgcacttc gatttcaata ctcagagatc gacttccctt ttctctagga gtattcgatc 3180

aaggcattaa catggaagga agctcaagct ctaaaggctt cacaatggaa cctgaaaaat 3240

ttcaacaagc ctaaactgaa atcgaagtca aatcacccaa ccgggagctc taaatcagca 3300

aacactcctc ctccacagta tccaatcatc gtgcacgatg ctccaggtat tgcaagccag 3360

gtattgcaag ctaggagtag gatagagacc ttaaacgtcg ttggtgtgaa gagtcatctt 3420

cagacctaat ggagatagat gtagacggcg gcacgaagac tctgaaacac cagaaaggct 3480

agtccaggat aaggatctgc tatcccaact gacctctcgt tagtcccaag gcctctcaac 3540

tagagcagga ggaaggatgg tcacaagact aggataatga tgtttccaat atgaacctga 3600

atgtccatag ctaatttttt tagtcttgct tctgcacttt ttgtttatta tgttctggtg 3660

actatgttat ttacccttgt ccgtatgctt gagggtaccc tagtagattg gttggttggt 3720

ttccatgtac cagaaggctt accctattag ttgaaagttg aaactttgtt ccctactcaa 3780

ttcctagttg tgtaaatgta tgtatatgta atgtgtataa aacgtagtac ttaaatgact 3840

aggagtggtt cttgagaccg atgagagatg ggagcagaac taaagatgat gacataatta 3900

agaacgaatt tgaaaggctc ttaggtttga atcctattcg agaatgtttt tgtcaaagat 3960

agtggcgatt ttgaaccaaa gaaaacattt aaaaaatcag tatccggtta cgttcatgca 4020

aatagaaagt ggtctaggat ctgattgtaa ttttagactt aaagagtctc ttaagattca 4080

atcctggctg tgtacaaaac tacaaataat atattttaga ctatttggcc ttaactaaac 4140

ttccactcat tatttactga ggttagagaa tagacttgcg aataaacaca ttcccgagaa 4200

atactcatga tcccataatt agtcagaggg tatgccaatc agatctaaga acacacattc 4260

cctcaaattt taatgcacat gtaatcatag tttagcacaa ttcaaaaata atgtagtatt 4320

aaagacagaa atttgtagac ttttttttgg cgttaaaaga agactaagtt tatacgtaca 4380

ttttatttta agtggaaaac cgaaattttc catcgaaata tatgaattta gtatatatat 4440

ttctgcaatg tactattttg ctattttggc aactttcagt ggactactac tttattacaa 4500

tgtgtatgga tgcatgagtt tgagtataca catgtctaaa tgcatgcttt gtaaaacgta 4560

acggaccaca aaagaggatc catacaaata catctcatag cttcctccat tattttccga 4620

cacaaacaga gcattttaca acaattacca acaacaacaa acaacaaaca acattacaat 4680

tacatttaca attaccatac catggaattc gcccagcctc ttgttgctat ggctcaagag 4740

caatacgctg ctatcgatgc tgttgttgct cctgctatct tctctgctac tgattctatc 4800

ggatggggac ttaagcctat ctcttctgct actaaggact tgcctcttgt tgagtctcct 4860

acacctctca tcctttcttt gcttgcttac ttcgctatcg ttggatctgg actcgtttac 4920

agaaaggttt tccctagaac cgtgaaggga caagatccat tccttttgaa ggctcttatg 4980

cttgctcaca acgtgttcct tatcggactt tctctttaca tgtgcctcaa gcttgtgtac 5040

gaggcttacg ttaacaagta ctctttctgg ggaaacgctt acaaccctgc tcaaactgag 5100

atggctaagg ttatctggat cttctacgtg agcaagatct acgagttcat ggataccttc 5160

atcatgctcc tcaagggaaa tgttaaccag gttagcttcc ttcacgttta ccatcacgga 5220

tctatctctg gaatctggtg gatgattact tacgctgctc ctggtggtga tgcttacttc 5280

tctgctgctc ttaactcttg ggttcacgtg tgtatgtaca cctactattt tatggctgcc 5340

gtgcttccta aggacgagaa aactaagaga aagtacctct ggtggggaag ataccttact 5400

caaatgcaga tgttccagtt cttcatgaac cttctccagg ctgtttacct tctctactct 5460

tcatctcctt accctaagtt tatcgctcag ctcctcgtgg tgtacatggt tactcttctc 5520

atgcttttcg gaaacttcta ctacatgaag caccacgcta gcaagtgatg aggcgcgccg 5580

ggccgccgcc atgtgacaga tcgaaggaag aaagtgtaat aagacgactc tcactactcg 5640

atcgctagtg attgtcattg ttatatataa taatgttatc tttcacaact tatcgtaatg 5700

catgtgaaac tataacacat taatcctact tgtcatatga taacactctc cccatttaaa 5760

actcttgtca atttaaagat ataagattct ttaaatgatt aaaaaaaata tattataaat 5820

tcaatcactc ctactaataa attattaatt attatttatt gattaaaaaa atacttatac 5880

taatttagtc tgaatagaat aattagattc tagtctcatc cccttttaaa ccaacttagt 5940

aaacgttttt ttttttaatt ttatgaagtt aagtttttac cttgttttta aaaagaatcg 6000

ttcataagat gccatgccag aacattagct acacgttaca catagcatgc agccgcggag 6060

aattgttttt cttcgccact tgtcactccc ttcaaacacc taagagcttc tctctcacag 6120

cacacacata caatcacatg cgtgcatgca ttattacacg tgatcgccat gcaaatctcc 6180

tttatagcct ataaattaac tcatccgctt cactctttac tcaaaccaaa actcatcgat 6240

acaaacaaga ttaaaaacat acacgaggat cttttacaac aattaccaac aacaacaaac 6300

aacaaacaac attacaatta catttacaat taccatacca tgcctccaag ggactcttac 6360

tcttatgctg ctcctccttc tgctcaactt cacgaagttg atactcctca agagcacgac 6420

aagaaagagc ttgttatcgg agatagggct tacgatgtta ccaacttcgt taagagacac 6480

cctggtggaa agatcattgc ttaccaagtt ggaactgatg ctaccgatgc ttacaagcag 6540

ttccatgtta gatctgctaa ggctgacaag atgcttaagt ctcttccttc tcgtcctgtt 6600

cacaagggat actctccaag aagggctgat cttatcgctg atttccaaga gttcaccaag 6660

caacttgagg ctgagggaat gttcgagcct tctcttcctc atgttgctta cagacttgct 6720

gaggttatcg ctatgcatgt tgctggtgct gctcttatct ggcatggata cactttcgct 6780

ggaatcgcta tgcttggagt tgttcaggga agatgtggat ggcttatgca tgagggtgga 6840

cattactctc tcactggaaa cattgctttc gacagagcta tccaagttgc ttgttacgga 6900

cttggatgtg gaatgtctgg tgcttggtgg cgtaaccagc ataacaagca ccatgctact 6960

cctcaaaagc ttcagcacga tgttgatctt gatacccttc ctctcgttgc tttccatgag 7020

agaatcgctg ctaaggttaa gtctcctgct atgaaggctt ggctttctat gcaagctaag 7080

cttttcgctc ctgttaccac tcttcttgtt gctcttggat ggcagcttta ccttcatcct 7140

agacacatgc tcaggactaa gcactacgat gagcttgcta tgctcggaat cagatacgga 7200

cttgttggat accttgctgc taactacggt gctggatacg ttctcgcttg ttaccttctt 7260

tacgttcagc ttggagctat gtacatcttc tgcaacttcg ctgtttctca tactcacctc 7320

cctgttgttg agcctaacga gcatgctact tgggttgagt acgctgctaa ccacactact 7380

aactgttctc catcttggtg gtgtgattgg tggatgtctt accttaacta ccagatcgag 7440

caccaccttt acccttctat gcctcaattc agacacccta agatcgctcc tagagttaag 7500

cagcttttcg agaagcacgg acttcactac gatgttagag gatacttcga ggctatggct 7560

gatactttcg ctaaccttga taacgttgcc catgctcctg agaagaaaat gcagtaatga 7620

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 7680

atgattatca tataatttct gttgaattac gttaagcacg taataattaa catgtaatgc 7740

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 7800

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 7860

atgttactag atcggtcgat taaaaatccc aattatattt ggtctaattt agtttggtat 7920

tgagtaaaac aaattcgaac caaaccaaaa tataaatata tagtttttat atatatgcct 7980

ttaagacttt ttatagaatt ttctttaaaa aatatctaga aatatttgcg actcttctgg 8040

catgtaatat ttcgttaaat atgaagtgct ccatttttat taactttaaa taattggttg 8100

tacgatcact ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt cttccatatt 8160

catatgtcaa aatctatcaa aattcttata tatctttttc gaatttgaag tgaaatttcg 8220

ataatttaaa attaaataga acatatcatt atttaggtat catattgatt tttatactta 8280

attactaaat ttggttaact ttgaaagtgt acatcaacga aaaattagtc aaacgactaa 8340

aataaataaa tatcatgtgt tattaagaaa attctcctat aagaatattt taatagatca 8400

tatgtttgta aaaaaaatta atttttacta acacatatat ttacttatca aaaatttgac 8460

aaagtaagat taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc 8520

ggcaaaaccg aaccaatcca aaccgatata gttggtttgg tttgattttg atataaaccg 8580

aaccaactcg gtccatttgc acccctaatc ataatagctt taatatttca agatattatt 8640

aagttaacgt tgtcaatatc ctggaaattt tgcaaaatga atcaagccta tatggctgta 8700

atatgaattt aaaagcagct cgatgtggtg gtaatatgta atttacttga ttctaaaaaa 8760

atatcccaag tattaataat ttctgctagg aagaaggtta gctacgattt acagcaaagc 8820

cagaatacaa agaaccataa agtgattgaa gctcgaaata tacgaaggaa caaatatttt 8880

taaaaaaata cgcaatgact tggaacaaaa gaaagtgata tattttttgt tcttaaacaa 8940

gcatcccctc taaagaatgg cagttttcct ttgcatgtaa ctattatgct cccttcgtta 9000

caaaaatttt ggactactat tgggaacttc ttctgaaaat agtgatagaa cccacacgag 9060

catgtgcttt ccatttaatt ttaaaaacca agaaacatac atacataaca ttccatcagc 9120

ctctctctct ttttattacg gttaatgact taaaacacat cttattatcc catccttaac 9180

acctagcagt gtctttatac gatctcatcg atcaccactt caaaaccatg cagactgctg 9240

ctgcccctgg agctggcatc ggctaggctg ggtgccgcac tgtcccggaa ggtccctagc 9300

gacttgttta gattgatggg accacctctc aacttcctgc tgctgtccct gctgctggat 9360

gtcctgcctc atctggccga ttgcacgctc cagtcccctg catgtgcact cgctcctcaa 9420

ttgcttaaga tcatcgcagc agctatcgaa gtgctggctc tgttgccctc ctccacggcc 9480

ttggttgtag tagtagctgc cgccgccctt ctggactttt tcccacagga accgccgaat 9540

aattcgatag aaccacacga gcatgtgctt tcatttattt taaaaaccaa gaaacataca 9600

taacatttca tcagcctctc tctctctctc tctctctctc tctctctctc tctctctctc 9660

tctctcttta ttacagctgt tacactaact taaaacacat tcatctcatt attattatta 9720

ttatccatcc ttaacaccta gcagtgtctt tgtacgatct cataatcgat caccccttca 9780

tcaggtatcc ttaggcttca ctccaacgtt gttgcagtta cggaacatgt acacaccatc 9840

atggttctca acgaactggc aagatctcca agttttccaa aggctaaccc acatgttctc 9900

atcggtgtgt ctgtagtgct ctcccataac tttcttgatg cactcggtag cttctctagc 9960

atggtagaat gggatccttg aaacgtagtg atggagcaca tgagtctcga tgatgtcatg 10020

gaagatgatt ccgaggattc cgaactctct atcgatagta gcagcagcac ccttagcgaa 10080

agtccactct tgagcatcgt aatgaggcat agaagaatcg gtgtgctgaa ggaaggtaac 10140

gaaaacaagc cagtggttaa caaggatcca aggacagaac catgtgatga aagtaggcca 10200

gaatccgaaa accttgtaag cggtgtaaac agaagtgagg gtagcaagga ttccaagatc 10260

agaaagaacg atgtaccagt agtccttctt atcgaaaaca gggctagaag gccagtagtg 10320

agacttgaag aacttagaaa caccagggta aggttgtcca gtagcgttag tagcaaggta 10380

aagagaaagt cctccaagct gttggaacaa gagagcgaaa acagagtaga taggagtttc 10440

ctcagcgata tcgtgaaggc tggtaacttg gtgcttctct ttgaattcct cggcggtgta 10500

aggaacgaaa accatatctc tggtcatgtg tccagtagcc ttatggtgct tagcatgaga 10560

gaacttccag ctgaagtaag gaaccataac aagagagtgg agaacccatc caacggtatc 10620

gttaacccat ccgtagttag agaaagcaga atgtccacac tcatgtccaa ggatccagat 10680

tccgaatccg aaacaagaga tagagaacac gtaagcagac caagcagcga atctaaggaa 10740

ttcgttaggg agaagaggga tgtaggtaag tccaacgtaa gcgatagcag agatagccac 10800

gatatctctc accacgtaag acatagactt cacgagagat ctctcgtaac agtgcttagg 10860

gatagcgtca aggatatcct tgatggtgta atctggcacc ttgaaaacgt ttccgaaggt 10920

atcgatagcg gtcttttgct gcttgaaaga tgcaacgttt ccagaacgcc taacggtctt 10980

agtagatccc tcaaggatct cagatccaga cacggtaacc ttagacatgg tatggtaatt 11040

gtaaatgtaa ttgtaatgtt gtttgttgtt tgttgttgtt ggtaattgtt gtaaaatttt 11100

tggtggtgat tggttcttta aggtgtgaga gtgagttgtg agttgtgtgg tgggtttggt 11160

gagattgggg atggtgggtt tatatagtgg agactgagga atggggtcgt gagtgttaac 11220

tttgcatggg ctacacgtgg gttcttttgg gcttacacgt agtattattc atgcaaatgc 11280

agccaataca tatacggtat tttaataatg tgtgggaata caatatgccg agtattttac 11340

taattttggc aatgacaagt gtacatttgg attatcttac ttggcctctc ttgctttaat 11400

ttggattatt tttattctct taccttggcc gttcatattc acatccctaa aggcaagaca 11460

gaattgaatg gtggccaaaa attaaaacga tggatatgac ctacatagtg taggatcaat 11520

taacgtcgaa ggaaaatact gattctctca agcatacgga caagggtaaa taacatagtc 11580

accagaacat aataaacaaa aagtgcagaa gcaagactaa aaaaattagc tatggacatt 11640

caggttcata ttggaaacat cattatccta gtcttgtgac catccttcct cctgctctag 11700

ttgagaggcc ttgggactaa cgagaggtca gttgggatag cagatcctta tcctggacta 11760

gcctttctgg tgtttcagag tcttcgtgcc gccgtctaca tctatctcca ttaggtctga 11820

agatgactct tcacaccaac gacgtttaag gtctctatcc tactcctagc ttgcaatacc 11880

tggcttgcaa tacctggagc atcgtgcacg atgattggat actgtggagg aggagtgttt 11940

gctgatttag agctcccggt tgggtgattt gacttcgatt tcagtttagg cttgttgaaa 12000

tttttcaggt tccattgtga agcctttaga gcttgagctt ccttccatgt taatgccttg 12060

atcgaatact cctagagaaa agggaagtcg atctctgagt attgaaatcg aagtgcacat 12120

tttttttcaa cgtgtccaat caatccacaa acaaagcaga agacaggtaa tctttcatac 12180

ttatactgac aagtaatagt cttaccgtca tgcataataa cgtctcgttc cttcaagagg 12240

ggttttccga catccataac gacccgaagc ctcatgaaag cattagggaa gaacttttgg 12300

ttcttcttgt catggccttt ataggtgtca gccgagctcg ccaattcccg tccgactggc 12360

tccgcaaaat attcgaacgg caagttatgg acttgcaacc ataactccac ggtattgagc 12420

aggacctatt gtgaagactc atctcatgga gcttcagaat gtggttgtca gcaaaccaat 12480

gaccgaaatc catcacatga cggacgtcca gtgggtgagc gaaacgaaac aggaagcgcc 12540

tatctttcag agtcgtgagc tccacaccgg attccggcaa ctacgtgttg ggcaggcttc 12600

gccgtattag agatatgttg aggcagaccc atctgtgcca ctcgtacaat tacgagagtt 12660

gttttttttg tgattttcct agtttctcgt tgatggtgag ctcatattct acatcgtatg 12720

gtctctcaac gtcgtttcct gtcatctgat atcccgtcat ttgcatccac gtgcgccgcc 12780

tcccgtgcca agtccctagg tgtcatgcac gccaaattgg tggtggtgcg ggctgccctg 12840

tgcttcttac cgatgggtgg aggttgagtt tgggggtctc cgcggcgatg gtagtgggtt 12900

gacggtttgg tgtgggttga cggcattgat caatttactt cttgcttcaa attctttggc 12960

agaaaacaat tcattagatt agaactggaa accagagtga tgagacggat taagtcagat 13020

tccaacagag ttacatctct taagaaataa tgtaacccct ttagacttta tatatttgca 13080

attaaaaaaa taatttaact tttagacttt atatatagtt ttaataacta agtttaacca 13140

ctctattatt tatatcgaaa ctatttgtat gtctcccctc taaataaact tggtattgtg 13200

tttacagaac ctataatcaa ataatcaata ctcaactgaa gtttgtgcag ttaattgaag 13260

ggattaacgg ccaaaatgca ctagtattat caaccgaata gattcacact agatggccat 13320

ttccatcaat atcatcgccg ttcttcttct gtccacatat cccctctgaa acttgagaga 13380

cacctgcact tcattgtcct tattacgtgt tacaaaatga aacccatgca tccatgcaaa 13440

ctgaagaatg gcgcaagaac ccttcccctc catttcttat gtggcgacca tccatttcac 13500

catctcccgc tataaaacac ccccatcact tcacctagaa catcatcact acttgcttat 13560

ccatccaaaa gatacccact tttacaacaa ttaccaacaa caacaaacaa caaacaacat 13620

tacaattaca tttacaatta ccataccatg ccacctagcg ctgctaagca aatgggagct 13680

tctactggtg ttcatgctgg tgttactgac tcttctgctt tcaccagaaa ggatgttgct 13740

gatagacctg atctcaccat cgttggagat tctgtttacg atgctaaggc tttcagatct 13800

gagcatcctg gtggtgctca tttcgtttct ttgttcggag gaagagatgc tactgaggct 13860

ttcatggaat accatagaag ggcttggcct aagtctagaa tgtctagatt ccacgttgga 13920

tctcttgctt ctactgagga acctgttgct gctgatgagg gataccttca actttgtgct 13980

aggatcgcta agatggtgcc ttctgtttct tctggattcg ctcctgcttc ttactgggtt 14040

aaggctggac ttatccttgg atctgctatc gctcttgagg cttacatgct ttacgctgga 14100

aagagacttc tcccttctat cgttcttgga tggcttttcg ctcttatcgg tcttaacatc 14160

cagcatgatg ctaaccatgg tgctttgtct aagtctgctt ctgttaacct tgctcttgga 14220

ctttgtcagg attggatcgg aggatctatg atcctttggc ttcaagagca tgttgttatg 14280

caccacctcc acactaacga tgttgataag gatcctgatc aaaaggctca cggtgctctt 14340

agactcaagc ctactgatgc ttggtcacct atgcattggc ttcagcatct ttaccttttg 14400

cctggtgaga ctatgtacgc tttcaagctt ttgttcctcg acatctctga gcttgttatg 14460

tggcgttggg agggtgagcc tatctctaag cttgctggat acctctttat gccttctttg 14520

cttctcaagc ttaccttctg ggctagattc gttgctttgc ctctttacct tgctccttct 14580

gttcatactg ctgtgtgtat cgctgctact gttatgactg gatctttcta cctcgctttc 14640

ttcttcttca tctcccacaa cttcgagggt gttgcttctg ttggacctga tggatctatc 14700

acttctatga ctagaggtgc tagcttcctt aagagacaag ctgagacttc ttctaacgtt 14760

ggaggacctc ttcttgctac tcttaacggt ggactcaact accaaattga gcatcacttg 14820

ttccctagag ttcaccatgg attctaccct agacttgctc ctcttgttaa ggctgagctt 14880

gaggctagag gaatcgagta caagcactac cctactatct ggtctaacct tgcttctacc 14940

ctcagacata tgtacgctct tggaagaagg cctagatcta aggctgagta atgacaagct 15000

tatgtgacgt gaaataataa cggtaaaata tatgtaataa taataataat aaagccacaa 15060

agtgagaatg aggggaaggg gaaatgtgta atgagccagt agccggtggt gctaattttg 15120

tatcgtattg tcaataaatc atgaattttg tggtttttat gtgttttttt aaatcatgaa 15180

ttttaaattt tataaaataa tctccaatcg gaagaacaac attccatatc catgcatgga 15240

tgtttcttta cccaaatcta gttcttgaga ggatgaagca tcaccgaaca gttctgcaac 15300

tatccctcaa aagctttaaa atgaacaaca aggaacagag caacgttcca aagatcccaa 15360

acgaaacata ttatctatac taatactata ttattaatta ctactgcccg gaatcacaat 15420

ccctgaatga ttcctattaa ctacaagcct tgttggcggc ggagaagtga tcggcgcggc 15480

gagaagcagc ggactcggag acgaggcctt ggaagatctg agtcgaacgg gcagaatcag 15540

tattttcctt cgacgttaat tgatcctaca ctatgtaggt catatccatc gttttaattt 15600

ttggccacca ttcaattctg tcttgccttt agggatgtga atatgaacgg ccaaggtaag 15660

agaataaaaa taatccaaat taaagcaaga gaggccaagt aagataatcc aaatgtacac 15720

ttgtcattgc caaaattagt aaaatactcg gcatattgta ttcccacaca ttattaaaat 15780

accgtatatg tattggctgc atttgcatga ataatactac gtgtaagccc aaaagaaccc 15840

acgtgtagcc catgcaaagt taacactcac gaccccattc ctcagtctcc actatataaa 15900

cccaccatcc ccaatctcac caaacccacc acacaactca caactcactc tcacacctta 15960

aagaaccaat caccaccaaa aattttacaa caattaccaa caacaacaaa caacaaacaa 16020

cattacaatt acatttacaa ttaccatacc atgagcgctg ttaccgttac tggatctgat 16080

cctaagaaca gaggatcttc tagcaacacc gagcaagagg ttccaaaagt tgctatcgat 16140

accaacggaa acgtgttctc tgttcctgat ttcaccatca aggacatcct tggagctatc 16200

cctcatgagt gttacgagag aagattggct acctctctct actacgtgtt cagagatatc 16260

ttctgcatgc ttaccaccgg ataccttacc cataagatcc tttaccctct cctcatctct 16320

tacacctcta acagcatcat caagttcact ttctgggccc tttacactta cgttcaagga 16380

cttttcggaa ccggaatctg ggttctcgct catgagtgtg gacatcaagc tttctctgat 16440

tacggaatcg tgaacgattt cgttggatgg acccttcact cttaccttat ggttccttac 16500

ttcagctgga agtactctca tggaaagcac cataaggcta ctggacacat gaccagagat 16560

atggttttcg ttcctgccac caaagaggaa ttcaagaagt ctaggaactt cttcggtaac 16620

ctcgctgagt actctgagga ttctccactt agaacccttt acgagcttct tgttcaacaa 16680

cttggaggat ggatcgctta cctcttcgtt aacgttacag gacaacctta ccctgatgtt 16740

ccttcttgga aatggaacca cttctggctt acctctccac ttttcgagca aagagatgct 16800

ctctacatct tcctttctga tcttggaatc ctcacccagg gaatcgttct tactctttgg 16860

tacaagaaat tcggaggatg gtcccttttc atcaactggt tcgttcctta catctgggtt 16920

aaccactggc tcgttttcat cacattcctt cagcacactg atcctactat gcctcattac 16980

aacgctgagg aatggacttt cgctaagggt gctgctgcta ctatcgatag aaagttcgga 17040

ttcatcggac ctcacatctt ccatgatatc atcgagactc atgtgcttca ccactactgt 17100

tctaggatcc cattctacaa cgctagacct gcttctgagg ctatcaagaa agttatggga 17160

aagcactaca ggtctagcga cgagaacatg tggaagtcac tttggaagtc tttcaggtct 17220

tgccaatacg ttgacggtga taacggtgtt ctcatgttcc gtaacatcaa caactgcgga 17280

gttggagctg ctgagaagta atgaaggggt gatcgattat gagatcgtac aaagacactg 17340

ctaggtgtta aggatggata ataataataa taatgagatg aatgtgtttt aagttagtgt 17400

aacagctgta ataaagagag agagagagag agagagagag agagagagag agagagagag 17460

agagaggctg atgaaatgtt atgtatgttt cttggttttt aaaataaatg aaagcacatg 17520

ctcgtgtggt tctatcgaat tattcggcgg ttcctgtggg aaaaagtcca gaagggccgc 17580

cgcagctact actacaacca aggccgtgga ggagggcaac agagccagca cttcgatagc 17640

tgctgcgatg atcttaagca attgaggagc gagtgcacat gcaggggact ggagcgtgca 17700

atcggccaga tgaggcagga catccagcag cagggacagc agcaggaagt tgagaggtgg 17760

tcccatcaat ctaaacaagt cgctagggac cttccgggac agtgcggcac ccagcctagc 17820

cgatgccagc tccaggggca gcagcagtct gcatggtttt gaagtggtga tcgatgagat 17880

cgtataaaga cactgctagg tgttaaggat gggataataa gatgtgtttt aagtcattaa 17940

ccgtaataaa aagagagaga ggctgatgga atgttatgta tgtatgtttc ttggttttta 18000

aaattaaatg gaaagcacat gctcgtgtgg gttctatctc gattaaaaat cccaattata 18060

tttggtctaa tttagtttgg tattgagtaa aacaaattcg aaccaaacca aaatataaat 18120

atatagtttt tatatatatg cctttaagac tttttataga attttcttta aaaaatatct 18180

agaaatattt gcgactcttc tggcatgtaa tatttcgtta aatatgaagt gctccatttt 18240

tattaacttt aaataattgg ttgtacgatc actttcttat caagtgttac taaaatgcgt 18300

caatctcttt gttcttccat attcatatgt caaaatctat caaaattctt atatatcttt 18360

ttcgaatttg aagtgaaatt tcgataattt aaaattaaat agaacatatc attatttagg 18420

tatcatattg atttttatac ttaattacta aatttggtta actttgaaag tgtacatcaa 18480

cgaaaaatta gtcaaacgac taaaataaat aaatatcatg tgttattaag aaaattctcc 18540

tataagaata ttttaataga tcatatgttt gtaaaaaaaa ttaattttta ctaacacata 18600

tatttactta tcaaaaattt gacaaagtaa gattaaaata atattcatct aacaaaaaaa 18660

aaaccagaaa atgctgaaaa cccggcaaaa ccgaaccaat ccaaaccgat atagttggtt 18720

tggtttgatt ttgatataaa ccgaaccaac tcggtccatt tgcaccccta atcataatag 18780

ctttaatatt tcaagatatt attaagttaa cgttgtcaat atcctggaaa ttttgcaaaa 18840

tgaatcaagc ctatatggct gtaatatgaa tttaaaagca gctcgatgtg gtggtaatat 18900

gtaatttact tgattctaaa aaaatatccc aagtattaat aatttctgct aggaagaagg 18960

ttagctacga tttacagcaa agccagaata caaagaacca taaagtgatt gaagctcgaa 19020

atatacgaag gaacaaatat ttttaaaaaa atacgcaatg acttggaaca aaagaaagtg 19080

atatattttt tgttcttaaa caagcatccc ctctaaagaa tggcagtttt cctttgcatg 19140

taactattat gctcccttcg ttacaaaaat tttggactac tattgggaac ttcttctgaa 19200

aatagtcctg caggctagta gattggttgg ttggtttcca tgtaccagaa ggcttaccct 19260

attagttgaa agttgaaact ttgttcccta ctcaattcct agttgtgtaa atgtatgtat 19320

atgtaatgcg tataaaacgt agtacttaaa tgactaggag tggttcttga gaccgatgag 19380

agatgggagc agaactaaag atgatgacat aattaagaac gaatttgaaa ggctcttagg 19440

tttgaatcct attcgagaat gtttttgtca aagatagtgg cgattttgaa ccaaagaaaa 19500

catttaaaaa atcagtatcc ggttacgttc atgcaaatag aaagtggtct aggatctgat 19560

tgtaatttta gacttaaaga gtctcttaag attcaatcct ggctgtgtac aaaactacaa 19620

ataatatatt ttagactatt tggccttaac taaacttcca ctcattattt actgaggtta 19680

gagaatagac ttgcgaataa acacattccc gagaaatact catgatccca taattagtca 19740

gagggtatgc caatcagatc taagaacaca cattccctca aattttaatg cacatgtaat 19800

catagtttag cacaattcaa aaataatgta gtattaaaga cagaaatttg tagacttttt 19860

tttggcgtta aaggaagact aagtttatac gtacatttta ttttaagtgg aaaaccgaaa 19920

ttttccatcg aaatatatga atttagtata tatatttctg caatgtacta ttttgctatt 19980

ttggcaactt tcagtggact actactttat tacaatgtgt atggatgcat gagtttgagt 20040

atacacatgt ctaaatgcat gctttgcaaa acgtaacgga ccacaaaaga ggatccatgc 20100

aaatacatct catagcttcc tccattattt tccgacacaa acagagcaga ctctagagga 20160

tccccccgtt ttacaacaat taccaacaac aacaaacaac aaacaacatt acaattacat 20220

ttacaattac catcccaaat cggcgcgcca tgtgtcctcc taagaccgat ggaagatctt 20280

ctcctagatc tcctctcacc aggtctaagt catctgctga ggctcttgat gctaaggatg 20340

cttctaccgc tcctgttgat cttaagaccc ttgagcctca tgaacttgct gctaccttcg 20400

agactagatg ggttagggtt gaggatgttg agtacgacgt gaccaacttc aaacatcctg 20460

gtggaagcgt gatcttctac atgcttgcta acactggtgc tgatgctact gaggctttca 20520

aagaatttca catgcgtagc ctcaaggctt ggaagatgct tagagctttg ccttctagac 20580

ctgctgagat caagagatct gagtctgagg atgctcctat gcttgaggat ttcgctaggt 20640

ggagagctga acttgagagg gacggattct tcaagccttc tatcacccat gttgcttacc 20700

gtcttttgga gcttcttgct actttcgctc ttggaaccgc tcttatgtac gctggatacc 20760

ctatcattgc tagcgttgtg tacggtgctt tcttcggagc tagatgtgga tgggttcaac 20820

atgagggtgg acacaactct cttaccggat ctgtgtacgt ggataagaga cttcaggcta 20880

tgacttgcgg attcggactt tctaccagcg gagagatgtg gaaccagatg cataacaagc 20940

accatgctac ccctcagaaa gttagacacg acatggatct tgataccact cctgctgtgg 21000

ctttcttcaa caccgctgtg gaggataata gacctagggg attctctaga gcttgggcta 21060

gacttcaagc ttggaccttc gttcctgtta cttctggact tctcgttcag gctttctgga 21120

tctacgttct ccatcctaga caggtgctca ggaagaagaa ctacgaggaa gcttcttgga 21180

tgctcgtttc tcacgttgtt agaaccgctg ttatcaagct tgctaccgga tactcttggc 21240

ctgttgctta ctggtggttc actttcggaa actggatcgc ttacatgtac ctcttcgctc 21300

acttctctac ttctcacact cacctccctg ttgttccatc tgacaagcac cttagctggg 21360

ttaactacgc tgttgatcac accgttgaca tcgatccttc tcgtggatac gttaactggc 21420

ttatgggata ccttaactgc caggttatcc accatctctt ccctgatatg cctcaattca 21480

gacagcctga ggtgtcaaga agattcgtcc ctttcgctaa gaagtgggga ctcaactaca 21540

aggtgctctc ttactacggt gcttggaagg ctactttcag caacctcgac aaagttggac 21600

agcactacta cgttaacgga aaggctgaga aggctcactg atgattaatt aaatttgggc 21660

tcgaaccggt tcgagcaagc ttatgtgacg tgaaataata acggtaaaat atatgtaata 21720

ataataataa taaagccaca aagtgagaat gaggggaagg ggaaatgtgt aatgagccag 21780

tagccggtgg tgctaatttt gtatcgtatt gtcaataaat catgaatttt gtggttttta 21840

tgtgtttttt taaatcatga attttaaatt ttataaaata atctccaatc ggaagaacaa 21900

cattccatat ccatgcatgg atgtttcttt acccaaatct agttcttgag aggatgaagc 21960

atcaccgaac agttctgcaa ctatccctca aaagctttaa aatgaacaac aaggaacaga 22020

gcaacgttcc aaagatccca aacgaaacat attatctata ctaatactat attattaatt 22080

actactgccc ggaatcacaa tccctgaatg attcctatta actacaagcc ttgttggcgg 22140

cggagaagtg atcggcgcgg cgagaagcag cggactcgga gacgaggcct tggaagatct 22200

cctgcagggc ggccgcggat cccatggagt caaagattca aatagaggac ctaacagaac 22260

tcgccgtaaa gactggcgaa cagttcatac agagtctctt acgactcaat gacaagaaga 22320

aaatcttcgt caacatggtg gagcacgaca cacttgtcta ctccaaaaat atcaaagata 22380

cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata tccggaaacc 22440

tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag 22500

gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg 22560

ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 22620

ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg 22680

acgcacaatc ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt 22740

tggagagaac acgggggact gaattaaata tgagccctga gaggcgtcct gttgaaatca 22800

gacctgctac tgctgctgat atggctgctg tttgtgatat cgtgaaccac tacatcgaga 22860

cttctaccgt taacttcaga actgagcctc aaactcctca agagtggatc gatgatcttg 22920

agagactcca agatagatac ccttggcttg ttgctgaggt tgagggtgtt gttgctggaa 22980

tcgcttacgc tggaccttgg aaggctagaa acgcttacga ttggactgtt gagtctaccg 23040

tttacgtttc acacagacat cagagacttg gacttggatc taccctttac actcaccttc 23100

tcaagtctat ggaagctcag ggattcaagt ctgttgttgc tgttatcgga ctccctaacg 23160

atccttctgt tagacttcat gaggctcttg gatacactgc tagaggaact cttagagctg 23220

ctggatacaa gcacggtgga tggcatgatg ttggattctg gcaaagagat ttcgagcttc 23280

ctgctcctcc tagacctgtt agaccagtta ctcagatctg aatttgcgtg atcgttcaaa 23340

catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat 23400

ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt 23460

tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa 23520

caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga 23580

tcactagtga tgtacggtta aaaccacccc agtacattaa aaacgtccgc aatgtgttat 23640

taagttgtct aagcgtcaat ttgtttacac cacaatatat cctgccacca gccagccaac 23700

agctccccga ccggcagctc ggcacaaaat caccactcga tacaggcagc ccatcagtcc 23760

<210> SEQ ID NO: 8

<211> LENGTH: 11042

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: pORE04+11ABGBEC_Cowpea_EPA_insert nucleotide

sequence

<400> SEQENCE: 8

tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60

aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120

tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180

tcacgacgtt gtaaaacggg cgccctagaa tctaattatt ctattcagac taaattagta 240

taagtatttt tttaatcaat aaataataat taataattta ttagtaggag tgattgaatt 300

tataatatat tttttttaat catttaaaga atcttatatc tttaaattga caagagtttt 360

aaatggggag agtgttatca tatcacaagt aggattaatg tgttatagtt tcacatgcat 420

tacgataagt tgtgaaagat aacattatta tatataacaa tgacaatcac tagcgatcga 480

gtagtgagag tcgtcttatt acactttctt ccttcgatct gtcacatggc ggcggcccga 540

attctcatca cttagaagcg tggtgcttca tgtagtagaa gtttccgaaa agcatgagaa 600

gagtcaccat gtacaccacg aggagctgag cgataaactt agggtaaggt gaagaagagt 660

agaggaggta cacagcctgg agaaggttca tgaagaactg gaacatttgc atctgggtga 720

ggtatcttcc ccaccagagg tactttctct tagttttctc atccttaggg agcacagcag 780

ccataaagta gtaggtgtac atgcacacgt gcacccaaga gttgagagca gcagagaaat 840

aagcatcacc acctggagca gcgtaggtga tcatccacca gattccagag atagatccgt 900

gatggtacac gtggaggaat gacacctggt tcacatttcc cttgaggagc atgatgaagg 960

tatccatgaa ctcgtagatc tttgacacgt agaaaatcca gatcacctta gccatctcgg 1020

tctgagcagg gttgtaagcg tttccccaga aagagtactt gttcacgtaa gcctcgtaca 1080

cgagcttgag gcacatgtag agtgagagtc cgatgaggaa cacgttatga gcgagcatga 1140

gagccttgag caagaatgga tcctgtccct tcacagttct agggaacacc tttctgtaca 1200

cgagtccaga tcccacgata gcgaagtaag cgaggagaga caagataaga ggggtaggag 1260

attccacgag agggagatcc ttagtagcag aagagatagg cttgagtccc catccgatag 1320

aatcggtagc agagaagata gcaggagcca caacagcatc gatagcagcg tattgttctt 1380

gagccatagc cacgagaggc tgagcaaatt ccatgaattc tgttcttctt tactctttgt 1440

gtgactgagg tttggtctag tgctttggtc atctatatat aatgataaca acaatgagaa 1500

caagctttgg agtgatcgga gggtctagga tacatgagat tcaagtggac taggatctac 1560

accgttggat tttgagtgtg gatatgtgtg aggttaattt tacttggtaa cggccacaaa 1620

ggcctaagga gaggtgttga gacccttatc ggcttgaacc gctggaataa tgccacgtgg 1680

aagataattc catgaatctt atcgttatct atgagtgaaa ttgtgtgatg gtggagtggt 1740

gcttgctcat tttacttgcc tggtggactt ggccctttcc ttatggggaa tttatatttt 1800

acttactata gagctttcat accttttttt taccttggat ttagttaata tataatggta 1860

tgattcatga ataaaaatgg gaaatttttg aatttgtact gctaaatgca taagattagg 1920

tgaaactgtg gaatatatat ttttttcatt taaaagcaaa atttgccttt tactagaatt 1980

ataaatatag aaaaatatat aacattcaaa taaaaatgaa aataagaact ttcaaaaaac 2040

agaactatgt ttaatgtgta aagattagtc gcacatcaag tcatctgtta caatatgtta 2100

caacaagtca taagcccaac aaagttagca cgtctaaata aactaaagag tccacgaaaa 2160

tattacaaat cataagccca acaaagttat tgatcaaaaa aaaaaaacgc ccaacaaagc 2220

taaacaaagt ccaaaaaaaa cttctcaagt ctccatcttc ctttatgaac attgaaaact 2280

atacacaaaa caagtcagat aaatctcttt ctgggcctgt cttcccaacc tcctacatca 2340

cttccctatc ggattgaatg ttttacttgt accttttccg ttgcaatgat attgatagta 2400

tgtttgtgaa aactaatagg gttaacaatc gaagtcatgg aatatggatt tggtccaaga 2460

ttttccgaga gctttctagt agaaagccca tcaccagaaa tttactagta aaataaatca 2520

ccaattaggt ttcttattat gtgccaaatt caatataatt atagaggata tttcaaatga 2580

aaacgtatga atgttattag taaatggtca ggtaagacat taaaaaaatc ctacgtcaga 2640

tattcaactt taaaaattcg atcagtgtgg aattgtacaa aaatttggga tctactatat 2700

atatataatg ctttacaaca cttggatttt tttttggagg ctggaatttt taatctacat 2760

atttgttttg gccatgcacc aactcattgt ttagtgtaat actttgattt tgtcaaatat 2820

atgtgttcgt gtatatttgt ataagaattt ctttgaccat atacacacac acatatatat 2880

atatatatat atattatata tcatgcactt ttaattgaaa aaataatata tatatatata 2940

gtgcattttt tctaacaacc atatatgttg cgattgatct gcaaaaatac tgctagagta 3000

atgaaaaata taatctattg ctgaaattat ctcagatgtt aagattttct taaagtaaat 3060

tctttcaaat tttagctaaa agtcttgtaa taactaaaga ataatacaca atctcgacca 3120

cggaaaaaaa acacataata aatttggggc ccctagaatc taattattct attcagacta 3180

aattagtata agtatttttt taatcaataa ataataatta ataatttatt agtaggagtg 3240

attgaattta taatatattt tttttaatca tttaaagaat cttatatctt taaattgaca 3300

agagttttaa atggggagag tgttatcata tcacaagtag gattaatgtg ttatagtttc 3360

acatgcatta cgataagttg tgaaagataa cattattata tataacaatg acaatcacta 3420

gcgatcgagt agtgagagtc gtcttattac actttcttcc ttcgatctgt cacatggcgg 3480

cggcccgcgg ccgctcatca gtgagccttc tcagcctttc cgttcacgta gtagtgctgt 3540

cccaccttat cgaggtttga gaaggtagcc ttccaagcac cgtagtaaga gagcaccttg 3600

tagttgagtc cccacttctt agcgaaagga acgaatcttc ttgacacctc aggctgtctg 3660

aactgtggca tatctgggaa gaggtgatgg atcacctggc agttgaggta tcccatgagc 3720

cagttcacgt aacccctaga aggatcgata tccacggtgt gatccacagc gtagttcacc 3780

caagaaaggt gcttatcaga tggcaccact gggagatggg tatgagaggt agagaagtga 3840

gcgaagaggt acatgtaagc gatccagttt ccgaaggtga accaccaata agcaacaggc 3900

caagagtatc cggtagcgag cttgataaca gcggttctca caacgtgaga cacgagcatc 3960

caagaagcct cttcgtagtt cttctttctg agcacctgtc taggatggag aacgtagatc 4020

cagaaagcct gcacgagaag tccagaagtc acaggaacga aagtccaagc ctgaagtcta 4080

gcccaagctc tagaaaatcc cctaggcctg ttatcctcaa cagcggtgtt gaagaaagcc 4140

acagcaggag tggtatcgag atccatatca tgcctcacct tttgtggggt tgcgtggtgc 4200

ttgttgtgca tctggttcca catctcacca gaggtagaaa gtccgaatcc gcaagtcata 4260

gcctggagcc tcttatccac atacacagat ccggtgagag agttatgacc accctcgtgt 4320

tgaacccatc cacatctagc tccgaagaaa gcaccgtaca ccacagaagc gataataggg 4380

tatccagcat acatgagagc agttccgaga gcgaaagtag caagaagctc gagaagtctg 4440

tatgccacgt gggtgataga aggcttgaag aatccatccc tctcaagctc agctctccac 4500

ctagcgaaat cttcgagcat aggagcatcc tcagactcag acctcttgat ctcagctggt 4560

ctagaaggca aagccctaag catcttccaa gccttgagag atctcatgtg aaattctttg 4620

aaagcctcag tagcatcagc accggtgtta gcgagcatgt agaagatcac agaaccacca 4680

gggtgcttga agttagtaac atcgtactca acatcctcaa ctctcaccca tctagtctcg 4740

aaggtagcag ccaactcatg aggctcaaga gtcttgagat ccacaggagc agtagaagca 4800

tccttagcat cgagagcctc agcagatgac ttagacctgg taagaggtga cctaggagaa 4860

gatcttccat cagtctttgg agggcacatg cggccgctgt tcttctttac tctttgtgtg 4920

actgaggttt ggtctagtgc tttggtcatc tatatataat gataacaaca atgagaacaa 4980

gctttggagt gatcggaggg tctaggatac atgagattca agtggactag gatctacacc 5040

gttggatttt gagtgtggat atgtgtgagg ttaattttac ttggtaacgg ccacaaaggc 5100

ctaaggagag gtgttgagac ccttatcggc ttgaaccgct ggaataatgc cacgtggaag 5160

ataattccat gaatcttatc gttatctatg agtgaaattg tgtgatggtg gagtggtgct 5220

tgctcatttt acttgcctgg tggacttggc cctttcctta tggggaattt atattttact 5280

tactatagag ctttcatacc ttttttttac cttggattta gttaatatat aatggtatga 5340

ttcatgaata aaaatgggaa atttttgaat ttgtactgct aaatgcataa gattaggtga 5400

aactgtggaa tatatatttt tttcatttaa aagcaaaatt tgccttttac tagaattata 5460

aatatagaaa aatatataac attcaaataa aaatgaaaat aagaactttc aaaaaacaga 5520

actatgttta atgtgtaaag attagtcgca catcaagtca tctgttacaa tatgttacaa 5580

caagtcataa gcccaacaaa gttagcacgt ctaaataaac taaagagtcc acgaaaatat 5640

tacaaatcat aagcccaaca aagttattga tcaaaaaaaa aaaacgccca acaaagctaa 5700

acaaagtcca aaaaaaactt ctcaagtctc catcttcctt tatgaacatt gaaaactata 5760

cacaaaacaa gtcagataaa tctctttctg ggcctgtctt cccaacctcc tacatcactt 5820

ccctatcgga ttgaatgttt tacttgtacc ttttccgttg caatgatatt gatagtatgt 5880

ttgtgaaaac taatagggtt aacaatcgaa gtcatggaat atggatttgg tccaagattt 5940

tccgagagct ttctagtaga aagcccatca ccagaaattt actagtaaaa taaatcacca 6000

attaggtttc ttattatgtg ccaaattcaa tataattata gaggatattt caaatgaaaa 6060

cgtatgaatg ttattagtaa atggtcaggt aagacattaa aaaaatccta cgtcagatat 6120

tcaactttaa aaattcgatc agtgtggaat tgtacaaaaa tttgggatct actatatata 6180

tataatgctt tacaacactt ggattttttt ttggaggctg gaatttttaa tctacatatt 6240

tgttttggcc atgcaccaac tcattgttta gtgtaatact ttgattttgt caaatatatg 6300

tgttcgtgta tatttgtata agaatttctt tgaccatata cacacacaca tatatatata 6360

tatatatata ttatatatca tgcactttta attgaaaaaa taatatatat atatatagtg 6420

cattttttct aacaaccata tatgttgcga ttgatctgca aaaatactgc tagagtaatg 6480

aaaaatataa tctattgctg aaattatctc agatgttaag attttcttaa agtaaattct 6540

ttcaaatttt agctaaaagt cttgtaataa ctaaagaata atacacaatc tcgaccacgg 6600

aaaaaaaaca cataataaat ttgggcgcgc cgcgtattgg ctagagcagc ttgccaacat 6660

ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca 6720

aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg 6780

cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca cctacaaatg 6840

ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca gtggtcccaa 6900

agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 6960

aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctcgtct actccaagaa 7020

tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac aaagggtaat 7080

atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca aaaggacagt 7140

agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca 7200

agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga 7260

aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga 7320

cgtaagggat gacgcacaat cccactatcc ttcgcaagac cttcctctat ataaggaagt 7380

tcatttcatt tggagaggac acgctgaaat caccagtctc tctctacaaa tctatctctg 7440

cgatcgcatg cctcctaggg attcttactc ttacgctgct cctccatctg ctcagctcca 7500

tgaagttgat actcctcaag agcacgataa gaaagaactc gtgatcggag atagggctta 7560

cgatgtgacc aacttcgtga agagacaccc tggtggaaag attatcgctt accaggttgg 7620

aactgatgct accgatgctt acaagcagtt ccacgtgaga tctgctaagg ctgataagat 7680

gctcaagtct ctcccatcta ggcctgtgca caagggatat tctccaagaa gggctgatct 7740

tatcgctgat ttccaagagt tcaccaagca gcttgaggct gagggaatgt tcgaaccttc 7800

tctccctcat gtggcttaca gactcgctga ggttatcgct atgcatgttg ctggtgctgc 7860

tctcatctgg cacggatata ctttcgctgg aatcgctatg ctcggagtgg ttcagggaag 7920

atgtggatgg cttatgcatg agggtggaca ctactctctc accggaaaca ttgctttcga 7980

tagggctatc caggtggcat gctatggact tggatgtgga atgtctggtg cttggtggag 8040

aaaccagcat aacaagcacc atgctacccc tcaaaagctc cagcatgatg tggatctcga 8100

tactctccct ctcgtggctt tccatgagag aatcgctgct aaggtgaagt ctcctgctat 8160

gaaggcttgg ctctctatgc aggctaagct tttcgctcct gtgactactc ttctcgttgc 8220

tcttggatgg cagctctacc tccatcctag acacatgctc aggaccaagc actacgatga 8280

gcttgctatg ctcggtatca gatacggact cgttggatac ctcgctgcta attacggtgc 8340

tggatacgtt ctcgcttgct accttcttta cgttcagctc ggagctatgt acatcttctg 8400

caacttcgct gtgtctcaca ctcatctccc tgtggttgaa cctaacgagc atgctacttg 8460

ggttgagtac gctgctaacc acactaccaa ctgctctcca tcttggtggt gtgattggtg 8520

gatgtcttac ctcaactacc agatcgagca ccacctctac ccttctatgc ctcagttcag 8580

acaccctaag atcgctccta gagtgaagca gcttttcgag aagcacggac tccactacga 8640

tgtgagagga tactttgagg ctatggctga taccttcgct aacctcgata atgtggctca 8700

cgctcctgag aagaaaatgc agtgatgagc gatcgcgatc gttcaaacat ttggcaataa 8760

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 8820

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 8880

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 8940

gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcc ctgcagggcg 9000

tattggctag agcagcttgc caacatggtg gagcacgaca ctctcgtcta ctccaagaat 9060

atcaaagata cagtctcaga agaccaaagg gctattgaga cttttcaaca aagggtaata 9120

tcgggaaacc tcctcggatt ccattgccca gctatctgtc acttcatcaa aaggacagta 9180

gaaaaggaag gtggcaccta caaatgccat cattgcgata aaggaaaggc tatcgttcaa 9240

gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa 9300

aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgataa catggtggag 9360

cacgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct 9420

attgagactt ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct 9480

atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat 9540

tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga 9600

cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 9660

gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg 9720

caagaccttc ctctatataa ggaagttcat ttcatttgga gaggacacgc tgaaatcacc 9780

agtctctctc tacaaatcta tctctctcga gatgattgaa caagatggat tgcacgcagg 9840

ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 9900

ctgctctgat gccgccgtgt tccggctgtc agcgcagggg aggccggttc tttttgtcaa 9960

gaccgacctg tccggtgccc tgaatgaact tcaagacgag gcagcgcggc tatcgtggct 10020

ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 10080

ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 10140

cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 10200

ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 10260

cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 10320

gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga ctcatggcga 10380

tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 10440

ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 10500

agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 10560

ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaaacg cgtgatcgtt 10620

caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 10680

tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 10740

tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 10800

aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 10860

tagatcgacg tccgtacggt taaaaccacc ccagtacatt aaaaacgtcc gcaatgtgtt 10920

attaagttgt ctaagcgtca atttgtttac accacaatat atcctgccac cagccagcca 10980

acagctcccc gaccggcagc tcggcacaaa atcaccactc gatacaggca gcccatcagt 11040

cc 11042

<210> SEQ ID NO: 9

<211> LENGTH: 1254

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Codon-optimized open reading frame for

expression of Lachancea kluyveri 12 desaturase in plants

<400> SEQENCE: 9

atgagcgctg ttaccgttac tggatctgat cctaagaaca gaggatcttc tagcaacacc 60

gagcaagagg ttccaaaagt tgctatcgat accaacggaa acgtgttctc tgttcctgat 120

ttcaccatca aggacatcct tggagctatc cctcatgagt gttacgagag aagattggct 180

acctctctct actacgtgtt cagagatatc ttctgcatgc ttaccaccgg ataccttacc 240

cataagatcc tttaccctct cctcatctct tacacctcta acagcatcat caagttcact 300

ttctgggccc tttacactta cgttcaagga cttttcggaa ccggaatctg ggttctcgct 360

catgagtgtg gacatcaagc tttctctgat tacggaatcg tgaacgattt cgttggatgg 420

acccttcact cttaccttat ggttccttac ttcagctgga agtactctca tggaaagcac 480

cataaggcta ctggacacat gaccagagat atggttttcg ttcctgccac caaagaggaa 540

ttcaagaagt ctaggaactt cttcggtaac ctcgctgagt actctgagga ttctccactt 600

agaacccttt acgagcttct tgttcaacaa cttggaggat ggatcgctta cctcttcgtt 660

aacgttacag gacaacctta ccctgatgtt ccttcttgga aatggaacca cttctggctt 720

acctctccac ttttcgagca aagagatgct ctctacatct tcctttctga tcttggaatc 780

ctcacccagg gaatcgttct tactctttgg tacaagaaat tcggaggatg gtcccttttc 840

atcaactggt tcgttcctta catctgggtt aaccactggc tcgttttcat cacattcctt 900

cagcacactg atcctactat gcctcattac aacgctgagg aatggacttt cgctaagggt 960

gctgctgcta ctatcgatag aaagttcgga ttcatcggac ctcacatctt ccatgatatc 1020

atcgagactc atgtgcttca ccactactgt tctaggatcc cattctacaa cgctagacct 1080

gcttctgagg ctatcaagaa agttatggga aagcactaca ggtctagcga cgagaacatg 1140

tggaagtcac tttggaagtc tttcaggtct tgccaatacg ttgacggtga taacggtgtt 1200

ctcatgttcc gtaacatcaa caactgcgga gttggagctg ctgagaagta atga 1254

<210> SEQ ID NO: 10

<211> LENGTH: 416

<212> TYPE: PRT

<213> ORGANISM: Lachancea kluyveri

<400> SEQENCE: 10

Met Ser Ala Val Thr Val Thr Gly Ser Asp Pro Lys Asn Arg Gly Ser

1 5 10 15

Ser Ser Asn Thr Glu Gln Glu Val Pro Lys Val Ala Ile Asp Thr Asn

20 25 30

Gly Asn Val Phe Ser Val Pro Asp Phe Thr Ile Lys Asp Ile Leu Gly

35 40 45

Ala Ile Pro His Glu Cys Tyr Glu Arg Arg Leu Ala Thr Ser Leu Tyr

50 55 60

Tyr Val Phe Arg Asp Ile Phe Cys Met Leu Thr Thr Gly Tyr Leu Thr

65 70 75 80

His Lys Ile Leu Tyr Pro Leu Leu Ile Ser Tyr Thr Ser Asn Ser Ile

85 90 95

Ile Lys Phe Thr Phe Trp Ala Leu Tyr Thr Tyr Val Gln Gly Leu Phe

100 105 110

Gly Thr Gly Ile Trp Val Leu Ala His Glu Cys Gly His Gln Ala Phe

115 120 125

Ser Asp Tyr Gly Ile Val Asn Asp Phe Val Gly Trp Thr Leu His Ser

130 135 140

Tyr Leu Met Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Gly Lys His

145 150 155 160

His Lys Ala Thr Gly His Met Thr Arg Asp Met Val Phe Val Pro Ala

165 170 175

Thr Lys Glu Glu Phe Lys Lys Ser Arg Asn Phe Phe Gly Asn Leu Ala

180 185 190

Glu Tyr Ser Glu Asp Ser Pro Leu Arg Thr Leu Tyr Glu Leu Leu Val

195 200 205

Gln Gln Leu Gly Gly Trp Ile Ala Tyr Leu Phe Val Asn Val Thr Gly

210 215 220

Gln Pro Tyr Pro Asp Val Pro Ser Trp Lys Trp Asn His Phe Trp Leu

225 230 235 240

Thr Ser Pro Leu Phe Glu Gln Arg Asp Ala Leu Tyr Ile Phe Leu Ser

245 250 255

Asp Leu Gly Ile Leu Thr Gln Gly Ile Val Leu Thr Leu Trp Tyr Lys

260 265 270

Lys Phe Gly Gly Trp Ser Leu Phe Ile Asn Trp Phe Val Pro Tyr Ile

275 280 285

Trp Val Asn His Trp Leu Val Phe Ile Thr Phe Leu Gln His Thr Asp

290 295 300

Pro Thr Met Pro His Tyr Asn Ala Glu Glu Trp Thr Phe Ala Lys Gly

305 310 315 320

Ala Ala Ala Thr Ile Asp Arg Lys Phe Gly Phe Ile Gly Pro His Ile

325 330 335

Phe His Asp Ile Ile Glu Thr His Val Leu His His Tyr Cys Ser Arg

340 345 350

Ile Pro Phe Tyr Asn Ala Arg Pro Ala Ser Glu Ala Ile Lys Lys Val

355 360 365

Met Gly Lys His Tyr Arg Ser Ser Asp Glu Asn Met Trp Lys Ser Leu

370 375 380

Trp Lys Ser Phe Arg Ser Cys Gln Tyr Val Asp Gly Asp Asn Gly Val

385 390 395 400

Leu Met Phe Arg Asn Ile Asn Asn Cys Gly Val Gly Ala Ala Glu Lys

405 410 415

<210> SEQ ID NO: 11

<211> LENGTH: 1251

<212> TYPE: DNA

<213> ORGANISM: Pichia pastoris

<400> SEQENCE: 11

atgtctaagg ttaccgtgtc tggatctgag atccttgagg gatctactaa gaccgttagg 60

cgttctggaa acgttgcatc tttcaagcag caaaagaccg ctatcgatac cttcggaaac 120

gttttcaagg tgccagatta caccatcaag gatatccttg acgctatccc taagcactgt 180

tacgagagat ctctcgtgaa gtctatgtct tacgtggtga gagatatcgt ggctatctct 240

gctatcgctt acgttggact tacctacatc cctcttctcc ctaacgaatt ccttagattc 300

gctgcttggt ctgcttacgt gttctctatc tcttgtttcg gattcggaat ctggatcctt 360

ggacatgagt gtggacattc tgctttctct aactacggat gggttaacga taccgttgga 420

tgggttctcc actctcttgt tatggttcct tacttcagct ggaagttctc tcatgctaag 480

caccataagg ctactggaca catgaccaga gatatggttt tcgttcctta caccgccgag 540

gaattcaaag agaagcacca agttaccagc cttcacgata tcgctgagga aactcctatc 600

tactctgttt tcgctctctt gttccaacag cttggaggac tttctcttta ccttgctact 660

aacgctactg gacaacctta ccctggtgtt tctaagttct tcaagtctca ctactggcct 720

tctagccctg ttttcgataa gaaggactac tggtacatcg ttctttctga tcttggaatc 780

cttgctaccc tcacttctgt ttacaccgct tacaaggttt tcggattctg gcctactttc 840

atcacatggt tctgtccttg gatccttgtt aaccactggc ttgttttcgt taccttcctt 900

cagcacaccg attcttctat gcctcattac gatgctcaag agtggacttt cgctaagggt 960

gctgctgcta ctatcgatag agagttcgga atcctcggaa tcatcttcca tgacatcatc 1020

gagactcatg tgctccatca ctacgtttca aggatcccat tctaccatgc tagagaagct 1080

accgagtgca tcaagaaagt tatgggagag cactacagac acaccgatga gaacatgtgg 1140

gttagccttt ggaaaacttg gagatcttgc cagttcgttg agaaccatga tggtgtgtac 1200

atgttccgta actgcaacaa cgttggagtg aagcctaagg atacctgatg a 1251

<210> SEQ ID NO: 12

<211> LENGTH: 415

<212> TYPE: PRT

<213> ORGANISM: Pichia pastoris

<400> SEQENCE: 12

Met Ser Lys Val Thr Val Ser Gly Ser Glu Ile Leu Glu Gly Ser Thr

1 5 10 15

Lys Thr Val Arg Arg Ser Gly Asn Val Ala Ser Phe Lys Gln Gln Lys

20 25 30

Thr Ala Ile Asp Thr Phe Gly Asn Val Phe Lys Val Pro Asp Tyr Thr

35 40 45

Ile Lys Asp Ile Leu Asp Ala Ile Pro Lys His Cys Tyr Glu Arg Ser

50 55 60

Leu Val Lys Ser Met Ser Tyr Val Val Arg Asp Ile Val Ala Ile Ser

65 70 75 80

Ala Ile Ala Tyr Val Gly Leu Thr Tyr Ile Pro Leu Leu Pro Asn Glu

85 90 95

Phe Leu Arg Phe Ala Ala Trp Ser Ala Tyr Val Phe Ser Ile Ser Cys

100 105 110

Phe Gly Phe Gly Ile Trp Ile Leu Gly His Glu Cys Gly His Ser Ala

115 120 125

Phe Ser Asn Tyr Gly Trp Val Asn Asp Thr Val Gly Trp Val Leu His

130 135 140

Ser Leu Val Met Val Pro Tyr Phe Ser Trp Lys Phe Ser His Ala Lys

145 150 155 160

His His Lys Ala Thr Gly His Met Thr Arg Asp Met Val Phe Val Pro

165 170 175

Tyr Thr Ala Glu Glu Phe Lys Glu Lys His Gln Val Thr Ser Leu His

180 185 190

Asp Ile Ala Glu Glu Thr Pro Ile Tyr Ser Val Phe Ala Leu Leu Phe

195 200 205

Gln Gln Leu Gly Gly Leu Ser Leu Tyr Leu Ala Thr Asn Ala Thr Gly

210 215 220

Gln Pro Tyr Pro Gly Val Ser Lys Phe Phe Lys Ser His Tyr Trp Pro

225 230 235 240

Ser Ser Pro Val Phe Asp Lys Lys Asp Tyr Trp Tyr Ile Val Leu Ser

245 250 255

Asp Leu Gly Ile Leu Ala Thr Leu Thr Ser Val Tyr Thr Ala Tyr Lys

260 265 270

Val Phe Gly Phe Trp Pro Thr Phe Ile Thr Trp Phe Cys Pro Trp Ile

275 280 285

Leu Val Asn His Trp Leu Val Phe Val Thr Phe Leu Gln His Thr Asp

290 295 300

Ser Ser Met Pro His Tyr Asp Ala Gln Glu Trp Thr Phe Ala Lys Gly

305 310 315 320

Ala Ala Ala Thr Ile Asp Arg Glu Phe Gly Ile Leu Gly Ile Ile Phe

325 330 335

His Asp Ile Ile Glu Thr His Val Leu His His Tyr Val Ser Arg Ile

340 345 350

Pro Phe Tyr His Ala Arg Glu Ala Thr Glu Cys Ile Lys Lys Val Met

355 360 365

Gly Glu His Tyr Arg His Thr Asp Glu Asn Met Trp Val Ser Leu Trp

370 375 380

Lys Thr Trp Arg Ser Cys Gln Phe Val Glu Asn His Asp Gly Val Tyr

385 390 395 400

Met Phe Arg Asn Cys Asn Asn Val Gly Val Lys Pro Lys Asp Thr

405 410 415

<210> SEQ ID NO: 13

<211> LENGTH: 1392

<212> TYPE: DNA

<213> ORGANISM: Micromonas pusilla

<400> SEQENCE: 13

atgtgcccgc cgaagacgga cggccgatcg tccccgcgat cgccgctgac gcgcagcaaa 60

tcctccgcgg aggcgctcga cgccaaggac gcgtcgaccg cgcccgtcga tctcaaaacg 120

ctcgagccgc acgagctcgc ggcgacgttc gagacgcgat gggtgcgcgt ggaggacgtc 180

gagtacgacg tcacaaactt caaacacccg ggaggcagcg tgatattcta catgctcgcg 240

aacacgggcg cggacgccac ggaggcgttc aaggagttcc acatgcgatc gcttaaggcg 300

tggaagatgc tcagagcgct gccgtcgcgc cccgcggaga tcaaacgcag cgagagcgag 360

gacgcgccga tgttggagga tttcgcgcgg tggcgcgcgg agctcgaacg cgacgggttc 420

tttaagccct cgataacgca cgtcgcgtat cggttactcg agctcctcgc gaccttcgcc 480

ctcggcaccg ccctcatgta cgccgggtac ccgatcatcg cgtccgtcgt gtacggcgcg 540

ttcttcggcg ctcggtgcgg ttgggtccag cacgagggcg ggcacaactc gctcacgggg 600

tccgtctacg tcgacaagcg cctccaagcg atgacgtgcg ggttcgggct gtccacgagc 660

ggggagatgt ggaaccagat gcacaataag caccacgcga cgccgcagaa agtgaggcac 720

gacatggacc tggacacgac ccccgcggtg gcgtttttta acaccgccgt ggaggacaac 780

cggccgaggg ggttctcccg cgcgtgggct cggcttcagg cgtggacgtt cgtcccggtg 840

acctccgggc tgctcgtcca ggcgttctgg atctacgtcc tgcacccgcg gcaggtgttg 900

cgaaagaaga actacgagga ggcgtcgtgg atgctcgtct ctcacgtcgt caggaccgcg 960

gtgattaaac tcgcgacggg gtactcgtgg cccgtcgcgt actggtggtt caccttcggc 1020

aactggatcg cgtacatgta cctcttcgcg cacttctcca cgagccacac gcacctcccg 1080

gtcgtgccct cggataagca cctgagctgg gtgaactacg cggtcgatca caccgtggac 1140

atcgacccgt cgcgcgggta cgtgaactgg ttgatgggat atctgaactg ccaggtcatt 1200

catcacctgt tcccggacat gccgcagttt cgccagccgg aggtgagccg gcggttcgtc 1260

ccgttcgcga agaagtgggg gctgaactac aaggtgctgt cctattacgg cgcctggaag 1320

gcgacgttct cgaacttgga taaggtcggg cagcactact acgtcaacgg caaggcggag 1380

aaggcgcact ga 1392

<210> SEQ ID NO: 14

<211> LENGTH: 1395

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Codon-optimized open reading frame for

expression of Micromonas pusilla 6-desaturase in plants

(version 1)

<400> SEQENCE: 14

atgtgccctc ctaagactga tggaagatct tctcctagat ctccacttac caggtctaaa 60

tcttctgctg aggctcttga tgctaaggat gcttctactg ctcctgttga tcttaagact 120

cttgagcctc atgagcttgc tgctactttc gagactagat gggttagagt tgaggacgtt 180

gagtacgatg tgactaactt caagcaccct ggtggatctg tgatcttcta catgcttgct 240

aacactggtg ctgatgctac tgaggctttc aaagaattcc acatgcgttc tctcaaggct 300

tggaagatgc ttagagcttt gccttctaga cctgctgaga tcaagagatc tgagtctgag 360

gatgctccta tgcttgagga tttcgctaga tggcgtgctg agcttgagag agatggattc 420

ttcaagcctt ctatcaccca tgtggcttac agacttctcg agcttcttgc tacattcgct 480

cttggaactg ctcttatgta cgctggatac cctatcattg cttctgttgt ttacggtgct 540

ttcttcggag ctagatgtgg atgggttcaa catgagggtg gacataactc tcttaccgga 600

tctgtttacg tggacaagag acttcaggct atgacttgtg gattcggact ttctacttct 660

ggtgagatgt ggaaccagat gcataacaag caccatgcta cccctcaaaa ggttagacac 720

gatatggatc ttgataccac tcctgctgtg gctttcttca acactgctgt tgaggataac 780

agacctagag gattctctag agcttgggct agacttcaag cttggacttt cgttcctgtt 840

acctctggac ttcttgttca agctttctgg atctacgttc tccaccctag acaagttctc 900

cgtaagaaga actacgaaga ggcttcttgg atgctcgttt ctcatgttgt tagaaccgct 960

gttatcaagc ttgctactgg atactcttgg cctgttgctt actggtggtt cactttcgga 1020

aactggatcg cttacatgta ccttttcgct cacttctcta cttctcatac tcacctccct 1080

gttgttccat ctgataagca cctttcttgg gttaactacg ctgttgatca caccgttgat 1140

atcgatcctt ctagaggata cgtgaactgg cttatgggat accttaactg tcaggttatc 1200

caccacctct tccctgatat gcctcaattc agacagcctg aggttagcag aagattcgtt 1260

cctttcgcta agaagtgggg actcaactac aaggtgctct cttactacgg tgcttggaag 1320

gctactttct ctaaccttga taaggtggga cagcactact acgttaacgg aaaggctgag 1380

aaggctcact aatga 1395

<210> SEQ ID NO: 15

<211> LENGTH: 1395

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Codon-optimized open reading frame for

expression of Micromonas pusilla 6-desaturase in plants

(version 2)

<400> SEQENCE: 15

atgtgtcctc ctaagaccga tggaagatct tctcctagat ctcctctcac caggtctaag 60

tcatctgctg aggctcttga tgctaaggat gcttctaccg ctcctgttga tcttaagacc 120

cttgagcctc atgaacttgc tgctaccttc gagactagat gggttagggt tgaggatgtt 180

gagtacgacg tgaccaactt caaacatcct ggtggaagcg tgatcttcta catgcttgct 240

aacactggtg ctgatgctac tgaggctttc aaagaatttc acatgcgtag cctcaaggct 300

tggaagatgc ttagagcttt gccttctaga cctgctgaga tcaagagatc tgagtctgag 360

gatgctccta tgcttgagga tttcgctagg tggagagctg aacttgagag ggacggattc 420

ttcaagcctt ctatcaccca tgttgcttac cgtcttttgg agcttcttgc tactttcgct 480

cttggaaccg ctcttatgta cgctggatac cctatcattg ctagcgttgt gtacggtgct 540

ttcttcggag ctagatgtgg atgggttcaa catgagggtg gacacaactc tcttaccgga 600

tctgtgtacg tggataagag acttcaggct atgacttgcg gattcggact ttctaccagc 660

ggagagatgt ggaaccagat gcataacaag caccatgcta cccctcagaa agttagacac 720

gacatggatc ttgataccac tcctgctgtg gctttcttca acaccgctgt ggaggataat 780

agacctaggg gattctctag agcttgggct agacttcaag cttggacctt cgttcctgtt 840

acttctggac ttctcgttca ggctttctgg atctacgttc tccatcctag acaggtgctc 900

aggaagaaga actacgagga agcttcttgg atgctcgttt ctcacgttgt tagaaccgct 960

gttatcaagc ttgctaccgg atactcttgg cctgttgctt actggtggtt cactttcgga 1020

aactggatcg cttacatgta cctcttcgct cacttctcta cttctcacac tcacctccct 1080

gttgttccat ctgacaagca ccttagctgg gttaactacg ctgttgatca caccgttgac 1140

atcgatcctt ctcgtggata cgttaactgg cttatgggat accttaactg ccaggttatc 1200

caccatctct tccctgatat gcctcaattc agacagcctg aggtgtcaag aagattcgtc 1260

cctttcgcta agaagtgggg actcaactac aaggtgctct cttactacgg tgcttggaag 1320

gctactttca gcaacctcga caaagttgga cagcactact acgttaacgg aaaggctgag 1380

aaggctcact gatga 1395

<210> SEQ ID NO: 16

<211> LENGTH: 463

<212> TYPE: PRT

<213> ORGANISM: Micromonas pusilla

<400> SEQENCE: 16

Met Cys Pro Pro Lys Thr Asp Gly Arg Ser Ser Pro Arg Ser Pro Leu

1 5 10 15

Thr Arg Ser Lys Ser Ser Ala Glu Ala Leu Asp Ala Lys Asp Ala Ser

20 25 30

Thr Ala Pro Val Asp Leu Lys Thr Leu Glu Pro His Glu Leu Ala Ala

35 40 45

Thr Phe Glu Thr Arg Trp Val Arg Val Glu Asp Val Glu Tyr Asp Val

50 55 60

Thr Asn Phe Lys His Pro Gly Gly Ser Val Ile Phe Tyr Met Leu Ala

65 70 75 80

Asn Thr Gly Ala Asp Ala Thr Glu Ala Phe Lys Glu Phe His Met Arg

85 90 95

Ser Leu Lys Ala Trp Lys Met Leu Arg Ala Leu Pro Ser Arg Pro Ala

100 105 110

Glu Ile Lys Arg Ser Glu Ser Glu Asp Ala Pro Met Leu Glu Asp Phe

115 120 125

Ala Arg Trp Arg Ala Glu Leu Glu Arg Asp Gly Phe Phe Lys Pro Ser

130 135 140

Ile Thr His Val Ala Tyr Arg Leu Leu Glu Leu Leu Ala Thr Phe Ala

145 150 155 160

Leu Gly Thr Ala Leu Met Tyr Ala Gly Tyr Pro Ile Ile Ala Ser Val

165 170 175

Val Tyr Gly Ala Phe Phe Gly Ala Arg Cys Gly Trp Val Gln His Glu

180 185 190

Gly Gly His Asn Ser Leu Thr Gly Ser Val Tyr Val Asp Lys Arg Leu

195 200 205

Gln Ala Met Thr Cys Gly Phe Gly Leu Ser Thr Ser Gly Glu Met Trp

210 215 220

Asn Gln Met His Asn Lys His His Ala Thr Pro Gln Lys Val Arg His

225 230 235 240

Asp Met Asp Leu Asp Thr Thr Pro Ala Val Ala Phe Phe Asn Thr Ala

245 250 255

Val Glu Asp Asn Arg Pro Arg Gly Phe Ser Arg Ala Trp Ala Arg Leu

260 265 270

Gln Ala Trp Thr Phe Val Pro Val Thr Ser Gly Leu Leu Val Gln Ala

275 280 285

Phe Trp Ile Tyr Val Leu His Pro Arg Gln Val Leu Arg Lys Lys Asn

290 295 300

Tyr Glu Glu Ala Ser Trp Met Leu Val Ser His Val Val Arg Thr Ala

305 310 315 320

Val Ile Lys Leu Ala Thr Gly Tyr Ser Trp Pro Val Ala Tyr Trp Trp

325 330 335

Phe Thr Phe Gly Asn Trp Ile Ala Tyr Met Tyr Leu Phe Ala His Phe

340 345 350

Ser Thr Ser His Thr His Leu Pro Val Val Pro Ser Asp Lys His Leu

355 360 365

Ser Trp Val Asn Tyr Ala Val Asp His Thr Val Asp Ile Asp Pro Ser

370 375 380

Arg Gly Tyr Val Asn Trp Leu Met Gly Tyr Leu Asn Cys Gln Val Ile

385 390 395 400

His His Leu Phe Pro Asp Met Pro Gln Phe Arg Gln Pro Glu Val Ser

405 410