Great research starts with great data.

Learn More
More >
Patent Analysis of

Transformant for expressing cis-prenyltranferase and Nogo B receptor

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10000774

Application Number

US15/107178

Application Date

06 January 2015

Publication Date

19 June 2018

Current Assignee

SUMITOMO RUBBER INDUSTRIES, LTD.

Original Assignee (Applicant)

SUMITOMO RUBBER INDUSTRIES, LTD.

International Classification

C12N5/04,C12N1/20,C12N15/00,C12N9/10,C12P5/00

Cooperative Classification

C12P5/007,C07K14/415,C07K14/705,C12N9/1085,C12N15/8201

Inventor

YAMAGUCHI, HARUHIKO

Patent Images

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

US10000774 Transformant expressing cis-prenyltranferase 1
See all images <>

Abstract

Provided are a transformant produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor, which are considered to be involved in polyisoprenoid biosynthesis, into a host to allow the host to express the cis-prenyltransferase and the Nogo-B receptor, and a method for producing a polyisoprenoid using the transformant. The present invention relates to a transformant produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor into a host to allow the host to express the cis-prenyltransferase and the Nogo-B receptor.

Read more

Claims

1. A transformant of a Hevea or Taraxacum cell, produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor into a Hevea or Taraxacum cell for the cell to express the cis-prenyltransferase and the Nogo-B receptor, wherein the gene coding for a Nogo-B receptor is either of the following DNAs: a DNA comprising the nucleotide sequence of SEQ ID NO: 5; and a DNA having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 5, wherein the gene coding for a cis-prenyltransferase is either of the following DNAs: a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3; and a DNA having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 1 or 3.

2. The transformant according to claim 1, wherein at least one of the gene coding for a cis-prenyltransferase or the gene coding for a Nogo-B receptor is derived from a plant.

3. The transformant according to claim 2, wherein at least one of the gene coding for a cis-prenyltransferase or the gene coding for a Nogo-B receptor is derived from an isoprenoid-producing plant.

4. The transformant according to claim 3, wherein at least one of the gene coding for a cis-prenyltransferase or the gene coding for a Nogo-B receptor is derived from Hevea brasiliensis.

5. The transformant according to claim 1, wherein the gene coding for a cis-prenyltransferase is either of the following DNAs: a DNA comprising the nucleotide sequence of SEQ ID NO: 1; and a DNA having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 1.

6. A method for producing a polyisoprenoid using the transformant according to claim 1.

Read more

Claim Tree

  • 1
    1. A transformant of a Hevea or Taraxacum cell, produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor into a Hevea or Taraxacum cell for the cell to express the cis-prenyltransferase and the Nogo-B receptor, wherein
    • the gene coding for a Nogo-B receptor is either of the following DNAs: a DNA comprising
    • 2. The transformant according to claim 1, wherein
      • at least one of the gene coding for a cis-prenyltransferase or the gene coding for a Nogo-B receptor is derived from a plant.
    • 5. The transformant according to claim 1, wherein
      • the gene coding for a cis-prenyltransferase is either of the following DNAs: a DNA comprising
  • 6
    6. A method for producing a polyisoprenoid using the transformant according to claim 1.
See all independent claims <>

Description

TECHNICAL FIELD

The present invention relates to a transformant produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor into a host to allow the host to express the cis-prenyltransferase and the Nogo-B receptor, and a method for producing a polyisoprenoid using the transformant.

BACKGROUND ART

Nowadays natural rubber (one example of polyisoprenoids) for use in industrial rubber products are harvested from isoprenoid-producing plants, such as para rubber tree (Hevea brasiliensis) belonging to the family Euphorbiaceae, or Indian rubber tree (Ficus elastica) belonging to the family Moraceae.

At present, Hevea brasiliensis is virtually the only source for the natural rubber used in industrial rubber products. Hevea brasiliensis is a plant that can only be grown in certain regions, including Southeast Asia and South America. Moreover, Hevea brasiliensis trees take about seven years from planting to grow mature enough to yield rubber, and yields natural rubber only for a period of 20 to 30 years. Demand for natural rubber is expected to grow in the future, especially in developing countries, but for the reasons discussed above it is difficult to greatly increase natural rubber production from Hevea brasiliensis. There is therefore concern that natural rubber sources will dry up, and needs exist to develop stable natural rubber sources other than mature Hevea brasiliensis trees and to improve productivity of natural rubber from Hevea brasiliensis.

Natural rubber has a cis-1,4-polyisoprene structure, with isopentenyl diphosphate (IPP) unit, and the nature of this structure suggests that cis-prenyltransferase (CPT) is involved in natural rubber biosynthesis. For example, several CPTs are found in Hevea brasiliensis, including Hevea rubber transferase 1 (HRT1) and Hevea rubber transferase 2 (HRT2) (see, for example, Non Patent Literatures 1 and 2). It is also known that rubber synthesis can be reduced in the dandelion species Taraxacum brevicorniculatum by suppressing CPT expression (see, for example, Non Patent Literature 3).

Previous studies of proteins associated with natural rubber biosynthesis have focused on rubber elongation factor (REF) and small rubber particle protein (SRPP) (see, for example, Non Patent Literatures 4 and 5). However, the associations between these proteins and CPT are not completely understood.

It has also been suggested that Nogo-B receptor (NgBr) is involved in dolichol biosynthesis by a human CPT (see, for example, Non Patent Literature 6).

CITATION LIST

Non Patent Literature

  • Non Patent Literature 1: Rahaman et al., BMC Genomics, 2013, vol. 14
  • Non Patent Literature 2: Asawatreratanakul et al, European Journal of Biochemistry, 2003, vol. 270, pp. 4671-4680
  • Non Patent Literature 3: Post et al., Plant Physiology, 2012, vol. 158, pp. 1406-1417
  • Non Patent Literature 4: Hillebrand et al., PLoS ONE, 2012, vol. 7
  • Non Patent Literature 5: Priya et al., Plant Cell Reports, 2007, vol. 26, pp. 1833-1838
  • Non Patent Literature 6: K. D. Harrison et al., The EMBO Journal, 2011, vol. 30, pp. 2490-2500

SUMMARY OF INVENTION

Technical Problem

As discussed above, needs exist to develop stable natural rubber sources other than mature Hevea brasiliensis trees and to improve productivity of natural rubber from Hevea brasiliensis. At present, however, the biosynthesis mechanism of natural rubber, and particularly the regulatory mechanism remains largely unclear, and there is still much room for improvement to greatly increase natural rubber production. In this context, one possible approach to solving these problems is to stabilize and enhance the activity of CPT in natural rubber biosynthesis in order to increase natural rubber production.

In view of these circumstances, the present invention aims to provide a transformant produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor, which are considered to be involved in polyisoprenoid biosynthesis, into a host to allow the host to express the cis-prenyltransferase and the Nogo-B receptor, and a method for producing a polyisoprenoid using the transformant.

Solution to Problem

The present invention relates to a transformant produced by introducing a gene coding for a cis-prenyltransferase and a gene coding for a Nogo-B receptor into a host to allow the host to express the cis-prenyltransferase and the Nogo-B receptor.

Preferably, at least one of the gene coding for a cis-prenyltransferase or the gene coding for a Nogo-B receptor is derived from a plant.

Preferably, at least one of the gene coding for a cis-prenyltransferase or the gene coding for a Nogo-B receptor is derived from an isoprenoid-producing plant.

Preferably, at least one of the gene coding for a cis-prenyltransferase or the gene coding fora Nogo-B receptor is derived from Hevea brasiliensis.

Preferably, the gene coding for a Nogo-B receptor is either of the following DNAs:

[3] a DNA having the nucleotide sequence of SEQ ID NO:5; and

[4] a DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:5 under stringent conditions.

Preferably, the gene coding for a cis-prenyltransferase is either of the following DNAs:

[1] a DNA having the nucleotide sequence of SEQ ID NO:1 or 3; and

[2] a DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 or 3 under stringent conditions, and coding for a protein having an enzyme activity that catalyzes a reaction of cis-chain elongation of an isoprenoid compound.

Preferably, the gene coding for a cis-prenyltransferase is either of the following DNAs:

[1-1] a DNA having the nucleotide sequence of SEQ ID NO: 1; and

[2-1] a DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 under stringent conditions, and coding for a protein having an enzyme activity that catalyzes a reaction of cis-chain elongation of an isoprenoid compound.

The host is preferably an isoprenoid-producing plant.

The present invention also relates to a method for producing a polyisoprenoid using the above-described transformant.

Advantageous Effects of Invention

The transformant of the present invention is produced by introducing a gene coding for a cis-prenyltransferase (CPT) and a gene coding for a Nogo-B receptor (NgBr) into a host to allow the host to express the CPT and NgBr. Since the CPT and NgBR are co-expressed in the host, the activity of CPT is expected to be stabilized and enhanced. Accordingly, it is expected that in the transformant, the amount of the products biosynthesized through the reactions catalyzed by CPT and therefore polyisoprenoid production is enhanced, and thus the use of such a transformant in polyisoprenoid production can result in increased polyisoprenoid yield.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows photographs illustrating the results of yeast two-hybrid analyses.

DESCRIPTION OF EMBODIMENTS

The inventor of the present invention made various studies for improving polyisoprenoid productivity. In the studies, they focused on cis-prenyltransferase (CPT) because it has been considered to be one of enzymes playing a key role in polyisoprenoid biosynthesis. According to a report (Non-Patent Literature 6), Nogo-B receptor (NgBr) interacts with CPT in humans to improve the stability of the CPT protein, thereby stabilizing and enhancing dolichol biosynthesis activity. This suggests the possible involvement of NgBr in the activity of CPT in organisms in general. In view of the above, the present inventor prepared a transformant engineered to express CPT and NgBr. In such a transformant, due to the coexistence of CPT and NgBr, the activity of CPT is expected to be stabilized and enhanced. Accordingly, it is expected that the amount of the products biosynthesized through the reactions catalyzed by CPT and therefore polyisoprenoid production is enhanced, resulting in increased polyisoprenoid yield.

The transformant of the present invention is produced by introducing a gene coding for a cis-prenyltransferase (CPT) and a gene coding for a Nogo-B receptor (NgBr) into a host to allow the host to express the CPT and NgBr.

The gene coding for a CPT and/or the gene coding for a NgBr may be of any origin, and is preferably derived from a plant, more preferably an isoprenoid-producing plant. The genes are still more preferably both derived from at least one isoprenoid-producing plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum, and Taraxacum kok-saghyz, particularly preferably Hevea brasiliensis, among others.

(Amino Acid Sequence of cis-prenyltransferase (CPT))

The following protein [1] is a specific example of the CPT:

  • [1] a protein having the amino acid sequence of SEQ ID NO:2 or 4.

It is known that proteins having one or more amino acid substitutions, deletions, insertions, or additions relative to the original amino acid sequence can have the inherent function. Considering this fact, another specific example of the CPT is the following protein [2]:

  • [2] a protein having an amino acid sequence containing one or more amino acid substitutions, deletions, insertions and/or additions relative to the amino acid sequence of SEQ ID NO:2 or 4, and having the inherent function thereof.

The inherent function of the protein of SEQ ID NO:2 or 4 herein refers to the inherent function of CPT, that is, an enzyme activity that catalyzes a reaction of cis-chain elongation of an isoprenoid compound.

In order to maintain the enzyme activity, the amino acid sequence preferably contains one or more, more preferably 1-58, still more preferably 1-44, further more preferably 1-29, particularly preferably 1-15, most preferably 1-6, yet most preferably 1-3 amino acid substitutions, deletions, insertions and/or additions relative to the amino acid sequence of SEQ ID NO:2.

Also in order to maintain the enzyme activity, the amino acid sequence preferably contains one or more, more preferably 1-57, still more preferably 1-43, further more preferably 1-29, particularly preferably 1-15, most preferably 1-6, yet most preferably 1-3 amino acid substitutions, deletions, insertions and/or additions relative to the amino acid sequence of SEQ ID NO:4.

Among other amino acid substitutions, conservative substitutions are preferred. Specific examples include substitutions within each of the following groups in the parentheses: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine), and the like.

It is also known that proteins with amino acid sequences having high sequence identity to the original amino acid sequence can also have similar function. Considering this fact, another specific example of the CPT is the following protein [3]:

  • [3] a protein having an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2 or 4, and having the inherent function thereof.

In order to maintain the inherent function, i.e. the enzyme activity described above, the sequence identity to the amino acid sequence of SEQ ID NO:2 or 4 is preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, particularly preferably at least 98%, most preferably at least 99%.

The sequence identity between amino acid sequences or nucleotide sequences may be determined using the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] developed by Karlin and Altschul or FASTA [Methods Enzymol., 183, 63 (1990)].

Whether it is a protein having the above enzyme activity may be determined by conventional techniques, such as by expressing a target protein in a transformant prepared by introducing a gene coding for the target protein into Escherichia coli or other host organisms, and determining the presence or absence of the function of the target protein by the corresponding activity measurement method.

The CPT is preferably any of the following proteins:

  • [1-1] a protein having the amino acid sequence of SEQ ID NO:2;
  • [2-1] a protein having an amino acid sequence containing one or more amino acid substitutions, deletions, insertions, and/or additions relative to the amino acid sequence of SEQ ID NO:2, and having the inherent function thereof; and
  • [3-1] a protein having an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, and having the inherent function thereof. The present inventor has found that, when the CPT is any of the above proteins, it interacts with NgBr. In this case, the activity of CPT is expected to be further stabilized and enhanced.

    (Amino Acid Sequence of Nogo-B Receptor (NgBr))

The following protein [4] is a specific example of the NgBr:

  • [4] a protein having the amino acid sequence of SEQ ID NO:6.

As described above, it is known that proteins having one or more amino acid substitutions, deletions, insertions, or additions relative to the original amino acid sequence can have the inherent function. Thus, another specific example of the NgBr is the following protein [5]:

  • [5] a protein having an amino acid sequence containing one or more amino acid substitutions, deletions, insertions and/or additions relative to the amino acid sequence of SEQ ID NO:6, and having the inherent function thereof.

The inherent function of the protein of SEQ ID NO: 6 herein refers to the inherent function of NgBr, that is, the functions of binding to a membrane via one or more transmembrane domains on the N-terminal side of the protein, and interacting with another protein on the C-terminal side thereof. As a function of Nogo-B receptor in neurons, NgBr is known to interact with a myelin inhibitory protein (for example, Nogo-A) or the like, thereby being involved in signaling leading to neuronal growth-cone collapse and neurite outgrowth inhibition. Herein, however, the interaction involved in the above signaling is not included in the inherent function of the protein of SEQ ID NO: 6.

In order to maintain the inherent function, i.e. the function of NgBr, the amino acid sequence preferably contains one or more, more preferably 1 to 52, still more preferably 1 to 39, further more preferably 1 to 26, particularly preferably 1 to 13, most preferably 1 to 6, yet most preferably 1 to 3 amino acid substitutions, deletions, insertions, and/or additions relative to the amino acid sequence of SEQ ID NO:6.

Similarly to the above, among other amino acid substitutions, conservative substitutions are preferred. Specific examples include substitutions within each of the following groups in the parentheses: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine), and the like.

As described above, it is also known that proteins with amino acid sequences having high sequence identity to the original amino acid sequence can also have similar function. Thus, another specific example of the NgBr is the following protein [6]:

  • [6] a protein having an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6, and having the inherent function thereof.

In order to maintain the inherent function, i.e. the function of NgBr, the sequence identity to the amino acid sequence of SEQ ID NO:6 is preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, particularly preferably at least 98%, most preferably at least 99%.

Whether it is a protein having a structure of NgBr may be determined by conventional techniques, such as by expressing a target protein in a transformant prepared by introducing a gene coding for the target protein into Escherichia coli or other host organisms, crushing the transformant followed by separation into fractions by centrifugation, and then observing strong expression in the membrane fraction by western blot analysis using a commercial anti-Nogo receptor antibody (e.g. Millipore, GeneTex).

(DNA Coding for cis-prenyltransferase (CPT))

The DNA coding for a CPT may be either of the following DNAs:

  • [1] a DNA having the nucleotide sequence of SEQ ID NO:1 or 3; and
  • [2] a DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 or 3 under stringent conditions, and coding for a protein having the inherent function thereof.

The inherent function of the protein encoded by the DNA having the nucleotide sequence of SEQ ID NO:1 or 3 herein is the same as the inherent function of the protein of SEQ ID NO:2 or 4.

As used herein, the term “hybridizing” means a process in which a DNA hybridizes to a DNA having a specific nucleotide sequence or a part of the DNA. Accordingly, the DNA having a specific nucleotide sequence or part of the DNA may have a nucleotide sequence long enough to be usable as a probe in northern or southern blot analysis or as an oligonucleotide primer in polymerase chain reaction (PCR) analysis. The DNA used as a probe may have a length of at least 100 bases, preferably at least 200 bases, more preferably at least 500 bases although it may be a DNA of at least 10 bases, preferably of at least 15 bases in length.

Techniques to perform DNA hybridization experiments are well known. The hybridization conditions under which experiments are carried out may be determined according to, for example, Molecular Cloning, 2nd ed. and 3rd ed. (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press (Molecular), and many other standard textbooks.

The stringent conditions may include, for example, an overnight incubation at 42° C. of a DNA-immobilized filter and a DNA probe in a solution containing 50% formamide, 5×SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/l denatured salmon sperm DNA, followed by washing the filter for example in a 0.2×SSC solution at approximately 65° C. Less stringent conditions may also be used. Changes in the stringency may be accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lower stringency), salt concentrations or temperature. For example, low stringent conditions include an overnight incubation at 37° C. in a solution containing 6×SSCE (20×SSCE: 3 mol/l sodium chloride, 0.2 mol/l sodium dihydrogen phosphate, 0.02 mol/l EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg/l denatured salmon sperm DNA, followed by washing in a 1×SSC solution containing 0.1% SDS at 50° C. In addition, to achieve even lower stringency, washes performed following hybridization may be done at higher salt concentrations (e.g. 5×SSC) in the above-mentioned low stringent conditions.

Variations in the above various conditions may be accomplished through the inclusion or substitution of blocking reagents used to suppress background in hybridization experiments. The inclusion of blocking reagents may require modification of the hybridization conditions for compatibility.

The DNA capable of hybridization under stringent conditions as described above may have a nucleotide sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, particularly preferably at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:1 or 3 as calculated using a program such as BLAST or FASTA with the parameters mentioned above.

Whether the DNA capable of hybridizing to the aforementioned DNA under stringent conditions codes for a protein having a predetermined enzyme activity may be determined by conventional techniques, such as by expressing a target protein in a transformant prepared by introducing a gene coding for the target protein into Escherichia coli or other host organisms, and determining the presence or absence of the function of the target protein by the corresponding activity measurement method.

The DNA coding for a CPT is preferably either of the following DNAs:

  • [1-1] a DNA having the nucleotide sequence of SEQ ID NO:1; and
  • [2-1] a DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 under stringent conditions, and coding for a protein having the inherent function thereof. The present inventor has found that, when the DNA coding for a CPT is either of the DNAs with above sequences, the expressed CPT interacts with NgBr. In this case, the activity of CPT is expected to be further stabilized and enhanced.

    (DNA Coding for Nogo-B Receptor (NgBr))

The DNA coding for a NgBr may be either of the following DNAs:

  • [3] a DNA having the nucleotide sequence of SEQ ID NO:5; and
  • [4] a DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:5 under stringent conditions, and coding for a protein having the inherent function thereof.

Herein, the inherent function of the protein encoded by the DNA having the nucleotide sequence of SEQ ID NO:5 is the same as the inherent function of the protein of SEQ ID NO:6.

The term “hybridizing” herein is as described above. Also, the stringent conditions are as described above.

The DNA capable of hybridization under stringent conditions as described above may have a nucleotide sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, particularly preferably at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:5 as calculated using a program such as BLAST or FASTA with the parameters mentioned above.

Whether the DNA capable of hybridizing to the aforementioned DNA under stringent conditions codes for a protein having a predetermined structure may be determined by conventional techniques, such as by expressing a target protein in a transformant prepared by introducing a gene coding for the target protein into Escherichia coli or other host organisms, crushing the transformant followed by separation into fractions by centrifugation, and then observing strong expression in the membrane fraction by western blot analysis using a commercial anti-Nogo receptor antibody (e.g. Millipore, GeneTex).

Conventional techniques may be employed to identify the amino acid sequence or the nucleotide sequence of the proteins. For example, total RNA is extracted from a growing plant, the mRNA is optionally purified, and a cDNA is synthesized by a reverse transcription reaction. Subsequently, degenerate primers are designed based on the amino acid sequence of a known protein corresponding to the target protein, a DNA fragment is partially amplified by RT-PCR, and the sequence is partially identified. Then, the RACE method or the like is performed to identify the full-length nucleotide sequence or amino acid sequence. The RACE method (rapid amplification of cDNA ends method) refers to a method in which, when the nucleotide sequence of a cDNA is partially known, PCR is performed based on the nucleotide sequence information of such a known region to clone an unknown region extending to the cDNA terminal, and is capable of cloning the full-length cDNA by PCR without preparing a cDNA library.

The degenerate primers may each preferably be prepared from a plant-derived sequence having a highly similar sequence part to the target protein.

If the nucleotide sequence coding for the protein is known, it is possible to identify the full-length nucleotide sequence or amino acid sequence by designing a primer containing an initiation codon and a primer containing a termination codon using the known nucleotide sequence, followed by performing RT-PCR using a synthesized cDNA as a template.

(Transformant)

The gene coding for a CPT and the gene coding for a NgBr are introduced into a host to produce an organism (transformant) that has been transformed to express the CPT and the NgBr. Since the CPT and NgBr are co-expressed in the transformant, the activity of CPT is expected to be stabilized and enhanced. Accordingly, it is expected that in the transformant, the amount of the products biosynthesized through the reactions catalyzed by CPT and therefore polyisoprenoid production is enhanced, suitably resulting in increased polyisoprenoid yield.

The following briefly describes how to prepare the organism (transformant) transformed to express the CPT and NgBr. Such a transformant can be prepared by conventionally known methods.

Specifically, for example, the transformant may be prepared as follows: A DNA containing the nucleotide sequence of SEQ ID NO: 1 or 3, and a DNA containing the nucleotide sequence of SEQ ID NO:5 are inserted downstream of the promoter of a suitable expression vector with suitable restriction enzymes and the like to prepare a recombinant DNA. This recombinant DNA may then be introduced into host cells which are compatible with the expression vector, to obtain transformed cells. Alternatively, an expression vector in which a DNA containing the nucleotide sequence of SEQ ID NO:1 or 3 is inserted downstream of the promoter with suitable restriction enzymes and the like, and an expression vector in which a DNA containing the nucleotide sequence of SEQ ID NO:5 is inserted downstream of the promoter with suitable restriction enzymes and the like are used to prepare recombinant DNAs, and these recombinant DNAs may then be introduced into host cells which are compatible with the expression vectors, to obtain transformed cells.

Any host (host cells) capable of expressing the genes of interest may be used such as microorganisms, yeasts, animal cells, insect cells, plant cells, and other organisms, preferably eukaryotes. Since improved polyisoprenoid productivity and increased polyisoprenoid yield can be expected particularly when the CPT and NgBR are expressed in organisms capable of polyisoprenoid biosynthesis, the host is preferably a plant, more preferably an isoprenoid-producing plant, among others, and the host cells are preferably plant cells, more preferably cells of an isoprenoid-producing plant. Thus, in another suitable embodiment of the present invention, the host is an isoprenoid-producing plant.

The isoprenoid-producing plant is not particularly limited as long as it is capable of producing an isoprenoid. Examples include plants of the genus Hevea, such as Hevea brasiliensis; plants of the genus Sonchus, such as Sonchus oleraceus, Sonchus asper, and Sonchus brachyotus; plants of the genus Solidago, such as Solidago altissima, Solidago virgaurea subsp. asiatica, Solidago virgaurea subsp. leipcarpa, Solidago virgaurea subsp. leipcarpa f. paludosa, Solidago virgaurea subsp. gigantea, and Solidago gigantea Ait. var. leiophylla Fernald; plants of the genus Helianthus, such as Helianthus annuus, Helianthus argophyllus, Helianthus atrorubens, Helianthus debilis, Helianthus decapetalus, and Helianthus giganteus; plants of the genus Taraxacum, such as dandelion (Taraxacum), Taraxacum venustum H. Koidz, Taraxacum hondoense Nakai, Taraxacum platycarpum Dahlst, Taraxacum japonicum, Taraxacum officinale Weber, and Taraxacum kok-saghyz; plants of the genus Ficus, such as Ficus carica, Ficus elastica, Ficus pumila L., Ficus erecta Thumb., Ficus ampelas Burm. f., Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarpa L.f., Ficus septica Burm. f., and Ficus benghalensis; plants of the genus Parthenium, such as Parthenium argentatum, Parthenium hysterophorus, and Ambrosia artemisiifolia (Parthenium hysterophorus); lettuce (Lactuca sativa) (Lactuca serriola), and Ficus benghalensis. The isoprenoid-producing plant is preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum, and Parhenium, more preferably at least one selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum, and Taraxacum kok-saghyz, among others.

The expression vector may be a vector that is capable of autonomous replication in the host cells or of being incorporated into the chromosome thereof, and contains a promoter at a position that permits transcription of the recombinant DNA.

In the case where plant cells are used as host cells, a pBI vector, a pUC vector, a Ti plasmid or tobacco mosaic virus vector, for example, may be used as an expression vector.

Any promoter that functions in plant cells can be used. Examples include cauliflower mosaic virus (CaMV) 35S promoter, rice actin-1 promoter, nopaline synthase gene promoter, tobacco mosaic virus 35S promoter, and rice actin gene promoter.

Preferred are expression vectors with promoters that are specifically expressed in tissues in which isoprenoid compounds are biosynthesized, such as laticifers. Plant growth retardation and other adverse effects can be reduced by expressing specifically in a tissue in which an isoprenoid is biosynthesized.

The recombinant vector can be introduced by any method that allows the DNA to be introduced into host cells. Examples include methods using Agrobacterium (JP S59-140885 A, JP S60-70080 A, WO94/00977), electroporation (JP S60-251887 A), and methods using particle guns (gene guns) (JP 2606856 B, JP 2517813 B).

The transformant (transgenic plant cells) can be prepared by the above or other methods.

The present invention also provides an isoprenoid-producing plant into which have been introduced a gene coding for a CPT and a gene coding for a NgBr. The isoprenoid-producing plant is not particularly limited, as long as it is an isoprenoid-producing plant containing the transgenic plant cells. The isoprenoid-producing plant conceptually includes not only transgenic plant cells prepared by the above-described methods, but also, for example, all of their progeny or clones and even progeny plants obtained by passaging these cells. Once transgenic plant cells into which the DNA or vector has been introduced in the genome are obtained, progeny or clones can be obtained from the transgenic plant cells by sexual or asexual reproduction, tissue culture, cell culture, cell fusion, or other techniques. Further, the transgenic plant cells, or their progeny or clones may be used to obtain reproductive materials (e.g. seeds, fruits, cuttings, stem tubers, root tubers, shoots, adventitious buds, adventitious embryos, calluses, protoplasts), which can then be used to produce the isoprenoid-producing plant on a large scale.

Techniques to regenerate plants from transgenic plant cells are already known; for example, Doi et al. disclose techniques for eucalyptus (JP H11-127025 A), Fujimura et al. disclose techniques for rice (Fujimura et al., (1995), Plant Tissue Culture Lett., vol. 2: p. 74-), Shillito et al. disclose techniques for corn (Shillito et al., (1989), Bio/Technology, vol. 7: p. 581-), Visser et al. disclose techniques for potato (Visser et al., (1989), Theor. Appl. Genet., vol. 78: p. 589-), and Akama et al. disclose techniques for Arabidopsis thaliana (Akama et al., (1992), Plant Cell Rep., vol. 12: p. 7-). Those skilled in the art can regenerate plants from the transgenic plant cells according to these documents.

Whether a target protein gene is expressed in a regenerated plant may be determined by well-known methods. For example, western blot analysis may be used to assess the expression of a target protein.

Seeds can be obtained from the transgenic plant, for example, as follows: the transgenic plant is rooted in an appropriate medium, transplanted to water-containing soil in a pot, and grown under proper cultivation conditions so as to finally produce seeds, which are then collected. Further, plants can be grown from seeds, for example, as follows: seeds obtained from the transgenic plant as described above are sown in water-containing soil, and grown under proper cultivation conditions into plants.

According to the present invention, it is expected that the isoprenoid-producing plant into which have been introduced a gene coding for a CPT and a gene coding for a NgBr can be used in polyisoprenoid production to improve polyisoprenoid productivity. Specifically, polyisoprenoid production may be carried out by culturing transgenic plant cells prepared as described above, calluses obtained from such transgenic plant cells, cells redifferentiated from such calluses, or the like in an appropriate medium, or by growing transgenic plants regenerated from the transgenic plant cells, plants grown from seeds collected from such transgenic plants, or the like under proper cultivation conditions. The transformant of the present invention is expected to have stabilized and enhanced CPT activity due to the proteins introduced therein. Accordingly, it is expected that the amount of the products biosynthesized through the reactions catalyzed by CPT and therefore polyisoprenoid production is enhanced, resulting in increased polyisoprenoid yield.

The term “polyisoprenoid” as used herein is a generic term used to refer to polymers having isoprene (C5H8) units. Examples of polyisoprenoids include polymers such as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40), and naturalrubber. Theterm“isoprenoid” as used herein refers to a compound having isoprene (C5H8) units, and conceptually includes polyisoprenoids.

As described above, since the CPT and NgBr are co-expressed in the present invention, the activity of CPT is expected to be stabilized and enhanced. Accordingly, it is expected that the amount of the products biosynthesized through the reactions catalyzed by CPT and therefore polyisoprenoid production is enhanced in the transformant, resulting in increased polyisoprenoid yield. Thus, another aspect of the present invention relates to a method for producing a polyisoprenoid using a transformant produced by introducing a gene coding for a CPT and a gene coding for a NgBr into a host to allow the host to express the CPT and the NgBr.

Possible methods for increasing polyisoprenoid yield in the presence of both CPT and NgBr include, in addition to the method of using a transformant engineered to express both CPT and NgBr in vivo as described above, a method involving the presence of both CPT and NgBr in vitro, for example, by extracting crude enzymes from cells, purifying the CPT and NgBr, and allowing them to be present together in a test tube.

In the method involving the presence of both CPT and NgBr in vitro, the CPT and NgBr may be produced, for example, by inserting a gene coding for the CPT and/or a gene coding for the NgBr into an appropriate vector, and using transformed Escherichia coli, yeasts, or plants, cell-free protein expression systems, or other means.

The origin of the CPT used in the method involving the presence of both CPT and NgBr in vitro is not particularly limited, and the CPT is preferably derived from a eukaryote, more preferably a plant, still more preferably an isoprenoid-producing plant. The CPT may also be modified by any method, such as by adding a modification group to the enzyme (e.g. phosphorylation, methylation, acetylation, palmitoylation, myristoylation, farnesylation, sugar chain addition, or ubiquitination), or by oxidation/reduction of the disulfide group, or by structural modification with protease.

The origin of the NgBr used in the method involving the presence of both CPT and NgBr in vitro is not particularly limited, and the NgBr is preferably derived from a eukaryote, more preferably a plant, still more preferably an isoprenoid-producing plant. The NgBr may also be modified by any method, such as by adding a modification group to the enzyme (e.g. phosphorylation, methylation, acetylation, palmitoylation, myristoylation, farnesylation, sugar chain addition, or ubiquitination), or by oxidation/reduction of the disulfide group, or by structural modification with protease.

As described above, the present inventor has found that the CPT encoded by the DNA having the nucleotide sequence of SEQ ID NO:1 or the DNA capable of hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 under stringent conditions, and coding for a protein having the inherent function thereof, interacts with NgBr. Further to the CPT, NgBr is also considered to interact with a third protein. Examples of the mode of such interaction include the following two modes:

(1) The CPT interacts with the third protein via NgBr, or in other words, NgBr, the CPT, and the third protein simultaneously interact with one another.

(2) The interaction between NgBr and the CPT and the interaction between NgBr and the third protein occur individually.

Non-limiting examples of the third protein include rubber elongation factor (REF), small rubber particle protein (SRPP), and farnesyl diphosphate (FPP) synthase.

In the method involving the presence of both CPT and NgBr in vitro, in addition to the CPT and NgBr, another enzyme may further be added so as to be present together. Examples of the enzyme include enzymes known to be present in latex, such as rubber elongation factor (REF), small rubber particle protein (SRPP), small GTP-binding protein, hevein, β-1,3-glucanase, farnesyl diphosphate (FPP) synthase, and protease inhibitor proteins. Among these, enzymes interacting with NgBr, such as REF, are especially preferred.

In the method involving the presence of both CPT and NgBr in vitro, further components that may be added so as to be present together in addition to the CPT and NgBr are not limited to enzymes and may include membranes that serve to incorporate reaction products. The type of membrane is not particularly limited, and may be a natural membrane such as a cell membrane or small rubber particle, or an artificial membrane such as liposome.

The lipid forming the membrane may be a lipid that can form a lipid bilayer membrane, and examples include known glyceroglycolipids, sphingoglycolipids, cholesterol, and phospholipids.

Examples of the glyceroglycolipids include sulfoxyribosylglycerides, diglycosyldiglycerides, digalactosyldiglycerides, galactosyldiglycerides, and glycosyldiglycerides. Examples of the sphingoglycolipids include galactosylcerebrosides, lactosylcerebrosides, and gangliosides.

Examples of the phospholipids include natural or synthetic phospholipids, such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, lysophosphatidylcholines, sphingomyelins, egg yolk lecithin, soybean lecithin, and hydrogenated phospholipids.

Examples of the phosphatidylcholines include soybean phosphatidylcholine, egg yolk phosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, and distearoylphosphatidylcholine.

Examples of the phosphatidylethanolamines include dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine.

Examples of the phosphatidylserines include dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, and distearoylphosphatidylserine.

Examples of the phosphatidylglycerols include dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and distearoylphosphatidylglycerol.

Examples of the phosphatidylinositols include dilauroylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, and distearoylphosphatidylinositol.

EXAMPLES

The present invention is specifically explained with reference to examples, but the present invention is not limited to these examples.

[Cloning]

(Identification of Amino Acid Sequence and Nucleotide Sequence of Target Protein)

Total RNA was extracted from Hevea brasiliensis latex by the hot phenol method. To 6 mL of the latex were added 6 mL of 100 mM sodium acetate buffer and 1 mL of a 10% SDS solution, and then 12 mL of water-saturated phenol pre-heated at 65° C. The mixture was incubated for 5 minutes at 65° C., agitated using a vortex mixer, and centrifuged for 10 minutes at room temperature at 7000 rpm. After the centrifugation, the supernatant was transferred to a new tube, 12 mL of a phenol:chloroform (1:1) solution was added, and the mixture was agitated by shaking for 2 minutes. After the agitation, the resulting mixture was centrifuged again for 10 minutes at room temperature at 7000 rpm, the supernatant was transferred to a new tube, 12 mL of a chloroform: isoamyl alcohol (24:1) solution was added, and the mixture was agitated by shaking for 2 minutes. After the agitation, the resulting mixture was centrifuged again for 10 minutes at room temperature at 7000 rpm, the supernatant was transferred to a new tube, 1.2 mL of a 3M sodium acetate solution and 13 mL of isopropanol were added, and the mixture was agitated using a vortex mixer. The resulting mixture was incubated for 30 minutes at −20° C. to precipitate total RNA. The incubated mixture was centrifuged for 10 minutes at 4° C. at 15000 rpm, and the supernatant was removed to collect a precipitate of total RNA. The collected total RNA was washed twice with 70% ethanol, and then dissolved in RNase-free water.

cDNA was synthesized from the collected total RNA. The cDNA synthesis was carried out using a PrimeScript II 1st strand cDNA synthesis kit (Takara) in accordance with the manual.

CPT and NgBr genes were obtained using the prepared 1st strand cDNA as a template. PCR was performed using a KOD-plus-Neo (Toyobo Co., Ltd.) in accordance with the manual. The PCR reaction involved 35 cycles with each cycle consisting of 10 seconds at 98° C., 30 seconds at 58° C., and 1 minute at 68° C.

The CPT gene was obtained using the following primers:


Primer 1:
(SEQ ID NO: 7)
5′-tttggccattacggccatggaattatacaacggtgagagg-3′,
Primer 2:
(SEQ ID NO: 8)
5′-tttggccgaggcggccttattttaagtattccttatgtttc-3′.
The NgBr gene was obtained using the
following primers:
Primer 3:
(SEQ ID NO: 9)
5′-tttggccattacggccatggatttgaaacctggag-3′,
Primer 4:
(SEQ ID NO: 10)
5′-tttggccgaggcggcctcatgtaccataattttgctgcac-3′.

Two types of CPT genes (HRT1 and HRT2) and one type of NgBr gene (NgBr) were obtained as above. The three types of genes were sequenced to identify the full-length nucleotide sequence and amino acid sequence. The nucleotide sequence of HRT1 is given by SEQ ID NO:1, the nucleotide sequence of HRT2 is given by SEQ ID NO:3, and the nucleotide sequence of NgBr is given by SEQ ID NO:5. The amino acid sequence of HRT1 is given by SEQ ID NO:2, the amino acid sequence of HRT2 is given by SEQ ID NO:4, and the amino acid sequence of NgBr is given by SEQ ID NO:6.

(Vector Construction)

The resulting amplified three types of DNA fragments were subjected to dA addition and then inserted into pGEM-T Easy vectors using a pGEM-T Easy Vector System (Promega) to prepare pGEM-HRT1, pGEM-HRT2, and pGEM-NgBr.

(Transformation of E. coli)

Escherichia coli DH5α was transformed with the prepared vectors, the transformant was cultured on LB agar medium containing ampicillin and X-gal, and E. coli cells carrying the introduced target genes were selected by blue/white screening.

The E. coli cells transformed with the plasmids with the target genes were cultured overnight at 37° C. on LB liquid medium. After the culture, the cells were collected, and the plasmids were collected. A FastGene Plasmid mini kit (Nippon Genetics Co., Ltd.) was used for plasmid collection.

It was confirmed by sequence analysis that there were no mutations in the nucleotide sequences of the genes inserted into the collected plasmids

[Preparation of Yeast Transformed to Express Gene Coding for CPT and Gene Coding for NgBr]

HRT1, HRT2, and NgBr genes to be inserted into yeast expression vectors were obtained by PCR using the vectors prepared in the above “Vector construction” as templates.

The HRT1 and HRT2 genes were obtained using the following primers:


Primer 5:
(SEQ ID NO: 11)
5′-gacgcccgggaggccatgaa-3′,
Primer 6:
(SEQ ID NO: 12)
5′-cagcttcctcccgggctttg-3′.
The NgBr gene was obtained using the
following primers:
Primer 7:
(SEQ ID NO: 13)
5′-tttctcgagatggatttgaaacctggagctg-3′,
Primer 8:
(SEQ ID NO: 14)
5′-tttctcgagtgtaccataattttgctgcac-3′.

The obtained DNA fragments were subjected to dA addition and then inserted into pGEM-T Easy vectors using a pGEM-T Easy Vector System (Promega) to prepare pGEM-HRT1 YE, pGEM-HRT2 YE, and pGEM-NgBr YE.

Transformation of E. coli, collection of plasmids, and nucleotide sequence confirmation were performed as described in the above “Transformation of E. coli” but using the prepared vectors. The pGEM-HRT1 YE and pGEM-HRT2 YE, which were confirmed to have no mutations in the nucleotide sequence, were treated with the restriction enzyme SmaI and inserted into pGK426 treated similarly with SmaI, to prepare pGK-HRT1 and pGK-HRT2.

Similarly, the pGEM-NgBr YE, which was confirmed to have no mutations in the nucleotide sequence, was treated with the restriction enzyme XbaI and inserted into pGK425 treated similarly with XbaI, to prepare pGK-NgBr.

The yeast strain SNH23-7D was transformed with the prepared plasmids. The PEG method was used for the double transformation of the yeast. The transformant cells were cultured at 23° C. for three days on Leu- and Ura-deficient SD medium for screening. The pairs used in the preparation of double transformants of the yeast are as follows.

(1) SNH23-7D/pGK426, pGK425 (with no genes introduced)

(2) SNH23-7D/pGK-HRT1, pGK425 (HRT1 alone expressing strain)

(3) SNH23-7D/pGK-HRT2, pGK425 (HRT2 alone expressing strain)

(4) SNH23-7D/pGK-HRT1, pGK-NgBr (HRT1/NgBr co-expressing strain)

(5) SNH23-7D/pGK-HRT2, pGK-NgBr (HRT2/NgBr co-expressing strain)

No SNH23-7D/pGK426, pGK-NgBr transformant (NgBr alone expressing strain) was obtained.

[Yeast Two-Hybrid Screening]

The target genes inserted in the plasmids collected in the above “Cloning” were cleaved, by treatment with the restriction enzyme SfiI in order to insert the target genes into plasmids for yeast two-hybrid screening.

(Vector Construction)

The cleaved genes were inserted into yeast two-hybrid plasmids using Ligation high ver. 2 (Toyobo). The yeast two-hybrid bait plasmid used was pBT3-SUC bait vector, and the yeast two-hybrid prey plasmid used was pPR3-N prey vector. HRT1 or HRT2 was inserted into the prey vector (pPR-HRT1, pPR-HRT2), and HRT1, HRT2, or NgBr was inserted into the bait vector (pBT-HRT1, pBT-HRT2, pBT-NgBr).

(Transformation of E. coli)

Escherichia coli DH5a was transformed with the yeast two-hybrid vectors prepared as above. The transformant cells were cultured on LB agar medium containing ampicillin, and E. coli cells carrying the introduce target genes were selected.

The E. coli cells transformed with the plasmids with the target genes were cultured overnight at 37° C. on LB liquid medium. After the culture, the cells were collected, and the plasmids were collected. A FastGene Plasmid mini kit (Nippon Genetics Co., Ltd.) was used for plasmid collection.

(Yeast Double Transformation)

The yeast strain NMY-51 was transformed with the collected plasmids. The PEG method was used for the double transformation of the yeast. The transformant cells were cultured at 30° C. for three days on Trp- and Leu-deficient SD medium for screening. Table 1 shows the pairs used in the preparation of double transformants of the yeast.


TABLE 1
No.
Prey
Bait
1
pPR3N
pBT3-SUC
2
pPR3N
pBT-HRT1
3
pPR3N
pBT-HRT2
4
pPR-HRT1
pBT3-SUC
5
pPR-HRT1
pBT-NgBr
6
pPR-HRT1
pBT-HRT2
7
pPR-HRT2
pBT3-SUC
8
pPR-HRT2
pBT-HRT1
9
pPR-HRT2
pBT-NgBr

The double transformants of the yeast were observed for the interactions between the enzymes through yeast two-hybrid screening by culturing on Trp-, Leu-, Ade- and His-deficient SD medium for three days at 30° C. In this experiment, the yeast can grow on the selective medium only when a protein encoded by the gene introduced in the bait plasmid interacts with a protein encoded by the gene introduced in the prey plasmid.

The results of yeast two-hybrid screening analyses are shown in FIG. 1. FIG. 1(a) shows the results demonstrating whether or not HRT1 interacted with HRT2 or NgBr. FIG. 1(b) shows the results demonstrating whether or not HRT2 interacted with HRT1 or NgBr. In FIG. 1(a), the sections clockwise from the upper right correspond to the results of the pairs Nos. 1, 4, 5, 6, and 3, respectively, in Table 1. In FIG. 1(b), the sections clockwise from the upper right correspond to the results of the pairs Nos. 1, 7, 8, 9, and 2, respectively, in Table 1.

As shown in FIG. 1, only the results of the pair No. 5 in Table 1, i.e. the pair of HRT1 and NgBr, in FIG. 1(a) exhibited interaction. This indicated that NgBr interacts with HRT1 which is one type of CPT present in latex but does not interact with HRT2. It is thus demonstrated that NgBr does not interact with all types of CPT but interacts with a specific CPT.

From these results it is considered that the activity of HRT1 can be further stabilized and enhanced when HRT1 is combined with NgBr.

REFERENCE SIGNS LIST

  • 1: a pair of pPR3N prey plasmid and pBT3-SUC bait plasmid
  • 2: a pair of pPR3N prey plasmid and pBT-HRT1 bait plasmid
  • 3: a pair of pPR3N prey plasmid and pBT-HRT2 bait plasmid
  • 4: a pair of pPR-HRT1 prey plasmid and pBT3-SUC bait plasmid
  • 5: a pair of pPR-HRT1 prey plasmid and pBT-NgBr bait plasmid
  • 6: a pair of pPR-HRT1 prey plasmid and pBT-HRT2 bait plasmid
  • 7: a pair of pPR-HRT2 prey plasmid and pBT3-SUC bait plasmid
  • 8: a pair of pPR-HRT2 prey plasmid and pBT-HRT1 bait plasmid
  • 9: a pair of pPR-HRT2 prey plasmid and pBT-NgBr bait plasmid

    (Sequence Listing Free Text)

  • SEQ ID NO:1: nucleotide sequence of gene coding for HRT1 from Hevea brasiliensis
  • SEQ ID NO:2: amino acid sequence of HRT1 from Hevea brasiliensis
  • SEQ ID NO:3: nucleotide sequence of gene coding for HRT2 from Hevea brasiliensis
  • SEQ ID NO:4: amino acid sequence of HRT2 from Hevea brasiliensis
  • SEQ ID NO:5: nucleotide sequence of gene coding for NgBr from Hevea brasiliensis
  • SEQ ID NO: 6: amino acid sequence of NgBr from Hevea brasiliensis
  • SEQ ID NO:7: Primer 1
  • SEQ ID NO:8: Primer 2
  • SEQ ID NO:9: Primer 3
  • SEQ ID NO:10: Primer 4
  • SEQ ID NO:11: Primer 5
  • SEQ ID NO:12: Primer 6
  • SEQ ID NO:13: Primer 7
  • SEQ ID NO:14: Primer 8

<160> NUMBER OF SEQ ID NOS: 14

<210> SEQ ID NO: 1

<211> LENGTH: 873

<212> TYPE: DNA

<213> ORGANISM: Hevea brasiliensis

<400> SEQENCE: 1

atggaattat acaacggtga gaggccaagt gtgttcagac ttttagggaa gtatatgaga 60

aaagggttat atagcatcct aacccagggt cccatcccta ctcatattgc cttcatattg 120

gatggaaaca ggaggtttgc taagaagcat aaactgccag aaggaggtgg tcataaggct 180

ggatttttag ctcttctgaa cgtactaact tattgctatg agttaggagt gaaatatgcg 240

actatctatg cctttagcat cgataatttt cgaaggaaac ctcatgaggt tcagtacgta 300

atggatctaa tgctggagaa gattgaaggg atgatcatgg aagaaagtat catcaatgca 360

tatgatattt gcgtacgttt tgtgggtaac ctgaagcttt taagtgagcc agtcaagacc 420

gcagcagata agattatgag ggctactgcc aacaattcca aatgtgtgct tctcattgct 480

gtatgctata cttcaactga tgagatcgtg catgctgttg aagaatcctc tgaattgaac 540

tccaatgaag tttgtaacaa tcaagaattg gaggaggcaa atgcaactgg aagcggtact 600

gtgattcaaa ttgagaacat ggagtcgtat tctggaataa aacttgtaga ccttgagaaa 660

aacacctaca taaatcctta tcctgatgtt ctgattcgaa cttctgggga gacccgtctg 720

agcaactact tactttggca gactactaat tgcatactgt attctcctca tgcactgtgg 780

ccagagattg gtcttcgaca cgtggtgtgg gcagtaatta acttccaacg tcattattct 840

tacttggaga aacataagga atacttaaaa taa 873

<210> SEQ ID NO: 2

<211> LENGTH: 290

<212> TYPE: PRT

<213> ORGANISM: Hevea brasiliensis

<400> SEQENCE: 2

Met Glu Leu Tyr Asn Gly Glu Arg Pro Ser Val Phe Arg Leu Leu Gly

1 5 10 15

Lys Tyr Met Arg Lys Gly Leu Tyr Ser Ile Leu Thr Gln Gly Pro Ile

20 25 30

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

35 40 45

Lys His Lys Leu Pro Glu Gly Gly Gly His Lys Ala Gly Phe Leu Ala

50 55 60

Leu Leu Asn Val Leu Thr Tyr Cys Tyr Glu Leu Gly Val Lys Tyr Ala

65 70 75 80

Thr Ile Tyr Ala Phe Ser Ile Asp Asn Phe Arg Arg Lys Pro His Glu

85 90 95

Val Gln Tyr Val Met Asp Leu Met Leu Glu Lys Ile Glu Gly Met Ile

100 105 110

Met Glu Glu Ser Ile Ile Asn Ala Tyr Asp Ile Cys Val Arg Phe Val

115 120 125

Gly Asn Leu Lys Leu Leu Ser Glu Pro Val Lys Thr Ala Ala Asp Lys

130 135 140

Ile Met Arg Ala Thr Ala Asn Asn Ser Lys Cys Val Leu Leu Ile Ala

145 150 155 160

Val Cys Tyr Thr Ser Thr Asp Glu Ile Val His Ala Val Glu Glu Ser

165 170 175

Ser Glu Leu Asn Ser Asn Glu Val Cys Asn Asn Gln Glu Leu Glu Glu

180 185 190

Ala Asn Ala Thr Gly Ser Gly Thr Val Ile Gln Ile Glu Asn Met Glu

195 200 205

Ser Tyr Ser Gly Ile Lys Leu Val Asp Leu Glu Lys Asn Thr Tyr Ile

210 215 220

Asn Pro Tyr Pro Asp Val Leu Ile Arg Thr Ser Gly Glu Thr Arg Leu

225 230 235 240

Ser Asn Tyr Leu Leu Trp Gln Thr Thr Asn Cys Ile Leu Tyr Ser Pro

245 250 255

His Ala Leu Trp Pro Glu Ile Gly Leu Arg His Val Val Trp Ala Val

260 265 270

Ile Asn Phe Gln Arg His Tyr Ser Tyr Leu Glu Lys His Lys Glu Tyr

275 280 285

Leu Lys

290

<210> SEQ ID NO: 3

<211> LENGTH: 855

<212> TYPE: DNA

<213> ORGANISM: Hevea brasiliensis

<400> SEQENCE: 3

atggaattat acaacggtga gaggccaagt gtgttcagac ttttagggaa gtatatgaga 60

aaagggttat atagcatcct aacccagggt cccatcccta ctcatattgc cttcatattg 120

gatggaaacg ggaggtttgc taagaagcat aaactgccag aaggaggtgg tcataaggct 180

ggatttttag ctcttctgaa cgtactaact tattgctatg agttaggagt gaaatatgcg 240

actatctatg cctttagcat cgataatttt cgaaggaaac ctcatgaggt tcagtacgta 300

atgaatctaa tgctggagaa gattgaaggg atgatcatgg aagaaagtat catcaatgca 360

tatgatattt gcgtgcgttt tgttggtaat ctgaagcttt tagatgagcc actcaagacc 420

gcagcagata agataatgag ggctactgcc aaaaattcca aatttgtgct tctccttgct 480

gtatgctaca cttcaactga tgagatcgtg catgctgttg aagaatcctc taaggataaa 540

ttgaaatccg atgaaatttg caacgatgga aacggagatt gtgtgattaa aattgaggag 600

atggagccat attctgaaat aaaacttgta gagcttgaga gaaacactta cataaatcct 660

tatcctgatg tcttgattcg aacttctggg gagacccgtc tgagcaacta cctactttgg 720

cagactacta attgcatact gtattctcct catgcactgt ggccagagat tggtcttcga 780

cacgtggtgt gggcagtaat taactgccaa cgtcattatt cttacttgga gaaacataag 840

gaatacttaa aataa 855

<210> SEQ ID NO: 4

<211> LENGTH: 284

<212> TYPE: PRT

<213> ORGANISM: Hevea brasiliensis

<400> SEQENCE: 4

Met Glu Leu Tyr Asn Gly Glu Arg Pro Ser Val Phe Arg Leu Leu Gly

1 5 10 15

Lys Tyr Met Arg Lys Gly Leu Tyr Ser Ile Leu Thr Gln Gly Pro Ile

20 25 30

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

35 40 45

Lys His Lys Leu Pro Glu Gly Gly Gly His Lys Ala Gly Phe Leu Ala

50 55 60

Leu Leu Asn Val Leu Thr Tyr Cys Tyr Glu Leu Gly Val Lys Tyr Ala

65 70 75 80

Thr Ile Tyr Ala Phe Ser Ile Asp Asn Phe Arg Arg Lys Pro His Glu

85 90 95

Val Gln Tyr Val Met Asn Leu Met Leu Glu Lys Ile Glu Gly Met Ile

100 105 110

Met Glu Glu Ser Ile Ile Asn Ala Tyr Asp Ile Cys Val Arg Phe Val

115 120 125

Gly Asn Leu Lys Leu Leu Asp Glu Pro Leu Lys Thr Ala Ala Asp Lys

130 135 140

Ile Met Arg Ala Thr Ala Lys Asn Ser Lys Phe Val Leu Leu Leu Ala

145 150 155 160

Val Cys Tyr Thr Ser Thr Asp Glu Ile Val His Ala Val Glu Glu Ser

165 170 175

Ser Lys Asp Lys Leu Lys Ser Asp Glu Ile Cys Asn Asp Gly Asn Gly

180 185 190

Asp Cys Val Ile Lys Ile Glu Glu Met Glu Pro Tyr Ser Glu Ile Lys

195 200 205

Leu Val Glu Leu Glu Arg Asn Thr Tyr Ile Asn Pro Tyr Pro Asp Val

210 215 220

Leu Ile Arg Thr Ser Gly Glu Thr Arg Leu Ser Asn Tyr Leu Leu Trp

225 230 235 240

Gln Thr Thr Asn Cys Ile Leu Tyr Ser Pro His Ala Leu Trp Pro Glu

245 250 255

Ile Gly Leu Arg His Val Val Trp Ala Val Ile Asn Cys Gln Arg His

260 265 270

Tyr Ser Tyr Leu Glu Lys His Lys Glu Tyr Leu Lys

275 280

<210> SEQ ID NO: 5

<211> LENGTH: 774

<212> TYPE: DNA

<213> ORGANISM: Hevea brasiliensis

<400> SEQENCE: 5

atggatttga aacctggagc tggagggcag agagttaatc gattagtgga tccgattagt 60

tatcattttc ttcaatttct gtggcgtact ctacatcttc ttgtcagctt atggtacctt 120

caagttagta tggtccaaat gatcgaaggc tttctaatct ctagtggact tgtgaaacgc 180

tatggagccc tcgatattga caaggtccgg taccttgcca ttgtggtaga tagtgaagaa 240

gcttaccaaa tttctaaagt tattcagctt ttgaaatggg tggaagatat gggtgtgaaa 300

catttatgcc tctatgattc aaaaggagtt ctcaagacaa acaagaaaac catcatggag 360

agtttgaaca atgctatgcc atttgaggaa gcagttgaaa aagatgtttt actggaccag 420

aaacagatga ctgtggaatt tgcttccagc tccgatggaa aggaagcaat aaccagggca 480

gctaacgtac tctttatgaa gtatttgaag tatgctaaaa ctggtgtagg aaaggaagaa 540

ccatgcttta cagaagatca aatggatgag gcactaaaag ctataggtta caaagggccg 600

gaacctgact tgctattaat ttatggacct gttagatgcc atctaggttt ctcaccgtgg 660

agacttcgat atactgagat ggtgcatatg ggacccttga ggtacatgaa cctcggttca 720

ctaaaaaagg ccattcacag gttcacaaca gtgcagcaaa attatggtac atga 774

<210> SEQ ID NO: 6

<211> LENGTH: 257

<212> TYPE: PRT

<213> ORGANISM: Hevea brasiliensis

<400> SEQENCE: 6

Met Asp Leu Lys Pro Gly Ala Gly Gly Gln Arg Val Asn Arg Leu Val

1 5 10 15

Asp Pro Ile Ser Tyr His Phe Leu Gln Phe Leu Trp Arg Thr Leu His

20 25 30

Leu Leu Val Ser Leu Trp Tyr Leu Gln Val Ser Met Val Gln Met Ile

35 40 45

Glu Gly Phe Leu Ile Ser Ser Gly Leu Val Lys Arg Tyr Gly Ala Leu

50 55 60

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

65 70 75 80

Ala Tyr Gln Ile Ser Lys Val Ile Gln Leu Leu Lys Trp Val Glu Asp

85 90 95

Met Gly Val Lys His Leu Cys Leu Tyr Asp Ser Lys Gly Val Leu Lys

100 105 110

Thr Asn Lys Lys Thr Ile Met Glu Ser Leu Asn Asn Ala Met Pro Phe

115 120 125

Glu Glu Ala Val Glu Lys Asp Val Leu Leu Asp Gln Lys Gln Met Thr

130 135 140

Val Glu Phe Ala Ser Ser Ser Asp Gly Lys Glu Ala Ile Thr Arg Ala

145 150 155 160

Ala Asn Val Leu Phe Met Lys Tyr Leu Lys Tyr Ala Lys Thr Gly Val

165 170 175

Gly Lys Glu Glu Pro Cys Phe Thr Glu Asp Gln Met Asp Glu Ala Leu

180 185 190

Lys Ala Ile Gly Tyr Lys Gly Pro Glu Pro Asp Leu Leu Leu Ile Tyr

195 200 205

Gly Pro Val Arg Cys His Leu Gly Phe Ser Pro Trp Arg Leu Arg Tyr

210 215 220

Thr Glu Met Val His Met Gly Pro Leu Arg Tyr Met Asn Leu Gly Ser

225 230 235 240

Leu Lys Lys Ala Ile His Arg Phe Thr Thr Val Gln Gln Asn Tyr Gly

245 250 255

Thr

<210> SEQ ID NO: 7

<211> LENGTH: 40

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Sense primer for CPT

<400> SEQENCE: 7

tttggccatt acggccatgg aattatacaa cggtgagagg 40

<210> SEQ ID NO: 8

<211> LENGTH: 41

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense primer for CPT

<400> SEQENCE: 8

tttggccgag gcggccttat tttaagtatt ccttatgttt c 41

<210> SEQ ID NO: 9

<211> LENGTH: 35

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Sense primer for NgBr

<400> SEQENCE: 9

tttggccatt acggccatgg atttgaaacc tggag 35

<210> SEQ ID NO: 10

<211> LENGTH: 40

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense primer for NgBr

<400> SEQENCE: 10

tttggccgag gcggcctcat gtaccataat tttgctgcac 40

<210> SEQ ID NO: 11

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Sense primer for HRT1,HRT2

<400> SEQENCE: 11

gacgcccggg aggccatgaa 20

<210> SEQ ID NO: 12

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense primer for HRT1,HRT2

<400> SEQENCE: 12

cagcttcctc ccgggctttg 20

<210> SEQ ID NO: 13

<211> LENGTH: 31

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Sense primer for NgBr

<400> SEQENCE: 13

tttctcgaga tggatttgaa acctggagct g 31

<210> SEQ ID NO: 14

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense primer for NgBr

<400> SEQENCE: 14

tttctcgagt gtaccataat tttgctgcac 30

Read more
PatSnap Solutions

Great research starts with great data.

Use the most comprehensive innovation intelligence platform to maximise ROI on research.

Learn More

Patent Valuation

$

Reveal the value <>

24.78/100 Score

Market Attractiveness

It shows from an IP point of view how many competitors are active and innovations are made in the different technical fields of the company. On a company level, the market attractiveness is often also an indicator of how diversified a company is. Here we look into the commercial relevance of the market.

38.0/100 Score

Market Coverage

It shows the sizes of the market that is covered with the IP and in how many countries the IP guarantees protection. It reflects a market size that is potentially addressable with the invented technology/formulation with a legal protection which also includes a freedom to operate. Here we look into the size of the impacted market.

71.47/100 Score

Technology Quality

It shows the degree of innovation that can be derived from a company’s IP. Here we look into ease of detection, ability to design around and significance of the patented feature to the product/service.

59.0/100 Score

Assignee Score

It takes the R&D behavior of the company itself into account that results in IP. During the invention phase, larger companies are considered to assign a higher R&D budget on a certain technology field, these companies have a better influence on their market, on what is marketable and what might lead to a standard.

22.89/100 Score

Legal Score

It shows the legal strength of IP in terms of its degree of protecting effect. Here we look into claim scope, claim breadth, claim quality, stability and priority.

Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
パラゴムノキラテックスにおけるイソペンテニル二リン酸の生合成に関与する遺伝子 イー·アイ·デュポン·ドウ·ヌムール·アンド·カンパニー 23 July 2002 13 January 2005
See full citation <>

More like this

Title Current Assignee Application Date Publication Date
Substitution mutant receptors and their use in an ecdysone receptor-based inducible gene expression system INTREXON CORPORATION 20 February 2002 02 February 2016
Nogo receptor-mediated blockade of axonal growth YALE UNIVERSITY 26 January 2010 12 March 2013
Legume isoprene synthase for production of isoprene THE GOODYEAR TIRE & RUBBER COMPANY 13 March 2013 25 November 2014
Prenyltransferase from Arabidopsis SUNGENE GMBH & CO.KGAA 02 July 2002 11 June 2003
Cold shock protein receptors and methods of use TWO BLADES FOUNDATION 07 October 2016 13 April 2017
Compositions and methods for suppressing axonal growth inhibition YALE UNIVERSITY 07 July 2006 26 February 2009
G protein-coupled receptors PHARMACIA & UPJOHN COMPANY,LIND, PETER 15 August 2001 30 May 2002
Nucleotide and protein sequences of Nogo genes and methods based thereon ZURICH UNIVERSITY OF 05 November 1999 24 August 2010
NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST THE NOGO-66 RECEPTOR (NgR) AND USES THEREOF ABBVIE DEUTSCHLAND GMBH & CO KG 21 November 2007 13 November 2008
Immunoglobulins directed against Nogo GLAXO GROUP LIMITED 14 December 2006 15 September 2010
Receptor for B anthracis toxin PRESIDENT AND FELLOWS OF HARVARD COLLEGE,WISCONSIN ALUMNI RESEARCH FOUNDATION 03 October 2001 11 July 2006
Method of identifying modulators of nogo-functions GLAXO GROUP LIMITED,SMITHKLINE BEECHAM PLC 18 January 2002 18 September 2007
Nogo-B receptor antagonists YALE UNIVERSITY 12 January 2007 27 December 2011
Isolation and use of ryanodine receptors E I DU PONT DE NEMOURS AND COMPANY 10 January 2012 08 October 2013
Methods and compositions for enhanced plant cell transformation PURDUE RESEARCH FOUNDATION,IOWA STATE UNIVERSITY RESEARCH FOUNDATION,GELVIN, STANTON, B.,MYSORE, KIRANKUMAR, S.,WANG, KAN 17 September 2004 19 May 2005
NOGO receptor-mediated blockade of axonal growth YALE UNIVERSITY 12 January 2001 24 December 2013
Polynucleotide compositions encoding broad spectrum delta-endotoxins MONSANTO TECHNOLOGY LLC 18 December 2003 04 July 2006
Novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system RHEOGENE, INC. (DE CORPORATION) 20 August 2007 27 December 2007
Insect retinoid-like receptor compositions and methods THE SALK INSTITUTE FOR BIOLOGICAL STUDIES 05 June 1995 18 November 1997
See all similar patents <>

More Patents & Intellectual Property

PatSnap Solutions

PatSnap solutions are used by R&D teams, legal and IP professionals, those in business intelligence and strategic planning roles and by research staff at academic institutions globally.

PatSnap Solutions
Search & Analyze
The widest range of IP search tools makes getting the right answers and asking the right questions easier than ever. One click analysis extracts meaningful information on competitors and technology trends from IP data.
Business Intelligence
Gain powerful insights into future technology changes, market shifts and competitor strategies.
Workflow
Manage IP-related processes across multiple teams and departments with integrated collaboration and workflow tools.
Contact Sales
Clsoe
US10000774 Transformant expressing cis-prenyltranferase 1