METHOD FOR PRODUCING TRANS-POLYISOPRENOID, VECTOR, TRANSGENIC PLANT, METHOD FOR PRODUCING PNEUMATIC TIRE AND METHOD FOR PRODUCING RUBBER PRODUCT
The present invention aims to provide a method for producing a trans-polyisoprenoid which can increase trans rubber production. The present invention is directed to a method for producing a trans-polyisoprenoid in vitro, which involves the use of a gene coding for a trans-prenyltransferase (tPT) family protein and further involves the use of rubber particles bound to a protein encoded by the gene, or a method for producing a trans-polyisoprenoid, which includes introducing into a plant a vector including a promoter having a promoter activity that drives laticifer-specific gene expression and a gene coding for a tPT family protein linked to the promoter to express a protein encoded by the gene specifically in laticifers.
1 . A method for producing a trans-polyisoprenoid, the method comprising
binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro.
2 . The method for producing a trans-polyisoprenoid according to claim 1 ,
wherein the trans-prenyltransferase (tPT) family protein comprises, at positions 183 to 187 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions,
the following amino acid sequence (A1):
DDX 1 X 2 D (A1)
wherein X 1 and X 2 are the same as or different from each other and each represent any amino acid residue, or
the following amino acid sequence (A2):
DDX 1 X 2 X 3 X 4 D (A2)
wherein X 1 , X 2 , X 3 , and X 4 are the same as or different from each other and each represent any amino acid residue, and
the trans-prenyltransferase (tPT) family protein comprises, at positions 310 to 314 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions,
the following amino acid sequence (B):
DDX 11 X 12 D (B)
wherein X 11 and X 12 are the same as or different from each other and each represent any amino acid residue.
3 . The method for producing a trans-polyisoprenoid according to claim 1 ,
wherein the gene coding for a trans-prenyltransferase (tPT) family protein is derived from a plant.
4 . The method for producing a trans-polyisoprenoid according to claim 3 ,
wherein the gene coding for a trans-prenyltransferase (tPT) family protein is derived from a rubber-producing plant.
5 . The method for producing a trans-polyisoprenoid according to claim 4 ,
wherein the gene coding for a trans-prenyltransferase (tPT) family protein is derived from Hevea brasiliensis.
6 . The method for producing a trans-polyisoprenoid according to claim 1 ,
wherein the binding comprises performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a trans-prenyltransferase (tPT) family protein to bind the tPT family protein to the rubber particles.
7 . The method for producing a trans-polyisoprenoid according to claim 6 ,
wherein the cell-free protein synthesis solution comprises a germ extract.
8 . The method for producing a trans-polyisoprenoid according to claim 7 ,
wherein the germ extract is derived from wheat.
9 . The method for producing a trans-polyisoprenoid according to claim 6 ,
wherein the rubber particles are present in the cell-free protein synthesis solution at a concentration of 5 to 50 g/L.
10 . A method for producing a pneumatic tire, the method comprising:
kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid according to claim 1 with an additive to obtain a kneaded mixture;
building a green tire from the kneaded mixture; and
vulcanizing the green tire.
11 . A method for producing a rubber product, the method comprising:
kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid according to claim 1 with an additive to obtain a kneaded mixture;
forming a raw rubber product from the kneaded mixture; and
vulcanizing the raw rubber product.
12 . A vector, comprising:
a promoter having a promoter activity that drives laticifer-specific gene expression; and
a gene coding for a trans-prenyltransferase (tPT) family protein functionally linked to the promoter.
13 . The vector according to claim 12 ,
wherein the promoter having a promoter activity that drives laticifer-specific gene expression is at least one selected from the group consisting of a promoter of a gene coding for rubber elongation factor (REF), a promoter of a gene coding for small rubber particle protein (R PP), a promoter of a gene coding for Hevein 2.1 (HEV2.1), and a promoter of a gene coding for MYC1 transcription factor (MYC1).
14 . A transgenic plant into which the vector according to claim 12 has been introduced.
15 . A method for enhancing trans-polyisoprenoid production in a plant by introducing the vector according to claim 12 into the plant.
16 . A method for producing a pneumatic tire, the method comprising:
kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing the vector according to claim 12 into a plant;
building a green tire from the kneaded mixture; and
vulcanizing the green tire.
17 . A method for producing a rubber product, the method comprising:
kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing the vector according to claim 12 into a plant;
forming a raw rubber product from the kneaded mixture; and
vulcanizing the raw rubber product.