Method for producing trans-polyisoprenoid, vector, transgenic plant, method for producing pneumatic tire and method for producing rubber product

Abstract

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.