Plant sorbitol biosynthetic

Abstract

This invention relates to an isolated nucleic acid fragment encoding a sorbitol biosynthetic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sorbitol biosynthetic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sorbitol biosynthetic enzyme in a transformed host cell.

What is claimed is:  
     
         1 . An isolated nucleic acid fragment encoding an aldehyde reductase comprising a member selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding an amino acid sequence with at least 300 contiguous amino acids and that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6 and 8;    (b) an isolated nucleic acid fragment that is complementary to (a).    
     
     
         2 . The isolated nucleic acid fragment of  claim 1  wherein nucleic acid fragment is a functional RNA.  
     
     
         3 . The isolated nucleic acid fragment of  claim 1  wherein the nucleotide sequence of the fragment comprises the sequence set forth in a member selected from the group consisting of SEQ ID NO:1, 3,5 and 7.  
     
     
         4 . A chimeric gene comprising the nucleic acid fragment of  claim 1  operably linked to suitable regulatory sequences.  
     
     
         5 . A transformed host cell comprising the chimeric gene of  claim 4 .  
     
     
         6 . An aldehyde reductase polypeptide comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, 4, 6 and 6.  
     
     
         7 . An isolated nucleic acid fragment encoding a NADPH-dependent mannose 6-phosphate reductase comprising a member selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:10, 12 and 14, wherein the amino acid sequence encodes a functional enzyme;    (b) an isolated nucleic acid fragment that is complementary to (a).    
     
     
         8 . The isolated nucleic acid fragment of  claim 7  wherein nucleic acid fragment is a functional RNA.  
     
     
         9 . The isolated nucleic acid fragment of  claim 7  wherein the nucleotide sequence of the fragment comprises the sequence set forth in a member selected from the group consisting of SEQ ID NO:9, 11 and 13.  
     
     
         10 . A chimeric gene comprising the nucleic acid fragment of  claim 7  operably linked to suitable regulatory sequences.  
     
     
         11 . A transformed host cell comprising the chimeric gene of  claim 10 .  
     
     
         12 . A NADPH-dependent mannose 6-phosphate reductase polypeptide comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:10, 12 and 14.  
     
     
         13 . An isolated nucleic acid fragment encoding a sorbitol dehydrogenase comprising a member selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding an amino acid sequence of at least 150 contiguous amino acids wherein the amino acid sequence is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:16, 18, 20 and 22;    (b) an isolated nucleic acid fragment that is complementary to (a).    
     
     
         14 . The isolated nucleic acid fragment of  claim 13  wherein nucleic acid fragment is a functional RNA.  
     
     
         15 . The isolated nucleic acid fragment of  claim 13  wherein the nucleotide sequence of the fragment comprises the sequence set forth in a member selected from the group consisting of SEQ ID NO:15, 17, 19 and 21.  
     
     
         16 . A chimeric gene comprising the nucleic acid fragment of  claim 13  operably linked to suitable regulatory sequences.  
     
     
         17 . A transformed host cell comprising the chimeric gene of  claim 16 .  
     
     
         18 . A sorbitol dehydrogenase polypeptide comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:16, 18, 20 and 22.  
     
     
         19 . A method of altering the level of expression of a sorbitol biosynthetic enzyme in a host cell comprising: 
 (a) transforming a host cell with the chimeric gene of any of claims  4 ,  10  and  16 ; and    (b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene    wherein expression of the chimeric gene results in production of altered levels of a sorbitol biosynthetic enzyme in the transformed host cell.    
     
     
         20 . A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a sorbitol biosynthetic enzyme comprising: 
 (a) probing a cDNA or genomic library with the nucleic acid fragment of any of claims  1 ,  7  and  13 ;    (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of any of claims  1 ,  7  and  13 ;    (c) isolating the DNA clone identified in step (b); and    (d) sequencing the cDNA or genomic fragment that comprises the clone isolated in step (c)    wherein the sequenced nucleic acid fragment encodes all or a substantial portion of the amino acid sequence encoding a sorbitol biosynthetic enzyme.    
     
     
         21 . A method of obtaining a nucleic acid fragment encoding a substantial portion of an amino acid sequence encoding a sorbitol biosynthetic enzyme comprising: 
 (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21; and    (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector    wherein the amplified nucleic acid fragment encodes a substantial portion of an amino acid sequence encoding a sorbitol biosynthetic enzyme.    
     
     
         22 . The product of the method of  claim 20 .  
     
     
         23 . The product of the method of  claim 21.