Plant polyphenol oxidase homologs

Abstract

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

What is claimed is:  
     
         1 . An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 112 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 14, 24, 32, 34, 36, 38, 42 and 44, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein the first nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:3, 5, 13, 23, 31, 33, 35, 37, 41 and 43 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 14, 24, 32, 34, 36, 38, 42 and 44.  
     
     
         3 . The isolated polynucleotide of  claim 1  wherein the nucleotide sequences are DNA.  
     
     
         4 . The isolated polynucleotide of  claim 1  wherein the nucleotide sequences are RNA.  
     
     
         5 . A chimeric gene comprising the isolated polynucleotide of  claim 1  operably linked to suitable regulatory sequences.  
     
     
         6 . An isolated host cell comprising the chimeric gene of  claim 5 .  
     
     
         7 . A host cell comprising an isolated polynucleotide of  claim 1 .  
     
     
         8 . The host cell of  claim 7  wherein the host cell is selected from the group consisting of yeast, bacteria, plant, and virus.  
     
     
         9 . A virus comprising the isolated polynucleotide of  claim 1 .  
     
     
         10 . A polypeptide of at least 112 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 14, 24, 32, 34, 36, 38, 42 and 44.  
     
     
         11 . An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 163 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 12, 28 and 40, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         12 . The isolated polynucleotide of  claim 11 , wherein the first nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 27 and 39 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:12, 28 and 40.  
     
     
         13 . The isolated polynucleotide of  claim 11  wherein the nucleotide sequences are DNA.  
     
     
         14 . The isolated polynucleotide of  claim 11  wherein the nucleotide sequences are RNA.  
     
     
         15 . A chimeric gene comprising the isolated polynucleotide of  claim 11  operably linked to suitable regulatory sequences.  
     
     
         16 . An isolated host cell comprising the chimeric gene of  claim 15 .  
     
     
         17 . A host cell comprising an isolated polynucleotide of  claim 11 .  
     
     
         18 . The host cell of  claim 17  wherein the host cell is selected from the group consisting of yeast, bacteria, plant, and virus.  
     
     
         19 . A virus comprising the isolated polynucleotide of  claim 11 .  
     
     
         20 . A polypeptide of at least 163 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 10, 20 and 40.  
     
     
         21 . A method of selecting an isolated polynucleotide that affects the level of expression of a polyphenol oxidase enzyme polypeptide in a plant cell, the method comprising the steps of: 
 (a) constructing an isolated polynucleotide comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from an isolated polynucleotide of any of claims  1  or 11;    (b) introducing the isolated polynucleotide into a plant cell;    (c) measuring the level of a polypeptide in the plant cell containing the polynucleotide; and    (d) comparing the level of polypeptide in the plant cell containing the isolated polynucleotide with the level of polypeptide in a plant cell that does not contain the isolated polynucleotide.    
     
     
         22 . The method of  claim 21  wherein the isolated polynucleotide consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs:3, 5, 11, 13, 23, 27, 31, 33, 35, 37, 39, 41 and 43 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 12, 14, 24, 28, 32, 34, 36, 38, 40, 42 and 44.  
     
     
         23 . A method of selecting an isolated polynucleotide that affects the level of expression of a polyphenol oxidase enzyme polypeptide in a plant cell, the method comprising the steps of: 
 (a) constructing an isolated polynucleotide of any of claims  1  or  11 ;    (b) introducing the isolated polynucleotide into a plant cell;    (c) measuring the level of polypeptide in the plant cell containing the polynucleotide; and    (d) comparing the level of polypeptide in the plant cell containing the isolated polynucleotide with the level of polypeptide in a plant cell that does not contain the polynucleotide.    
     
     
         24 . A method of obtaining a nucleic acid fragment encoding a polyphenol oxidase enzyme polypeptide comprising the steps of: 
 (a) synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:3, 5, 11, 13, 23, 27, 31, 33, 35, 37, 39, 41 and 43 and the complement of such nucleotide sequences; and    (b) amplifying a nucleic acid sequence using the oligonucleotide primer.    
     
     
         25 . A method of obtaining a nucleic acid fragment encoding a polyphenol oxidase enzyme polypeptide comprising the steps of: 
 (a) probing a cDNA or genomic library with an isolated polynucleotide comprising at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:3, 5, 11, 13, 23, 27, 31, 33, 35, 37, 39, 41 and 43 and the complement of such nucleotide sequences;    (b) identifying a DNA clone that hybridizes with the isolated polynucleotide;    (c) isolating the identified DNA clone; and    (d) sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.    
     
     
         26 . A composition comprising the isolated polynucleotide of any of  claim 1  or  11 .  
     
     
         27 . A composition comprising the isolated polypeptide of any of claims  10  or  20 .  
     
     
         28 . An isolated polynucleotide comprising the nucleotide sequence having at least one of 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs:3, 5, 11, 13, 23, 27, 31, 33, 35, 37, 39, 41 and 43 and the complement of such sequences.  
     
     
         29 . An expression cassette comprising an isolated polynucleotide of any of claims  1  or  11  operably linked to a promoter.  
     
     
         30 . A method for positive selection of a transformed cell comprising: 
 (a) transforming a host cell with the chimeric gene of any of claims  5  or  15 ; and    (b) growing the transformed host cell under conditions which allow expression of the polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.    
     
     
         31 . A method for positive selection of a transformed cell comprising: 
 (a) transforming a host cell with the expression cassette of  claim 29;  and    (b) growing the transformed host cell under conditions which allow expression of the polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.    
     
     
         32 . The method of any of claims  21  or  23  wherein the plant cell is a monocot.  
     
     
         33 . The method of any of claims  21  or  23  wherein the plant cell is a dicot.  
     
     
         34 . An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 8, 12, 16, 18, 22, 30 and 46.