Plant diacylglycerol acyltransferases

Abstract

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

What is claimed is: 
     
         1 . An isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 50 amino acids that has at least 60% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 8, 10, 14, 20 and 22,
 or an isolated polynucleotide comprising the complement of the nucleotide sequence.   
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein the isolated nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 7, 13, 15, and 21 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 8, 14, 16, and 22. 
     
     
         3 . The isolated polynucleotide of  claim 1  wherein the isolated polynucleotide is DNA. 
     
     
         4 . The isolated polynucleotide of  claim 1  wherein the isolated polynucleotide is 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 . An isolated host cell comprising the isolated polynucleotide of  claim 1 . 
     
     
         8 . The isolated host cell of  claim 7  wherein the isolated host is selected from the group consisting of yeast, bacteria, plant, and virus. 
     
     
         9 . A virus comprising the isolated polynucleotide of  claim 1 . 
     
     
         10 . A method of selecting an isolated polynucleotide that affects the level of expression of a diacylglycerol acyltransferase 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, and the complement of such nucleotide sequences;   (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.   
     
     
         11 . The method of  claim 10  wherein the isolated polynucleotide consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 11, 13, 15, 17, 19 and 21 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22. 
     
     
         12 . A method of selecting an isolated polynucleotide that affects the level of expression of a diacylglycerol acyltransferase polypeptide in a plant cell, the method comprising the steps of:
 (a) constructing the isolated polynucleotide of  claim 1 ;   (b) introducing the isolated polynucleotide into a plant cell; and   (c) measuring the level of diacylglycerol acyltransferase polypeptide in the plant cell containing the polynucleotide.   
     
     
         13 . A method for evaluating at least one compound for its ability to inhibit the activity of a diacylglycerol acyltransferase, the method comprising the steps of:
 (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a diacylglycerol acyltransferase, operably linked to suitable regulatory sequences;   (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the diacylglycerol acyltransferase encoded by the operably linked nucleic acid fragment in the transformed host cell;   (c) optionally purifying the diacylglycerol acyltransferase expressed by the transformed host cell;   (d) treating the diacylglycerol acyltransferase with a compound to be tested; and   (e) comparing the activity of the diacylglycerol acyltransferase that has been treated with a test compound to the activity of an untreated diacylglycerol acyltransferase, thereby selecting compounds with potential for inhibitory activity.   
     
     
         14 . A composition comprising the isolated polynucleotide of  claim 1 . 
     
     
         15 . An expression cassette comprising the isolated polynucleotide of  claim 1  operably linked to a promoter. 
     
     
         16 . A method for positive selection of a transformed cell comprising:
 (a) transforming a plant cell with the expression cassette of  claim 15 ; and   (b) growing the transformed plant cell under conditions allowing expression of the polynucleotide in an amount sufficient to modify oil content in the plant cell to provide a positive selection means.