Fatty acid desaturase genes from plants

Abstract

The preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes are described. The invention permits alteration of plant lipid composition. Chimeric genes incorporating such nucleic acid fragments with suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids.

1 . An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a fatty acid desaturase or a fatty acid desaturase-related enzyme with an amino acid identity of 50% or greater to the polypeptide encoded by SEQ ID NOS:1, 4, 6, 8, 10, 12, 14 or 16. 
     
     
         2 . The isolated nucleic acid fragment of  claim 1  wherein the amino acid identity is 65% or greater to the polypeptide encoded by SEQ ID NOS:1, 4, 6, 8, 10, 12, 14 or 16. 
     
     
         3 . The isolated nucleic acid fragment of  claim 1  wherein the nucleic acid identity is 90% or greater to SEQ ID NOS:1, 4, 6, 8, 10, 12, 14 or 16. 
     
     
         4 . An isolated nucleic acid fragment of  claim 1  wherein said fragment is isolated from a plant selected from the group consisting of soybean, oilseed  Brassica  species,  Arabidopsis thaliana  and corn. 
     
     
         5 . A chimeric gene capable of causing altered levels of linolenic acid in a transformed plant cell, the gene comprising a nucleic acid fragment of any of  claims 1 ,  2 , or  3 , the fragment operably linked to suitable regulatory sequences. 
     
     
         6 . Plants containing the chimeric genes of  claim 5 . 
     
     
         7 . Oil obtained from seeds of the plants containing the chimeric genes of  claim 5 . 
     
     
         8 . A method of producing seed oil containing altered levels of linolenic (18:3) acid comprising:
 (a) transforming a plant cell of an oil-producing species with a chimeric gene of  claim 5 ;   (b) growing fertile plants from the transformed plant cells of step (a);   (c) screening progeny seeds from the fertile plants of step (b) for the desired levels of linolenic (18:3) acid; and   (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of linolenic (18:3) acid.   
     
     
         9 . The product of the method of  claim 8 . 
     
     
         10 . A method of  claim 8  wherein said plant cell of an oil-producing species is selected from the group consisting of  Arabidonsis thaliana , soybean, oilseed  Brassica  species, sunflower, cotton, cocoa, peanut, safflower, and corn. 
     
     
         11 . A method of breeding plant species producing altered levels of linolenic acid in the seed oil of oil-producing plant species comprising:
 (a) making a cross between two varieties of oil-producing species differing in the linolenic acid trait;   (b) making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step (a); and   (c) hybridizing the Southern blot with a radiolabelled nucleic acid fragment of  claim 1 .   
     
     
         12 . The product of the method of  claim 11 . 
     
     
         13 . A method of RFLP mapping in a genomic RFLP marker comprising:
 (a) making a cross between two varieties of plants;   (b) making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step (a); and   (c) hybridizing the Southern blot with a radiolabelled nucleic acid fragments of  claim 1 .   
     
     
         14 . A method to isolate nucleic acid fragments encoding fatty acid desaturases and fatty acid desaturase-related enzymes, comprising:
 (a) comparing SEQ ID NOS:2, 5, 7, 9, 11, 13, 15 and 17 with other fatty acid desaturase polypeptide sequences;   (b) identifying the conserved sequence(s) of 4 or more amino acids obtained in step (a);   (c) making region-specific nucleotide probe(s) or oligomer(s) based on the conserved sequences identified in step b; and   (d) using the nucleotide probe(s) or oligomers(s) of step c to isolate sequences encoding fatty acid desaturases and fatty-acid desaturase-related enzymes by sequence-dependent protocols.   
     
     
         15 . The product of the method of  claim 14 . 
     
     
         16 . The isolated genomic DNA of  Arabidopsis thaliana  identified by accession number ATCC 75167. 
     
     
         17 . An isolated cDNA clone which encodes for soybean delta-15 desaturase, the clone designated pXF1 comprising the DNA sequence of SEQ ID NO 10 and identified by accession number ATCC 68874. 
     
     
         18 . An isolated cDNA clone which encodes for oilseed  Brassica  species delta-15 desaturase, the clone designated pBNSF3 comprising the DNA sequence of SEQ ID NO:6 and identified by accession number ATCC 68854. 
     
     
         19 . An isolated Polymerase Chain Reaction Product for  Zea mays  delta-15 desaturase, the clone designated pcr20 comprising the DNA sequence of SEQ ID NO:14.