Genes for microsomal delta-12 fatty acid desaturases and related enzymes 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, 3, 5, 7, 9, 11, or 15.  
     
     
         2 . The isolated nucleic acid fragment of  claim 1  wherein the amino acid identity is 60% or greater to the polypeptide encoded by SEQ ID NOS:1, 3, 5, 7, 9, 11, or 15.  
     
     
         3 . The isolated nucleic acid fragment of  claim 1  wherein the nucleic acid identity is 90% or greater to SEQ ID NOS:1, 3, 5, 7, 9, 11, or 15.  
     
     
         4 . The isolated nucleic acid fragment of  claim 1  wherein said fragment is isolated from an oil-producing plant species.  
     
     
         5 . An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a delta-12 fatty acid hydroxylase.  
     
     
         6 . A chimeric gene capable of causing altered levels of ricinoleic acid in a transformed plant cell, said chimeric gene comprising a nucleic acid fragment of  claim 5 , said fragment operably linked to suitable regulatory sequences.  
     
     
         7 . A chimeric gene capable of causing altered levels of fatty acids in a transformed plant cell, said chimeric gene comprising a nucleic acid fragment of any of claims  1 ,  2 ,  3 , said fragment operably linked to suitable regulatory sequences.  
     
     
         8 . Plants containing a chimeric gene of  claim 6  or  claim 7 .  
     
     
         9 . Oil obtained from seeds of the plants of  claim 8 .  
     
     
         10 . A method of producing seed oil containing altered levels of unsaturated fatty acids 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 unsaturated fatty acids; and    (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of unsaturated fatty acids.    
     
     
         11 . A method of molecular breeding to obtain altered levels of a fatty acid in seed oil of oil-producing plant species comprising: 
 (a) making a cross between two varieties of oil-producing species differing in the fatty 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 the radiolabelled nucleic acid fragment of  claim 1 .    
     
     
         12 . A method of RFLP mapping 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 the radiolabelled nucleic acid fragments of  claim 1 .    
     
     
         13 . A method to isolate nucleic acid fragments encoding fatty acid desaturases and related enzymes, comprising: 
 (a) comparing SEQ ID NOS:2, 4, 6, 8, 10, or 12 and other fatty acid desaturase polypeptide sequences;    (b) identifying the conserved sequences of 4 or more amino acids obtained in step a;    (c) designing degenerate oligomers based on the conserved sequences identified in step b; and    (d) using the degenerate oligomers of step c to isolate sequences encoding fatty acid desaturases and desaturase-related enzymes by sequence dependent protocols.    
     
     
         14 . An isolated nucleic acid fragment of  claim 1  comprising a nucleic acid sequence encoding a plant microsomal delta-12 fatty acid desaturase.  
     
     
         15 . A method for altering fatty acids composition in seeds comprising: 
 (a) making a cross between a mutant line with altered fatty acid composition with a plant containing the chimeric gene of  claim 7;     (b) growing fertile plants from seeds obtained from the cross; and    (c) screening progeny seeds from the fertile plants of step (b) for seeds containing altered fatty acid levels.    
     
     
         16 . A method for reducing polyunsaturated fatty acids in rapeseed oil comprising: 
 (a) making a cross between a rapeseed variety with increased oleic acid content or reduced linolenic acid content with a plant containing the chimeric gene of  claim 7;     (b) growing fertile plants from seeds obtained from the cross; and    (c) screening progeny seeds from the fertile plants of step (b) for seeds containing reduced polyunsaturated fatty acids.    
     
     
         17 . The method of  claim 16  wherein the cross in (a) is between a progeny plant derived from a seed comprising the  Brassica  variety having an oleic acid content of about 69% to 77%, based upon total extractable oil and belonging to a line in which the said oleic acid content has been stabilized for both the generation to which the seed belongs and its parent generation.  
     
     
         18 . A method for reducing saturated fatty acids in rapeseed seeds comprising: 
 (a) making a cross between a rapeseed variety with increased oleic acid content with a plant containing the chimeric gene of  claim 7;     (b) growing fertile plants from seeds obtained from the cross; and    (c) screening progeny seeds from the fertile plants of step (b) for seeds containing reduced saturated fatty acids.    
     
     
         19 . A method for reducing polyunsaturated fatty acids in soybean oil comprising: 
 (a) making a cross between a soybean variety with increased oleic acid content or reduced linolenic acid content with a plant containing the chimeric gene of  claim 7;     (b) growing fertile plants from seeds obtained from the cross; and    (c) screening progeny seeds from the fertile plants of step (b) for seeds containing reduced polyunsaturated fatty acids.    
     
     
         20 . A method for reducing saturated fatty acids in soybean seeds comprising: 
 (a) making a cross between a soybean variety with increased oleic acid content with a plant containing the chimeric gene of  claim 7;     (b) growing fertile plants from seeds obtained from the cross; and    (c) screening progeny seeds from the fertile plants of step (b) for seeds containing reduced saturated fatty acids.    
     
     
         21 . A  Brassica  sp. plant with seed palmitic acid of 2.7% or lower of total fatty acid.  
     
     
         22 . A  Brassica  sp. plant with seed stearic acid of 1.1% or lower of total fatty acid.  
     
     
         23 . A  Brassica  sp. plant with a combined seed palmitic acid and stearic acid content of 3.9% or lower of total fatty acids.  
     
     
         24 . A soybean plant with seed palmitic acid of 6.7% or lower of total fatty acid.  
     
     
         25 . A soybean plant with seed stearic acid of 2.1% or lower of total fatty acid.  
     
     
         26 . A soybean plant with a combined seed palmitic acid and stearic acid content of 9.2% or lower total of fatty acids.  
     
     
         27 . Oil obtained from the plants of claims  21 - 26 .