Genes for plant fatty acid modifying enzymes associated with conjugated double bond formation

Abstract

The preparation and use of nucleic acid fragments encoding plant fatty acid modifying enzymes associated with conjugated double bond formation or functionally equivalent subfragments thereof are disclosed. Chimeric genes incorporating such nucleic acid fragments or functionally equivalent subfragments thereof or complement thereof and suitable regulatory sequences can be used to create transgenic plants having altered lipid profiles.

What is claimed is:  
     
         1 . An isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to any of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 19, 23, or 29 under conditions of moderate stringency or (b) is at least 45% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 19, 23, or 29 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences.  
     
     
         2 . An isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation wherein said fragment or a functionally equivalent subfragment thereof encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOS: 2, 4, 20, 24, or 30.  
     
     
         3 . An isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to the isolated nucleic acid fragment of  claim 2  under conditions of moderate stringency or (b) is at least 45% identical to a polypeptide encoded by any of the isolated nucleic acid fragments of  claim 2  or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences.  
     
     
         4 . The isolated nucleic acid fragment of  claim 1 ,  2 , or  3  wherein said fragment is isolated from  Impatiens balsamina, Momordica charantia, Chrysobalanus icaco, Licania michauxii,  and  Aleurites fordii.    
     
     
         5 . The isolated nucleic acid fragment of  claim 1 ,  2  or  3  wherein the plant fatty acid modifying enzyme is associated with the formation of at least one fatty acid selected from the group consisting of eleostearic acid and parinaric acid.  
     
     
         6 . The isolated nucleic acid fragment of  claim 4  wherein the plant fatty acid modifying enzyme is associated with the formation of at least one fatty acid selected from the group consisting of eleostearic acid and parinaric acid.  
     
     
         7 . The isolated nucleic acid fragment of  claim 1 ,  2 , or  3  wherein the plant is selected from the group consisting of soybean, oilseed Brassica species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, and cocoa.  
     
     
         8 . The isolated nucleic acid fragment of  claim 4  wherein the plant is selected from the group consisting of soybean, oilseed Brassica species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, and cocoa.  
     
     
         9 . The isolated nucleic acid fragment of  claim 5  wherein the plant is selected from the group consisting of soybean, oilseed Brassica species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, and cocoa.  
     
     
         10 . The isolated nucleic acid fragment of  claim 6  wherein the plant is selected from the group consisting soybean, oilseed Brassica species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, and cocoa.  
     
     
         11 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 1 ,  2  or  3  or a functionally equivalent subfragment thereof or a complement thereof operably linked to suitable regulatory sequences.  
     
     
         12 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 4  or a functionally equivalent subfragment thereof or a complement thereof operably linked to suitable regulatory sequences.  
     
     
         13 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 5  or functionally equivalent subfragment thereof or a complement thereof operably linked to suitable regulatory sequences.  
     
     
         14 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 6  or a functionally equivalent subfragment thereof or a complement thereof operably linked to suitable regulatory sequences.  
     
     
         15 . A transformed host cell comprising the chimeric gene of  claim 11 .  
     
     
         16 . A transformed host cell comprising the chimeric gene of  claim 12 .  
     
     
         17 . A transformed host cell comprising the chimeric gene of  claim 13 .  
     
     
         18 . A transformed host cell comprising the chimeric gene of  claim 14 .  
     
     
         19 . The host cell of  claim 15  wherein said cell is selected from the group consisting of plant cells and microorganisms.  
     
     
         20 . The host cell of  claim 16  wherein said cell is selected from the group consisting of plant cells and microorganisms.  
     
     
         21 . The host cell of  claim 17  wherein said cell is selected from the group consisting of plant cells and microorganisms.  
     
     
         22 . The host cell of  claim 18  wherein said cell is selected from the group consisting of plant cells and microorganisms.  
     
     
         23 . Seeds obtained from a plant cell transformed with the chimeric gene of  claim 11 .  
     
     
         24 . Seeds obtained from a plant cell transformed with the chimeric gene of  claim 12 .  
     
     
         25 . Seeds obtained from a plant cell transformed with the chimeric gene of  claim 13 .  
     
     
         26 . Seeds obtained from a plant cell transformed with the chimeric gene of  claim 14 .  
     
     
         27 . Oil obtained from the seeds of  claim 23 .  
     
     
         28 . Oil obtained from the seeds of  claim 24 .  
     
     
         29 . Oil obtained from the seeds of  claim 25 .  
     
     
         30 . Oil obtained from the seeds of  claim 26 .  
     
     
         31 . A method of altering the level of fatty acids with conjugated double bonds in a host cell which comprises: 
 (a) transforming a host cell with the chimeric gene of  claim 11;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells having altered levels of fatty acids with conjugated double bonds.    
     
     
         32 . A method of altering the level of fatty acids with conjugated double bonds in a host cell which comprises: 
 (a) transforming a host cell with the chimeric gene of  claim 12;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells having altered levels of fatty acids with conjugated double bonds.    
     
     
         33 . A method of altering the level of fatty acids with conjugated double bonds in a host cell which comprises: 
 (a) transforming a host cell with the chimeric gene of  claim 13;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells having altered levels of fatty acids with conjugated double bonds.    
     
     
         34 . A method of altering the level of fatty acids with conjugated double bonds in a host cell which comprises: 
 (a) transforming a host cell with the chimeric gene of  claim 14;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells having altered levels of fatty acids with conjugated double bonds.    
     
     
         35 . The method of  claim 31 ,  32 ,  33  or  34  wherein the host cell is selected from the group consisting of plant cells and microorganisms.  
     
     
         36 . The method of  claim 31 ,  32 ,  33  or  34  wherein levels of eleostearic and/or parinaric fatty acid are altered.  
     
     
         37 . A method for producing seed oil containing fatty acids with conjugated double bonds in the seeds of plants which comprises: 
 (a) transforming a plant cell with the chimeric gene of  claim 11;     (b) growing a fertile mature plant from the transformed plant cell of step (a);    (c) screening progeny seeds from the fertile plants of step (b) for altered levels of fatty acids with conjugated double bonds; and    (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels fatty acids with conjugated double bonds.    
     
     
         38 . A method for producing seed oil containing fatty acids with conjugated double bonds in the seeds of plants which comprises: 
 (a) transforming a plant cell with the chimeric gene of  claim 12;     (b) growing a fertile mature plant from the transformed plant cell of step (a);    (c) screening progeny seeds from the fertile plants of step (b) for altered levels of fatty acids with conjugated double bonds; and    (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels fatty acids with conjugated double bonds.    
     
     
         39 . A method for producing seed oil containing fatty acids with conjugated double 10 bonds in the seeds of plants which comprises: 
 (a) transforming a plant cell with the chimeric gene of  claim 13;     (b) growing a fertile mature plant from the transformed plant cell of step (a);    (c) screening progeny seeds from the fertile plants of step (b) for altered levels of fatty acids with conjugated double bonds; and    (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels fatty acids with conjugated double bonds.    
     
     
         40 . A method for producing seed oil containing fatty acids with conjugated double bonds in the seeds of plants which comprises: 
 (a) transforming a plant cell with the chimeric gene of  claim 14;     (b) growing a fertile mature plant from the transformed plant cell of step (a);    (c) screening progeny seeds from the fertile plants of step (b) for altered levels of fatty acids with conjugated double bonds; and    (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels fatty acids with conjugated double bonds.    
     
     
         41 . The method of  claim 37 ,  38 ,  39  or  40  wherein the plants are selected from the group consisting of soybean, oilseed Brassica species, corn, peanut, rice, wheat, sunflower, safflower, cotton, and cocoa.  
     
     
         42 . The method of  claim 37 ,  38 ,  39  or  40  wherein the seed oil contains altered levels of eleostearic and/or parinaric fatty acids.  
     
     
         43 . A method for producing fatty acid modifying enzymes associated with conjugated double bond formation which comprises: 
 (a) transforming a microbial host cell with the chimeric gene of  claim 11;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells containing altered levels of protein encoded by the chimeric gene.    
     
     
         44 . A method for producing fatty acid modifying enzymes associated with conjugated double bond formation which comprises: 
 (a) transforming a microbial host cell with the chimeric gene of  claim 12;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells containing altered levels of protein encoded by the chimeric gene.    
     
     
         45 . A method for producing fatty acid modifying enzymes associated with conjugated double bond formation which comprises: 
 (a) transforming a microbial host cell with the chimeric gene of  claim 13;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells containing altered levels of protein encoded by the chimeric gene.    
     
     
         46 . A method for producing fatty acid modifying enzymes associated with conjugated double bond formation which comprises: 
 (a) transforming a microbial host cell with the chimeric gene of  claim 14;     (b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and    (c) selecting those transformed host cells containing altered levels of protein encoded by the chimeric gene.    
     
     
         47 . The method of  claim 43 ,  44 ,  45  or  46  wherein the fatty acid modifying enzyme is associated with the formation of at least one fatty acid selected from the group consisting of eleostearic and parinaric acid.  
     
     
         48 . An isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation.  
     
     
         49 . The isolated nucleic acid fragment of  claim 48  wherein the plant fatty acid modifying enzyme is associated with the formation of at least one fatty acid selected from the group consisting of eleostearic acid and parinaric acid.  
     
     
         50 . The isolated nucleic acid fragment of  claim 48  wherein said fragment is substantially similar to the nucleotide sequence set forth in SEQ ID NOS: 1, 3, 19, 23, or 29.  
     
     
         51 . The isolated nucleic acid fragment of  claim 1  or  claim 3  wherein the computer program is selected from the group consisting of: the BLAST computational method (Basic Local Alignment Search Tool) which includes BLASTN (nucleotide, both strands), BLASTX (nucleotide, six-frame translation), BLASTP (protein), TBLASTN (protein, from six-frame translation), and TBLASTX (nucleotide, six-frame translation); the Megalign program of the LASARGENE bioinformatics computing method (DNASTAR), or the Clustal method of multiple sequence alignment.  
     
     
         52 . A method to isolate nucleic acid fragments and functionally equivalent subfragments thereof encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation comprising: 
 (a) comparing SEQ ID NOS: 2, 4, 20, 24, and 30 and other plant fatty acid modifying enzyme polypeptide sequences;    (b) identifying 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 (s) to isolate sequences encoding a plant fatty acid modifying enzyme or a portion thereof associated with conjugated double bond formation by sequence dependent protocols.    
     
     
         53 . Animal feed comprising an ingredient derived from the processing of any of the seeds of claims  23 ,  24 ,  25  or  26 .  
     
     
         54 . Animal feed comprising any of the seeds of claims  23 ,  24 ,  25  or  26 .  
     
     
         55 . Animal feed comprising the oil of any of claims  27 ,  28 ,  29  or  30 .  
     
     
         56 . A method of improving the carcass quality of an animal by supplementing a diet of the animal with the feed of  claim 53 .  
     
     
         57 . A method of improving the carcass quality of an animal by supplementing a diet of the animal with the feed of  claim 54 .  
     
     
         58 . A method of improving the carcass quality of an animal by supplementing a diet of the animal with the feed of  claim 55 .  
     
     
         59 . Animal feed comprising at least one conjugated linolenic acid derived from oil extracted from a natural source selected from the group consisting of tung, bittermelon, pot marigold, jacaranda, catalpa, and pomegranate.  
     
     
         60 . The animal feed of  claim 59  wherein said conjugated linolenic acid is present in a carcass quality improving amount.  
     
     
         61 . A method of improving the carcass quality of an animal by supplementing a diet of the animal with the feed of claims  59  or  60 .  
     
     
         62 . The complement of the isolated nucleic acid fragment of any of claims  1 ,  2 ,  3 , or  48 .