Canola acetyl-CoA carboxylase compositions and methods of use

Abstract

The present invention provides isolated and purified polynucleotides that encode plant and cyanobacterial polypeptides that participate in the carboxylation of acetyl-CoA. Isolated cyanobacterial and plant polypeptides that catalyze acetyl-CoA carboxylation are also provided. Processes for altering acetyl-CoA carboxylation, increasing herbicide resistance of plants and identifying herbicide resistant variants of acetyl-CoA carboxylase are also provided.

What is claimed is:  
     
         1 . An isolated plant acetyl-CoA carboxylase enzyme.  
     
     
         2 . The enzyme according to    claim 1   , wherein said enzyme is isolated from a dicotyledonous plant.  
     
     
         3 . The enzyme according to    claim 2   , wherein said enzyme is isolated from soybean, rape, sunflower, tobacco, Arabidopsis, petunia, canola, pea, bean, tomato, potato, lettuce, spinach, carrot, alfalfa, or cotton.  
     
     
         4 . The enzyme according to    claim 3   , wherein said enzyme is isolated from canola.  
     
     
         5 . The enzyme according to    claim 1   , comprising the amino acid sequence of SEQ ID NO:20.  
     
     
         6 . The enzyme according to    claim 1   , wherein said enzyme is isolated from a monocotyledonous plant.  
     
     
         7 . The enzyme according to    claim 6   , wherein said enzyme is isolated from wheat, rice, maize, barley, rye, oats or timothy grass.  
     
     
         8 . The enzyme according to    claim 7   , wherein said enzyme is isolated from wheat.  
     
     
         9 . The enzyme according to    claim 1    comprising the amino acid sequence of SEQ ID NO:10.  
     
     
         10 . The enzyme according to    claim 1    comprising a portion of a dicotyledonous acetyl-CoA carboxylase functionally linked to a portion of a monocotyledonous acetyl-CoA carboxylase.  
     
     
         11 . An isolated and purified plant acetyl-CoA carboxylase enzyme having the ability to catalyze the carboxylation of acetyl-CoA.  
     
     
         12 . A purified DNA segment encoding plant or cyanobacterial acetyl-CoA carboxylase.  
     
     
         13 . The DNA segment of    claim 12   , wherein said segment encodes canola acetyl-CoA carboxylase.  
     
     
         14 . The DNA segment of    claim 13   , further defined as encoding the amino acid sequence of SEQ ID NO:20.  
     
     
         15 . The DNA segment of    claim 14   , fuirther defined as comprising SEQ ID NO:19.  
     
     
         16 . The DNA segment of    claim 12   , wherein said segment encodes wheat acetyl-CoA carboxylase.  
     
     
         17 . The DNA segment of    claim 16   , further defined as encoding the amino acid sequence of SEQ ID NO:10.  
     
     
         18 . The DNA segment of    claim 17   , further defined as SEQ ID NO:9.  
     
     
         19 . The DNA segment of    claim 12   , defined further as a recombinant vector.  
     
     
         20 . The DNA segment of    claim 12   , wherein said DNA is operatively linked to a promotor, said promoter expressing the DNA segment.  
     
     
         21 . The DNA segment of    claim 12   , wherein said DNA encodes a portion of a dicotyledonous acetyl-CoA carboxylase functionally linked to a portion of a monocotyledonous acetyl-CoA carboxylase.  
     
     
         22 . A recombinant host cell comprising the DNA segment of    claim 12   .  
     
     
         23 . The recombinant host cell of    claim 22   , defined furder as being a prokaryotic cell.  
     
     
         24 . The recombinant host cell of    claim 23   , firther defied as a bacterial or cyanobacterial host cell.  
     
     
         25 . The recombinant host cell of    claim 22   , defined further as being a eukaryotic cell.  
     
     
         26 . The recombinant host cell of    claim 25   , further defined as a yeast cell or a plant host cell.  
     
     
         27 . The recombinant host cell of    claim 26   , wherein said cell is a monocotyledonous plant cell.  
     
     
         28 . The recombinant host cell of    claim 24   , wherein the bacterial host cell is E. coli.  
     
     
         29 . The recombinant host cell of    claim 24   , wherein the cyanobacterial host cell is Synechococcus or Anabaena.  
     
     
         30 . The recombinant host cell of    claim 22   , wherein the DNA segment is introduced into the cell by means of a recombinant vector.  
     
     
         31 . The recombinant host cell of    claim 22   , wherein the host cell expresses the DNA segment to produce the encoded acetyl-CoA carboxylase protein or peptide.  
     
     
         32 . The recombinant host cell of    claim 22   , wherein the expressed acetyl-CoA carboxylase protein or peptide includes a contiguous amino acid sequence from SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ ID NO:20.  
     
     
         33 . A method of using a DNA segment that encodes an isolated acetyl-CoA carboxylase, comprising the steps of: 
 (a) preparing a recombinant vector in which an acetyl-CoA carboxylase-encoding DNA segment is positioned under the control of a promoter;    (b) introducing said recombinant vector into a recombinant host cell;    (c) culturing the recombinant host cell under conditions effective to allow expression of an encoded acetyl-CoA carboxylase protein or peptide; and    (d) collecting said expressed acetyl-CoA carboxylase protein or peptide.    
     
     
         34 . An isolated nucleic acid segment characterized as: 
 (a) a nucleic acid segrnent comprising a sequence region that consists of at least 14 contiguous nucleotides that have the same sequence as, or are complementary to, 14 contiguous nucleotides of SEQ ID NO:l; SEQ ID NO:3; SEQ ID NQ:S; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; or SEQ ID NO:19; or    (b) a nucleic acid segment of from 14 to about 10,000 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; or SEQ ID NO:19; or the complements thereof, under standard hybridization conditions.    
     
     
         35 . The nucleic acid segment of claimn 34, further defined as comprising a sequence region that consists of at least 14 contiguous nucleotides that have the same sequence as, or are complementary to, 14 contiguous nucleotides of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:II; or SEQ ID NO:19.  
     
     
         36 . The nucleic acid segment of    claim 34   , further defined as comprising a nucleic acid segment of from 14 to about 10,000 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:S; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; or SEQ ID NO:19, or the complements thereof, under standard hybridization conditions.  
     
     
         37 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least 14 contiguous nucleotides from SEQ ID NO:19, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:19, or the complement thereof, under standard hybridization conditions.  
     
     
         38 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least 14 contiguous nudleotides from SEQ ID NO:11, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:11, or the complement thereof, under standard hybridization conditions.  
     
     
         39 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least 14 contiguous nucleotides from SEQ ID NO:l9, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:9, or the complement thereof, under standard hybridization conditions.  
     
     
         40 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least 14 contiguous nucleotides from SEQ ID NO:7, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:7, or the complement thereof, under standard hybridization conditions.  
     
     
         41 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least 14 contiguous nucleotides from SEQ ID NO:3, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:3, or the complement thereof, under standard hybridization conditions.  
     
     
         42 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 20 nucleotides; or wherein the segment is about 20 nucleotides in length.  
     
     
         43 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 30 nucleotides; or wherein the segment is about 30 nucleotides in length.  
     
     
         44 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 50 nucleotides; or wherein the segment is about 50 nucleotides in length.  
     
     
         45 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 100 nucleotides; or wherein the segment is about 100 nucleotides in length.  
     
     
         46 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 200 nucleotides; or wherein the segment is about 200 nucleotides in length.  
     
     
         47 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 500 nucleotides; or wherein the segment is about 500 nucleotides in length.  
     
     
         48 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of at least about 1000 nucleotides; or wherein the segment is about 1000 nucleotides in length.  
     
     
         49 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.  
     
     
         50 . The nucleic acid segment of    claim 34   , wherein the segment comprises a sequence region of SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:19.  
     
     
         51 . The nucleic acid segment of    claim 34   , wherein the segment is up to 10,000 basepairs in length.  
     
     
         52 . The nucleic acid segment of    claim 34   , wherein the segment is up to 5,000 basepairs in length.  
     
     
         53 . The nucleic acid segment of    claim 34   , wherein the segment is up to 3,000 basepairs in length.  
     
     
         54 . The nucleic acid segment of    claim 34   , wherein the segment is up to 1,000 basepairs in length.  
     
     
         55 . A method for detecting a nucleic acid sequence encoding a plant acetyl-CoA carboxylase, comprising the steps of: 
 (a) obtaining sample nucleic acids suspected of encoding a plant acetyl-CoA carboxylase;    (b) contacting said sample nucleic acids with an isolated nucleic acid segment encoding acetylCoA carboxylase under conditions effective to allow hybridization of substantially complementary nucleic acids; and    (c) detecting the hybridized complementary nucleic acids thus formed.    
     
     
         56 . The method of    claim 55   , wherein the sample nucleic acids contacted are located within a cell.  
     
     
         57 . The method of    claim 55   , wherein the sample nucleic acids are separated from a cell prior to contact.  
     
     
         58 . The method of    claim 55   , wherein the isolated plant acetyl-CoA carboxylase-encoding nucleic acid segment comprises a detectable label and the hybridized complementary nucleic acids are detected by detecting said label.  
     
     
         59 . A nucleic acid detection kit comprising, in suitable container means, an isolated plant or cyanobacterial acetyl-CoA carboxylase-encoding nucleic acid segment and a detection reagent.  
     
     
         60 . The nucleic acid detection kit of    claim 59   , wherein the detection reagent is a detectable label that is linked to said acetyl-CoA carboxylase nucleic acid segment.  
     
     
         61 . An enzyme composition, free from total cells, comprising a purified acetyl-CoA carboxylase that includes a contiguous amino acid sequence from SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ ID NO:20.  
     
     
         62 . The composition of    claim 61   , comprising a peptide that includes a 15 to about 50 amino acid long sequence from SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ ID NO:20.  
     
     
         63 . The composition of    claim 61   , comprising a peptide that includes a iS to about 150 anio acid long sequence from SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ ID NO:20.  
     
     
         64 . The composition of    claim 61   , wherein the protein or peptide is a recombinant protein or peptide.  
     
     
         65 . A purified antibody that binds to a plant or cyanobacterial acetyl-CoA carboxylase protein or peptide.  
     
     
         66 . The antibody of    claim 65   , wherein the antibody is a monoclonal antibody.  
     
     
         67 . A method for detecting an acetyl-CoA carboxylase peptide in a biological sample, comprising the steps of: 
 (a) obtaining a biological sample suspected of containing an acetyl-CoA carboxylase peptide;    (b) contacting said sample with a first antibody that binds to a plant acetyl-CoA carboxylase protein or peptide, under conditions effective to allow the formation of immune complexes; and    (c) detecting the immune complexes so formed.    
     
     
         68 . The method of    claim 67   , wherein said first antibody is linked to a detectable label and the immune complexes are detected by detecting the presence of the label.  
     
     
         69 . The method of    claim 67   , wherein said immune complexes are detected by means of a second antibody linked to a detectable label, the second antibody having binding affinity for said first protein, peptide or antibody.  
     
     
         70 . An immunodetection kit comprising, in suitable container means, a first antibody that binds to an acetyl-CoA carboxylase protein or peptide, and an immunodetection reagent.  
     
     
         71 . A process for determining resistance to herbicides of the aryloxyphenoxypropionate or cyclohexanedione class in a plant, comprising: 
 (a) obtaining a sample from said plant; and    (b) testing for the presence of an acetyl-CoA carboxylase enzyme capable of conferring resistance to said plant in said sample.    
     
     
         72 . The process according to    claim 71   , wherein the presence of an acetyl-CoA carboxylase enzyme conferring said resistance is determined by identifying the presence of an acetyl-CoA carboxylase polypeptide in said plant.  
     
     
         73 . The process according to    claim 71   , wherein the presence of an acetyl-CoA carboxylase enzyme conferring said resistance is determined by identifying the presence of an acetyl-CoA carboxylase-encoding nucleic acid segment in said plant.  
     
     
         74 . The process according to    claim 71   , wherein said sample is obtained from a progeny plant of a parent plant that includes a herbicide-resistant acetyl-CoA carboxylase transgene.  
     
     
         75 . The process according to    claim 71   , wherein said sample is suspected of containing a fusion protein comprising a portion of a dicotyledonous plant acetyl-CoA carboxylase functionally linked to a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA carboxylase.  
     
     
         76 . A process for identifying herbicide resistant variants of a plant acetyl-CoA carboxylase enzyme, comprising the steps of: 
 (a) transforming a cyanobacterium or a yeast cell with a candidate DNA molecule that encodes an engineered plant acetyl-CoA carboxylase enzyme suspected of conferring herbicide resistance to form a transformed cyanobacterium;    (b) inactivating cyanobacterial or yeast acetyl-CoA carboxylase;    (c) exposing said transformed cyanobacterium or said transformed yeast cell to a herbicide that inhibits acetyl-CoA carboxylase activity;    (d) identifying transformed cyanobacteria or transformed yeast cells that are resistant to said herbicide; and    (e) characterizing DNA that encodes acetyl-CoA carboxylase from the cyanobacteria or yeast cells of step (d).    
     
     
         77 . The process of    claim 76   , wherein said acetyl-CoA carboxylase enzyme is a fusion protein comprising a portion of a dicotyledonous plant acetyl-CoA carboxylase functionally linked to a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA carboxylase.  
     
     
         78 . The process of    claim 76   , wherein said acetyl-CoA carboxylase enzyme is an engineered dicotyledonous plant acetyl-CoA carboxylase, or a portion of an engineered dicotyledonous plant acetyl-CoA carboxylase functionally linked to a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA carboxylase, or an engineered cyanobacterial acetyl-CoA carboxylase enzyme.  
     
     
         79 . A process of modifying the oil content of a plant cell, comprising expressing in a plant cell a DNA segment that encodes a plant or cyanobacterial acetyl-CoA carboxylase or the complement of said DNA segment.  
     
     
         80 . The process according to    claim 79   , comprising incorporating into said plant cell a DNA segment that encodes a plant or cyanobacterial acetyl-CoA carboxylase polypeptide, wherein said cell expresses the acetyl-CoA carboxylase enzyme.  
     
     
         81 . The process according to    claim 80   , wherein said plant cell is a monocotyledonous plant cell.  
     
     
         82 . A process of increasing the herbicide resistance of a monocotyledonous plant, comprising incorporating into said plant a trarsgene comprising a DNA segment encoding a plant or cyanobacterial acetyl-CoA carboxylase polypeptide resistant to herbicide inactivation, the plant expressing the polypeptide.  
     
     
         83 . The process according to    claim 82   , wherein said acetyl-CoA carboxylase polypeptide is a dicotyledonous plant acetyl-CoA carboxylase polypeptide.  
     
     
         84 . The process according to    claim 81   , wherein said plant acetyl-CoA carboxylase polypeptide comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:20.  
     
     
         85 . The process according to    claim 81   , wherein said plant acetyl-CoA carboxylase polypeptide is encoded by the DNA sequence comprising SEQ ID NO:9 or SEQ ID NO:19.  
     
     
         86 . The process according to    claim 81   , wherein said cyanobacterial acetyl-CoA carboxylase polypeptide comprises the amino acid sequence of SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; or SEQ ID NO:12.  
     
     
         87 . The process according to    claim 81   , wherein said cyanobacterial acetyl-CoA carboxylase polypeptide is encoded by the DNA sequence comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; or SEQ ID NO:11.  
     
     
         88 . A transgenic plant having incorporated into its genome a transgene that encodes a plant or cyanobacterial acetyl-CoA carboxylase.