WO1999067367A1 - ADN CODANT ACETYLE CoA CARBOXYLASE DE L'AVOINE - Google Patents

ADN CODANT ACETYLE CoA CARBOXYLASE DE L'AVOINE Download PDF

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WO1999067367A1
WO1999067367A1 PCT/US1999/014022 US9914022W WO9967367A1 WO 1999067367 A1 WO1999067367 A1 WO 1999067367A1 US 9914022 W US9914022 W US 9914022W WO 9967367 A1 WO9967367 A1 WO 9967367A1
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plant
coa carboxylase
accase
cells
acetyl coa
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PCT/US1999/014022
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WO1999067367A9 (fr
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Margaret A. Egli
Susanne Groh
Shahryar F. Kianian
Ronald L. Phillips
David A. Somers
R. Gary Fulcher
Howard W. Rines
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Regents Of The University Of Minnesota
United States Of America Represented By The Secretary, Department Of Agriculture
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Application filed by Regents Of The University Of Minnesota, United States Of America Represented By The Secretary, Department Of Agriculture filed Critical Regents Of The University Of Minnesota
Priority to CA002332643A priority Critical patent/CA2332643A1/fr
Priority to EP99930517A priority patent/EP1092011A1/fr
Priority to AU47044/99A priority patent/AU4704499A/en
Publication of WO1999067367A1 publication Critical patent/WO1999067367A1/fr
Publication of WO1999067367A9 publication Critical patent/WO1999067367A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • Acetyl CoA carboxylase is an enzyme involved in many important metabolic pathways in plant, animal and bacterial cells. The enzyme is especially important in fatty acid synthesis in plants and is sensitive to inhibition by some types of herbicides. Structurally, ACCases are biotinylated and are quite large enzymes consisting of one or more subunits. For example, most ACCases of animals, higher plants, and yeast are dimers of 420 to 700 kD native MW and contain subunits of 200 to 280 kD. Diatom and algal ACCases are 700 to 740 kD tetramers of 160 to 180 kD subunits.
  • Bacterial ACCase consists of three dissociable proteins; biotin carboxylase (51 kD), biotin carboxyl carrier protein (22.5 kD), and biotin transcarboxylase (130 kD).
  • Acetyl CoA Carboxylase catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate in animal, plant, and bacterial cells.
  • Malonyl-CoA is an essential substrate for (i) de novo fatty acid (FA) synthesis, (ii) fatty acid elongation, (iii) synthesis of secondary metabolites such as flavonoids and anthocyanins, and (iv) malonylation of some amino acids and secondary metabolites.
  • malonyl-CoA is the first committed step of flavonoid and fatty acid synthesis and current evidence suggests that ACCase catalyzes the rate-limiting step of fatty acid synthesis. Formation of malonyl-CoA by ACCase occurs via two partial reactions and requires a biotin prosthetic group:
  • ACCase activity In plants, most ACCase activity is located in plastids of green and non-green plant tissues including leaves and oil seeds. Leaf ACCase activity is primarily located in mesophyll cells, but lesser amounts have been found in C-4 bundle sheath cells and in epidermal cells. The subcellular location of ACCase activity in epidermal cells is unknown, but since synthesis of very long-chain fatty acids (VLCFA) for formation of waxes, cutin, and suberin occurs on the endoplasmic reticulum (ER), malonyl-CoA might also be derived from a cytosolic ACCase. In contrast, rat ACCase is primarily cytosolic or associated with the outer mitochondrial membrane.
  • VLCFA very long-chain fatty acids
  • ER endoplasmic reticulum
  • malonyl-CoA might also be derived from a cytosolic ACCase.
  • rat ACCase is
  • fatty acid synthesis in chloroplasts involves successive 2-carbon additions to acetate, using malonate as the 2-C donor. All intermediates are attached to acyl carrier protein (ACP).
  • ACP acyl carrier protein
  • Synthesis in plastids resembles that in in that the fatty acid synthesis complex can be dissociated into separate enzymes: ⁇ -ketoacyl-ACP synthase (KAS), ⁇ -ketoacyl- ACP reductase, ⁇ -hydroxyl-ACP dehydratase, and enoyl-ACP reductase, acetyl-CoA: ACP transacylase, and malonyl-CoA:ACP transacylase.
  • KAS ⁇ -ketoacyl-ACP synthase
  • KAS ⁇ -ketoacyl- ACP reductase
  • ⁇ -hydroxyl-ACP dehydratase enoyl-ACP reductase
  • a highly active KASIII isozyme catalyzes the condensation of acetyl-CoA and malonyl-ACP. Successive additions of malonyl-CoA to acyl-ACPs catalyzed by KAS I form C16 acyl- ACP, some of which is converted to C18 acyl- ACP by KAS II and then to C 18:1 -ACP. Fatty acid metabolism then diverges; de- esterification allows movement to the cytoplasm (eukaryotic path) where fatty acids may be further unsaturated and/or elongated by additions of malonyl-CoA in the ER. Alternatively, fatty acids are linked to glycerol-3-phosphate
  • Condensation of malonyl-CoA with phenylpropionyl-CoAs or acetyl-CoA leads to synthesis of fiavonoids, anthocyanins, or to polyacetates. Condensation is increased by light, elicitors, or pathogens and may be the rate- limiting step in synthesis of some phytoalexins.
  • malonyl conjugates of flavonoid glycosides formed by malonyl-CoA:flavonoid glycoside malonyltransferase, D-amino acids and 1-amino-carboxyl-cyclopropane (ethylene precursor) are found in plants. Malonylated compounds accumulate in vacuoles, probably after synthesis in the cytoplasm.
  • ACCase activity is the rate-limiting step for de novo fatty acid synthesis in plants.
  • ACCase in most monocots is also inhibited by several herbicides.
  • [ I4 C]acetate incorporation into maize lipids is strongly inhibited by fluazifop and sethoxydim due to inhibition of plastid ACCase.
  • fluazifop had little effect on [ 14 C]acetate incorporation into very long-chain fatty acids. Since synthesis of very long- chain fatty acids occurs in the cytosol on the ER, and de novo fatty acid synthesis occurs in the plastids, cytosolic malonyl-CoA might be supplied by a herbicide insensitive ACCase isozyme.
  • Tissue culture methods have been used to select for resistance (or tolerance) using a variety of herbicides and plant species (see review by Meredith and Carlson, 1982, in Herbicide Resistance in Plants, eds. Lebaron and Gressel, pp. 275-291, John Wiley and Sons, NY).
  • P. C. Anderson et al. in U.S. Patent No. 4,761,373, disclose the use of tissue culture methods to produce maize plants resistant to herbicidal imidazolidones and sulfonamides. The resistance is due to the presence of altered acetohydroxy acid synthase which is resistant to deactivation by these herbicides.
  • 1,3-cyclohexanediones exhibit general and selective herbicidal activity against plants.
  • One such cyclohexanedione is sethoxydim ⁇ 2- [l-(ethoxyimino)-butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-l- one ⁇ .
  • Sethoxydim is commercially available from BASF (Parsippany, New Jersey) under the designation POASTTM.
  • herbicidal cyclohexanediones include clethodim, (E,E)-( ⁇ )- 2-[ 1 -[[(3-chloro-2-propenyl)oxy] imino]propyl]-5-[2-(ethylthio)propyl]-3- hydroxy-2-cyclohexen-l-one; available as SELECTTM from Chevron Chemical (Valent) (Fresno, California); cloproxydim, (E,E)-2-[l-[[(3-chloro-2- propenyl)oxy]imino] butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-l- one; available as SELECTONETM from Chevron Chemical (Valent) (Fresno, California); and tralkoxydim, 2-[l-(ethoxyimino)propyl]-3-hydroxy-5- mesitylcyclohex
  • Certain aryloxyphenoxypropanoic acids exhibit general and selective herbicidal activity against plants.
  • the aryloxy group may be phenoxy, pyridinyloxy or quinoxalinyl.
  • One such herbicidal aryloxyphenoxypropanoic acid is haloxyfop, ⁇ 2-[4-[[3-chloro-5- (trifluoromethyl)-2-pyridinyl]oxy]phenoxy]- propanoic acid ⁇ , which is available as VERDICTTM from Dow Chemical USA (Midland, Michigan).
  • herbicides referred to in the two preceding paragraphs and other structurally related herbicidal compounds are collectively referred to as herbicidal aryloxyphenoxypropanoic acids.
  • the present invention provides an isolated and purified DNA molecule comprising a DNA segment encoding a plant acetyl CoA carboxylase such as a monocot acetyl CoA carboxylase, e.g., a maize or oat acetyl CoA carboxylase, and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in the plant cells.
  • a plant acetyl CoA carboxylase such as a monocot acetyl CoA carboxylase, e.g., a maize or oat acetyl CoA carboxylase
  • the DNA molecule encoding a plant acetyl CoA carboxylase can encode an unaltered plant acetyl CoA carboxylase or an altered plant acetyl CoA carboxylase substantially tolerant to inhibition by cyclohexanedione or aryloxyphenoxypropanoic acid herbicides as well as encoding an antisense DNA sequence that is substantially complementary to a plant acetyl CoA carboxylase gene or to a portion thereof.
  • a DNA molecule of the invention can also further comprise an amino terminal plant chloroplast transit peptide sequence operably linked to the plant acetyl CoA carboxylase gene.
  • the DNA molecule of the invention preferably encodes the plastidic form of plant acetyl CoA carboxylase.
  • the invention also provides for probes, e.g., a probe which is detectably labeled or which binds to a detectable label, or primers, e.g., a single-strand DNA molecule having at least seven nucleotide bases, that hybridize to a DNA encoding a plant acetyl CoA carboxylase, e.g., a DNA molecule comprising SEQ ID NO:23 or its complement.
  • probes e.g., a probe which is detectably labeled or which binds to a detectable label
  • primers e.g., a single-strand DNA molecule having at least seven nucleotide bases, that hybridize to a DNA encoding a plant acetyl CoA carboxylase, e.g., a DNA molecule comprising SEQ ID NO:23 or its complement.
  • the present invention provides an isolated and purified DNA molecule of at least seven nucleotide bases which hybridizes under high stringency conditions to a DNA molecule comprising a DNA segment encoding a plant acetyl CoA carboxylase and provides a hybridization probe comprising an isolated and purified DNA molecule of at least seven nucleotide bases, which is detectably labeled or which binds to a detectable label, which DNA molecule hybridizes under high stringency conditions to the non-coding strand of a DNA molecule comprising a DNA segment encoding a plant acetyl CoA carboxylase.
  • High stringency conditions are defined as: hybridization at 65 °C for at least 16 hours in 5xSSC, lx Denhardt's solution, 50 raM Tris-HCl, pH 8, 0.2% SDS, 10 mM EDTA, 0.1 mg/ml salmon sperm DNA, followed by washing twice for 5 minutes in 2xSSC, 0.5% SDS at 25°C, once for 10 minutes in 0.2xSSC, 0.1% SDS at 25°C and twice for 30 minutes in 0.2xSSC, 0.1% SDS at 65°C.
  • the method of imparting cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant includes the steps of introducing a chimeric or recombinant DNA molecule comprising a gene coding for a plant acetyl CoA carboxylase or an altered or a functional mutant thereof operably linked to a promoter functional in a plant cell into cells of a susceptible plant, and regenerating the transformed plant cells to provide a differentiated plant.
  • the promoter can be an inducible or tissue specific promoter or provide for overexpression of at least about a 2-fold amount of a native plant acetyl CoA carboxylase.
  • the functional linkage of a promoter to the chimeric DNA molecule results in an expression cassette.
  • Expression of the chimeric DNA molecule is in an amount effective to render the acetyl CoA carboxylase and/or the plant tissue substantially tolerant to the herbicides relative to the native acetyl CoA carboxylase present in said plant.
  • Herbicide tolerance can be achieved in the plants by at least two methods, including increasing the level of gene expression of a native or unaltered acetyl CoA carboxylase, or by introducing an altered gene coding for an acetyl CoA carboxylase that is less sensitive to herbicide inhibition.
  • the level of gene expression can be increased by either combining a plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression, such as a 35S cauliflower mosaic virus promoter (CaMV), or by introducing multiple copies of the gene into the cell so that the multiple copies of the gene are integrated into the genome of transformed plant cells.
  • a promoter that provides for a high level of gene expression such as a 35S cauliflower mosaic virus promoter (CaMV)
  • CaMV 35S cauliflower mosaic virus promoter
  • the preferred plant cells into which to introduce the expression cassette of the invention, to achieve herbicide tolerance are monocot plant cells. Preferably, the cells are not oat cells. Once transformed cells exhibiting herbicide tolerance are obtained, transgenic plants and seeds can then be regenerated therefrom, and evaluated for stability of the inheritance of the herbicide tolerance trait.
  • the invention also provides a method for altering, e.g., lowering or raising, the oil content in a plant.
  • a locus having a major effect on oat (Avena sativa L.) groat oil content was located on linkage group 11 by quantitative trait locus (QTL) dissection methods.
  • QTL quantitative trait locus
  • a partial oat acetyl-CoA carboxylase (ACCase) cDNA with >84% identity to plastidic ACCase sequences from wheat or maize was linked to this locus. Similar QTL and ACCase placements were obtained with two recombinant inbred populations (n ⁇ 137) with one common parent.
  • locus on linkage group 11 linked to the ACCase locus accounted for up to 48% of the phenotypic variance for groat oil content. Other loci were identified which accounted for an additional 14% of the phenotypic variance.
  • the major locus plus additional QTLs represent three potentially homoeologous genomic segments in oat that can be followed by as few as three RFLP markers to produce a large shift in groat oil content.
  • Oat acetyl CoA carboxylase DNA is also useful in molecular marker-assisted selection to facilitate breeding high- or low-oil oat, genetic dissection of the groat oil trait, and to identify specific genes with large effects whose activity may be manipulated via genetic transformation.
  • the invention provides a method which includes the steps of introducing a chimeric or recombinant DNA molecule comprising a gene coding for a plant, e.g., oat, acetyl CoA carboxylase or an altered or a functional mutant thereof operably linked to a promoter functional in a plant cell into the cells of plant tissue and expressing the gene in an amount effective to alter the oil content of the plant cell.
  • An alteration in oil content can include a change in total oil content over that normally present in that type of plant cell, a change in the location of the oil in the plant, plant tissue, seed or plant part, and/or a change in the type of oil present in the cell.
  • low-oil oat includes, but is not limited to, an oat plant, plant part, plant tissue or groat having about 8 to about 40, preferably about 8 to about 30, and more preferably about 20 to about 30, percent dry weight of oil relative to the corresponding parental, e.g., nontransformed or inbred, oat plant, plant part, plant tissue or groat.
  • High-oil oat includes, but is not limited to, an oat plant, plant part, plant tissue or groat having about 2 to about 8, preferably 1 to about 4, and more preferably about 2 to about 4, percent dry weight of oil relative to the corresponding parental oat plant, plant part, plant tissue or groat.
  • An alteration in oil content in the plant cell can be achieved by at least two methods including:
  • an increase or decrease in expression of an unaltered or altered plant acetyl CoA carboxylase gene or (2) by introducing an unaltered, altered or functional mutant plant acetyl CoA carboxylase gene.
  • the level of gene expression of an unaltered plant acetyl CoA carboxylase gene can be increased by either combining an unaltered plant acetyl CoA carboxylase with a promoter that provides for a high level of gene expression, or by introducing multiple copies of an expression cassette into cells so that multiple copies of the gene are integrated into the genome.
  • an altered or a functional mutant plant acetyl CoA carboxylase gene codes for an enzyme that exhibits an increase in specific activity, it can lead to an increase in total oil content of the plant cell.
  • acetyl CoA carboxylase gene codes for an enzyme having a decrease in specific activity, it may lead to a decrease in the total oil content of the plant cell.
  • the expression cassette is introduced into dicot plants such as soybeans, canola, and sunflower.
  • transformed cells exhibiting about a 1.2- to 5-fold increase in total oil content and/or expression or specific activity of acetyl CoA carboxylase are selected for and used to generate transgenic plants and seeds exhibiting a substantial increase in oil content.
  • a substantial increase in oil content depends on the oil content normally present in the plant or seed and can range from about a 1.2 to a 20-fold increase.
  • the invention also provides for a method of producing plant acetyl CoA carboxylase in a host cell.
  • the method includes the steps of introducing an expression cassette comprising a chimeric gene encoding a plant acetyl CoA carboxylase or an altered or a functional plant acetyl CoA carboxylase operably linked to a promoter into a host cell and expressing the gene in an amount sufficient to permit crystallization of the plant acetyl CoA carboxylase.
  • An expression cassette can include a promoter that is functional in either a eukaryotic or a prokaryotic cell.
  • the expression cassette is introduced into a prokaryotic cell, such as E.
  • Recombinantly produced and crystallized plant acetyl CoA carboxylase can then be used to identify other herbicides and that bind to and inhibit acetyl CoA carboxylase in plants.
  • the availability of large amounts of purified enzyme can permit the screening of the efficacy of such herbicides in terms of their ability to bind to, or otherwise inhibit, the activity of the enzyme.
  • the present invention also provides a method of introducing an exogenous plant acetyl CoA carboxylase gene into a host cell comprising transforming host cells in vitro with an expression cassette comprising a chimeric or recombinant DNA molecule encoding a plant acetyl CoA carboxylase gene operably linked to a promoter functional in the host cell, expanding the transformed host cells in vitro, and identifying a transformed host cell which expresses the chimeric DNA molecule. Further provided is a method to identify a plant, e.g., an oat plant, having altered characteristics, e.g., altered groat oil content.
  • the method comprises contacting a probe comprising at least a portion of a nucleic acid sequence encoding oat acetyl CoA carboxylase with a sample from an oat plant in an amount sufficient to form complexes, wherein the sample comprises plant genomic nucleic acid.
  • the amount of complex formation is then detected or determined.
  • the plant cells in the sample are monocot plant cells, e.g., wheat, rice, maize, barley, millet (e.g., foxtail or finger), sorghum, sugarcane, rye, oat or timothy cells.
  • the DNA molecule encodes a polypeptide comprising SEQ ID NO:24, e.g., a DNA molecule comprising SEQ ID NO:23.
  • the invention provides a nucleic acid fragment useful in marker-assisted breeding programs such as those which identify plants having altered groat oil content or composition.
  • an expression cassette comprising an endosperm-specific transcriptional control region, e.g., the DylO or zein transcriptional control region, operably linked to a DNA encoding a plant acetyl CoA carboxylase, or subunit thereof.
  • endosperm-specific promoters are also useful in the compositions and methods of the invention. These promoters include, but are not limited to, ltrl promoter, a synthetic promoter with endosperm box of HOD (Vincentecarbajosa et al., Plant J., 13., 629 (1998)); rice glutelin gene promoter (Sindhu et al., Plant Sci.
  • nucleotide sequence encodes an ACCase, and that nucleotide sequences are not present which encode proteins other than ACCase or functional equivalents thereof.
  • Figure 1 A schematic depiction of the fatty acid biosynthesis pathway in plants.
  • Figure 2 A graph depicting the effect of sethoxydim on the growth of mutant maize callus.
  • Figure 3 A graph depicting the shoot length growth of maize seedlings seven days after treatment with sethoxydim.
  • Figure 4 A graph depicting the shoot length growth of maize seedlings fourteen days after treatment with sethoxydim.
  • Figure 5 Total soluble and biotinylated polypeptides in ACCase purification fractions from seedling leaves of maize inbred A619. Proteins were separated by SDS-PAGE in 7.5% gels and then silver-stained (Panel A). An identical gel was Western-blotted and a longitudinal section of each lane was probed with avidin (Panel B). Lanes were 1: crude (10 ⁇ g); 2: (NH 4 ) 2 SO 4 (10 ⁇ g); 3: S-300 (5 ⁇ g); 4: Blue Sepharose (2 ⁇ g); 5: Mono-Q ACCase II (5 ⁇ g); and 6: Mono-Q ACCase I (5 ⁇ g).
  • Diagonal lines between lanes indicate position of molecular weight markers shown on the left.
  • Figure 6 mimunoprecipitation of ACCase activity from B73 leaf, embryo, endosperm, and BMS suspension cultured cells. Equal activities (0.58 nmol min "1 ) were incubated with 16 ⁇ L serum (immune plus preimmune), immune complexes were precipitated with Protein A-agarose, and ACCase activity remaining in the resulting supernatant fraction was measured relative to the preimmune serum control.
  • Figure 7 Herbicide inhibition of acetyl-(AcCoA) or propionyl-CoA (Prop-CoA)-dependent H 14 CO 3 -incorporation into acid-stable product by ACCase I and II Mono-Q fractions. Activities in the presence of haloxyfop (1 ⁇ M) are expressed relative to the minus herbicide control.
  • Figure 8 Comparison of the peptide sequence of maize cDNA clones #15-14 and #18-5 with chicken ACCase. The approximate locations of the biotin carboxylase, biotin binding site, and biotin transcarboxylase functional domains are indicated for the chicken sequence. The percentages of amino acid identity are indicated by cross-hatched boxes for the maize coding sequence. Regions of genomic DNA Type I and Type II clone sequences that align with cDNA #18-5 are indicated by solid heavy lines. The approximate locations of subclone #28 and #16 from genomic Type I and subclone #34 from genomic Type II clones are indicated.
  • Figure 9 Northern blot of total RNA from leaf, immature embryo and endosperm tissue (16 days after pollination), and Black Mexican Sweet corn (BMS) cells. Lanes contain 10 ⁇ g total RNA and were probed with the 2 kb EcoRI fragment of lambda clone #15-14.
  • Figure 10 DNA sequence (SEQ ID NO. 1) of a 2 kb EcoRI fragment of lambda clone #15-14 including a portion of a maize ACCase gene located at bases 2883 to 83 from the 3' stop codon.
  • Figure 11 Graph of ACCase activity during seed development in two high oil soybean cell lines and one low oil soybean cell line.
  • Figure 12 Cloning strategy to obtain the complete coding sequence of the maize ACCase gene.
  • Figure 13 DNA sequence (SEQ LD NO:5) of a 7470 base pair cDNA of a maize ACCase gene. (Genbank Accession No. U19183).
  • Figure 14 Predicted amino acid sequence of the complete
  • Figure 15 Restriction map of ACCase genomic clones.
  • Figure 16 Partial nucleotide sequence of a Type A ⁇ ACCase genomic clone (SEQ ID NO: 12).
  • Figure 17 Partial nucleotide sequence of clone 5 A, a Type A
  • Figure 18 Partial nucleotide sequence of six Type A 2 ACCase genomic clones (A-F) (SEQ ID NOs 14, 15, 16, 17, 18, and 19).
  • Figure 19 Partial nucleotide sequence of three Type B ACCase clones (SEQ ID NOs 20, 21 and 22).
  • Figure 20 SDS/PAGE analysis of chloroplast importation of 35 S- labeled ACCase polypeptides.
  • A Pea and maize chloroplasts incubated with - 35 S-labeled ACCase polypeptides for 30 minutes.
  • B A time course analysis of the importation of 35 S-labeled ACCase polypeptides into maize chloroplasts.
  • Figure 21 Partial DNA sequence encoding oat ACCase (SEQ LD NO:23, Genbank Accession No. AF072737).
  • Figure 22 Partial amino acid sequence of an oat ACCase (SEQ ID NO:24).
  • Figure 23 Scan of test statistics generated by MQTL for the two recombinant inbred populations.
  • a and B are the main effect and QTL x E interaction graphs for the Kanota X Ogle (KO) population, respectively.
  • C and D are the main effect and QTL x E interaction graphs for the Kanota X Marion (KM) population, respectively.
  • Thresholds for type I error rate of 5% after 1000 replications are reported above the graphs.
  • Figure 24 Histogram of % groat oil concentration for the mean of all environments relative to the number of recombinant inbred lines (RILs) with each parental genotype for the XaccaseA locus.
  • RILs recombinant inbred lines
  • Figure 25 Phylogram of ACCases.
  • the present invention provides a DNA molecule encoding a plant acetyl CoA carboxylase gene and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in the plant cells.
  • acetyl CoA carboxylase plays a central role in regulating fatty acid synthesis and in the sensitivity of monocots to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides.
  • a plant acetyl CoA carboxylase gene is identified, isolated and combined with a promoter functional in a plant cell to provide a recombinant expression cassette.
  • a plant acetyl CoA carboxylase gene can be introduced and expressed in a plant cell.
  • introduction of a plant acetyl CoA carboxylase gene into the plant cell can confer herbicide tolerance and/or alteration of the oil of the plant cell.
  • an exogenously introduced plant acetyl CoA carboxylase gene can be expressed at a level effective to render the cells of the plant tissue substantially tolerant to cyclohexanedione or aryloxyphenoxypropanoic acid herbicide levels which normally inhibit a native or endogenous acetyl CoA carboxylase.
  • a native acetyl CoA carboxylase is an enzyme that is normally encoded and expressed in the plant cell prior to transformation.
  • An exogenously introduced plant acetyl CoA carboxylase gene is a gene which has been isolated and amplified from either the same or different type of cell.
  • Exogenous introduction and expression of a plant acetyl CoA carboxylase gene in both monocots and dicots can result in alteration of the oil content and quality of plant tissue and seeds.
  • Exogenous introduction and expression in a host cell such as a bacteria, can provide for sufficient amounts of plant acetyl CoA carboxylase to allow for crystallization and isolation of the enzyme. Crystallized plant acetyl CoA carboxylase is useful to identify other herbicides that bind to and can inhibit plant acetyl CoA carboxylases. The enzyme could also be used to screen potential herbicidal compounds for efficacy.
  • An expression cassette of the invention can comprise a chimeric DNA molecule encoding a plant acetyl CoA carboxylase gene or an altered or functional mutant thereof operably linked to a promoter functional in a plant cell.
  • the gene can code for a plant acetyl CoA carboxylase that is substantially tolerant to herbicides, preferably cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides.
  • An expression cassette of the invention can also include an antisense DNA sequence that is substantially complementary to an acetyl CoA carboxylase gene or a portion thereof operably linked to a promoter functional in a plant cell.
  • a gene encoding a plant acetyl CoA carboxylase can be identified and isolated by standard methods, as described by Sambrook et al., Guide to Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). The gene can be obtained either from monocot or dicot plant cells. When the gene encoding a plant acetyl CoA carboxylase is obtained from a dicot plant, the enzyme encoded by the gene exhibits tolerance to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides. The gene can also be obtained from herbicide-tolerant maize cell lines, prepared as described in U.S. Patent No. 5,162,602.
  • a gene encoding a plant acetyl CoA carboxylase can be identified by screening of a DNA or cDNA library generated from plant cells. Screening for DNA fragments that encode all or a portion of the gene encoding a plant acetyl CoA carboxylase can be accomplished by complementation of an auxotrophic mutant of acetyl CoA carboxylase in E ⁇ ⁇ oli (fahE) (Bachman, Microbiological Reviews, 42:180 (1983)) or yeast (acc ) (Michionada, EiirJL Biochem., 111:79 (1980)) or by screening of plaques for binding to antibodies that specifically recognize a plant acetyl CoA carboxylase.
  • DNA fragments that can restore ACCase activity in E. co i or yeast and/or plaques carrying DNA fragments that are immunoreactive with antibodies to a plant ACCase can be subcloned into a vector and sequenced and/or used as probes to identify other cDNA or genomic sequences encoding all or a portion of a plant acetyl CoA carboxylase gene.
  • cDNA sequences encoding a portion of a plant acetyl CoA carboxylase gene include DNA fragments that include a DNA sequence that substantially corresponds to the coding sequence for the transcarboxylase active site of a plant acetyl CoA carboxylase, DNA fragments that include a DNA sequence that substantially corresponds to a coding sequence for the biotin binding site of a plant acetyl CoA carboxylase, a DNA fragment encoding the 5' transcriptional start sequence of a plant acetyl CoA carboxylase gene, and a DNA fragment encoding the 3' transcriptional stop sequence for the acetyl CoA carboxylase gene.
  • Substantially corresponding DNA sequences share about 90% to about 100% DNA sequence homology.
  • Especially preferred cDNA probes can be obtained from lambda clone #18-5 which include DNA sequences corresponding to the transcarboxylase active site domain and the biotin binding site domain.
  • Lambda clone #18-5 includes EcoRI subclones of 3.9 kb, 1.2 kb, or 0.23 kb.
  • Lambda subclone #18-51 is an 3.9 kb EcoRI subclone. The lambda subclone #18-51 has been deposited with the American Type Culture Collection, Rockville, MD, and given Accession No. 69236.
  • a plant acetyl CoA carboxylase gene is identified and isolated from an herbicide tolerant maize cell line prepared as described in Example II.
  • a cDNA library can be prepared by oligo dT priming. Plaques containing DNA fragments can be screened with antibodies specific for maize acetyl CoA carboxylase. DNA fragments encoding a portion of an acetyl CoA carboxylase gene can be subcloned and sequenced and used as probes to identify a genomic acetyl CoA carboxylase gene.
  • DNA fragments encoding a portion of a maize acetyl CoA carboxylase can be verified by determining sequence homology with other known acetyl CoA carboxylases, such as chicken or yeast acetyl CoA carboxylase, or by hybridization to acetyl CoA carboxylase specific messenger RNA. Once DNA fragments encoding portions of the 5', middle and 3 ' ends as well as the transcarboxylase active site or biotin binding site of a plant acetyl CoA carboxylase are obtained, they can be used to identify and clone a complete genomic copy of a maize acetyl CoA carboxylase gene.
  • a maize genomic library can then be probed with cDNA probes prepared as described above. Portions of the genomic copy or copies of a plant acetyl CoA carboxylase gene can be sequenced and the 5' end of the gene are identified by standard methods including either DNA sequence homology to other acetyl CoA carboxylase genes or by RNAase protection analysis, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989).
  • a plant acetyl CoA carboxylase gene can be obtained by standard methods, including by cloning or by polymerase chain reaction (PCR) synthesis using oligonucleotide primers complementary to the DNA sequence at the 5' end of the gene.
  • PCR polymerase chain reaction
  • the presence of an isolated full-length copy of a plant acetyl CoA carboxylase gene can be verified by hybridization, partial sequence analysis, or by expression of a plant acetyl CoA carboxylase enzyme.
  • the maize acetyl CoA carboxylase gene cloned and expressed from a maize herbicide tolerant cell line can be assessed for tolerance to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides by standard methods, as described in Example I.
  • An expression cassette of the invention can also contain an antisense DNA sequence.
  • a antisense DNA sequence is a sequence that is substantially complementary to all or a portion of a coding sequence of a plant acetyl CoA carboxylase gene.
  • a substantially complementary sequence has about 90% to about 100% DNA sequence homology with that of the coding sequence of all or a portion of a plant acetyl CoA carboxylase.
  • the antisense DNA sequence when expressed can act to inhibit the synthesis and expression of a native plant acetyl CoA carboxylase.
  • Antisense sequences are preferably about 200 to 1000 nucleotides long in order to provide sufficient inhibition of synthesis and/or expression of a native acetyl CoA carboxylase. The inhibition of acetyl CoA carboxylase synthesis and gene expression by antisense DNA sequences can be confirmed in a transformed plant cell by standard methods for measuring the presence and/or activity of the enzyme such as described in Examples I and V.
  • An expression cassette of the invention can also include a functional mutant of a plant acetyl CoA carboxylase gene. Mutants of a plant acetyl CoA carboxylase gene are substantially homologous to a plant acetyl CoA carboxylase gene and are functional if the acetyl CoA carboxylase expressed retains significant enzyme activity. A mutant substantially homologous to a plant acetyl CoA carboxylase can share about 90% to 99.99% DNA sequence with that gene.
  • a mutant acetyl CoA carboxylase gene can code for a herbicide tolerant acetyl CoA carboxylase, or for an acetyl CoA carboxylase with altered substrate specificity so that the total amount of oil content in the plants or seeds is increased, or for an enzyme with an altered substrate specificity so that synthesis of secondary metabolites such as flavonoids or anthocyanins is decreased.
  • a preferred mutant is a gene coding for an acetyl CoA carboxylase that is substantially tolerant to cyclohexanedione or aryloxyphenoxypropanoic acid herbicide
  • Altered or functional mutants of a gene coding for a plant acetyl CoA carboxylase can be obtained by several methods.
  • the alteration or mutation of the ACCase gene can be accomplished by a variety of means including, but not limited to, the following methods.
  • a functional mutant gene encoding for a plant acetyl CoA carboxylase tolerant to cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides is isolated from a maize herbicide tolerant cell line.
  • the maize herbicide tolerant cell line was obtained as described in U.S. Patent No. 5,162,602, issued November 10, 1992, the disclosure of which is incorporated in Examples I-IIL. Briefly, partially differentiated cell cultures are grown and subcultured with continuous exposures to low herbicide levels. Herbicide concentrations are then gradually increased over several subculture intervals. Maize cells or tissues growing in the presence of normally toxic herbicide levels are repeatedly subcultured in the presence of the herbicide and characterized. Stability of the herbicide tolerance trait of the cultured cells may be evaluated by growing the selected cell lines in the absence of herbicides for various periods of time and then analyzing growth after exposing the tissue to herbicide.
  • Maize cell lines which are tolerant by virtue of having an altered acetyl CoA carboxylase enzyme can be selected by identifying cell lines having enzyme activity in the presence of normally toxic levels of sethoxydim or haloxyfop. The tolerant maize cells can be further evaluated for whether acetyl CoA carboxylase is altered to a less sensitive form or increased in its level of expression. Maize cell lines with a acetyl CoA carboxylase less sensitive to herbicide inhibition can be used to isolate a functional mutant gene of a plant acetyl CoA carboxylase.
  • a DNA library from a maize cell line tolerant to herbicides can be generated and DNA fragments encoding all or a portion of an acetyl CoA carboxylase gene can be identified by hybridization to a cDNA probe encoding a portion of the maize ACCase gene.
  • a complete copy of the altered gene can be obtained either by cloning and ligation or by PCR synthesis using appropriate primers.
  • the isolation of the altered gene coding for acetyl CoA carboxylase can be confirmed in transformed plant cells by determining whether the acetyl CoA carboxylase being expressed retains enzyme activity when exposed to normally toxic levels of herbicides. 2.
  • a plant acetyl CoA carboxylase gene or functional mutant thereof or an antisense DNA sequence is obtained and amplified, it is combined with a promoter functional in a plant cell to form an expression cassette.
  • Most genes have regions of DNA sequence that are known as promoters and which regulate gene expression. Promoter regions are typically found in the flanking DNA sequence upstream from the coding sequence in both procaryotic and eukaryotic cells.
  • a promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression.
  • Some isolated promoter sequences can provide for gene expression of heterologous genes, that is a gene different from the native or homologous gene.
  • Promoter sequences are also known to be strong or weak or inducible.
  • a strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression.
  • An inducible promoter is a promoter that provides for turning on and off of gene expression in response to an exogenously added agent or to an environmental or developmental stimulus. Promoters can also provide for tissue specific or developmental regulation.
  • An isolated promoter sequence that is a strong promoter for heterologous genes is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
  • the promoter in an expression cassette of the invention can provide for overexpression of acetyl CoA of a plant acetyl CoA carboxylase gene or functional mutant thereof.
  • Overexpression of the gene is that amount of gene expression that results in an increase in tolerance of the plant cells to an herbicide or that results in an increase in the total oil content of the cells.
  • Overexpression of an acetyl CoA carboxylase gene is preferably about a 2- to 20-fold increase in expression of an acetyl CoA carboxylase over the expression level of the native acetyl CoA carboxylase.
  • the promoter can also be inducible so that gene expression can be turned on or off by an exogenously added agent.
  • a bacterial promoter such as the P toc promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells. It may also be preferable to combine the gene with a promoter that provides tissue specific expression or developmentally regulated gene expression in plants.
  • Specific promoters functional in plant cells include the 35S cauliflower mosaic virus promoter, nopaline synthase (NOS) promoter and the like. Currently, a preferred promoter for expression in monocots is the 35S cauliflower mosaic virus promoter.
  • acetyl CoA carboxylase gene can be combined with the promoter by standard methods as described in Sambrook cited supra. Briefly, a plasmid containing a promoter such as the 35S cauliflower mosaic virus promoter can be constructed as described in Jefferson, Plant Molecular Biology Reporter. 5_,387 (1987) or obtained from Clontech Lab in Palo Alto, CA (e.g. pBI121 or pBI221). Typically these plasmids are constructed to provide for multiple cloning sites having specificity for different restriction enzymes downstream from the promoter.
  • a gene for plant acetyl CoA carboxylase can be subcloned downstream from the promoter using restriction enzymes to ensure that the gene is inserted in proper orientation with respect to the promoter so that the gene can be expressed.
  • a maize acetyl CoA carboxylase is operably linked to a 35 S Came promoter in a plasmid such as pBI121 or pBI221.
  • the expression cassette can also optionally contain other DNA sequences.
  • the expression cassette can further be comprised of a chloroplast transit peptide sequence operably linked between a promoter and a plant acetyl CoA carboxylase gene. If the expression cassette is to be introduced into a plant cell, the expression cassette can also contain plant transcriptional termination and polyadenylation signals and translational signals linked to the 3 ' terminus of a plant acetyl CoA carboxylase gene.
  • the expression cassette can also optionally be further comprised of a plasmid.
  • an expression cassette of the invention can be combined with an exogenous DNA sequence coding for a chloroplast transit peptide, if necessary.
  • An exogenous chloroplast transit peptide is one which is not encoded within the plant acetyl CoA carboxylase gene.
  • a chloroplast transit peptide is typically 40 to 70 amino acids in length and functions post-translationally to direct the protein to the chloroplast. The transit peptide is cleaved either during or just after import into the chloroplast to yield the mature protein.
  • the complete copy of a gene encoding a plant acetyl CoA carboxylase may contain a chloroplast transit peptide sequence. In that case, it may not be necessary to combine an exogenously obtained chloroplast transit peptide sequence into the expression cassette.
  • Exogenous chloroplast transit peptide encoding sequences can be obtained from a variety of plant nuclear genes, so long as the products of the genes are expressed as preproteins comprising an amino terminal transit peptide and transported into chloroplast.
  • Examples of plant gene products known to include such transit peptide sequences are the small subunit of ribulose biphosphate carboxylase, ferredoxin, chlorophyll a/b binding protein, chloroplast ribosomal proteins encoded by nuclear genes, certain heatshock proteins, amino acid biosynthetic enzymes such as acetohydroxy acid synthase, 3- enolpyruvylphosphoshikimate synthase, dihydrodipicolinate synthase, and the like.
  • the DNA fragment coding for the transit peptide may be chemically synthesized either wholly or in part from the known sequences of transit peptides such as those listed above.
  • the DNA fragment coding for the transit peptide should include a translation initiation codon and an amino acid sequence that is recognized by and will function properly in chloroplasts of the host plant. Attention should also be given to the amino acid sequence at the junction between the transit peptide and the plant acetyl CoA carboxylase enzyme where it is cleaved to yield the mature enzyme. Certain conserved amino acid sequences have been identified and may serve as a guideline. Precise fusion of the transit peptide coding sequence with the acetyl CoA carboxylase coding sequence may require manipulation of one or both DNA sequences to introduce, for example, a convenient restriction site. This may be accomplished by methods including site directed mutagenesis, insertion of chemically synthesized oligonucleotide linkers and the like.
  • the chloroplast transit peptide sequence can be appropriately linked to the promoter and a plant acetyl CoA carboxylase gene in an expression cassette using standard methods. Briefly, a plasmid containing a promoter functional in plant cells and having multiple cloning sites downstream can be constructed as described in Jefferson cited supra. The chloroplast transit peptide sequence can be inserted downstream from the promoter using restriction enzymes. A plant acetyl CoA carboxylase gene can then be inserted immediately downstream from and in frame with the 3 ' terminus of the chloroplast transit peptide sequence so that the chloroplast transit peptide is linked to the amino terminus of the plant acetyl CoA carboxylase. Once formed, the expression cassette can be subcloned into other plasmids or vectors.
  • the expression cassette can also optionally include 3 ' nontranslated plant regulatory DNA sequences.
  • the 3' nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequence.
  • Specific examples of 3 ' nontranslated regulatory DNA sequences functional in plant cells include about 500 base pairs of the 3' flanking DNA sequence of the pea ribulose biphosphate carboxylase small subunit E9 gene, the 3 ' flanking DNA sequence of the octopine synthase gene, and the 3' flanking DNA sequence of the nopaline synthase gene.
  • 3' nontranslated regulatory sequences can be obtained as described in An, Methods in Enzymology, 153:292 (1987) or are already present in plasmids available from commercial sources such as Clontech, Palo Alto, CA.
  • the 3 ' nontranslated regulatory sequences can be operably linked to the 3 ' terminus of a plant acetyl CoA carboxylase gene by standard methods.
  • An expression cassette of the invention can also be further comprised of a plasmid. Plasmid vectors included additional DNA sequences that provide for easy selection, amplification and transformation of the expression cassette in procaryotic and eukaryotic cells.
  • the additional DNA sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette, and sequences that enhance transformation of prokaryotic and eukaryotic cells.
  • the preferred vectors of the invention are plasmid vectors.
  • the especially preferred vector is the pBI121 or pBI221 vector formed as described by Jefferson cited supra.
  • Another vector that is useful for expression in both plant and procaryotic cells is the binary Ti vector PGA582. This binary Ti vector has been previously characterized by An, cited supra., and is available from Dr. An. This binary Ti vector can be replicated in procaryotic bacteria such as E.
  • the Agrobacterium plasmid vectors can be used to transfer the expression cassette to plant cells.
  • the binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the colEl replication of origin and a wide host range replicon.
  • the binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells, but is preferably used to transform plant cells.
  • a method for screening for expression or overexpression of a plant acetyl CoA carboxylase gene is also provided by the invention. Once formed, an expression cassette comprising an acetyl CoA carboxylase gene can be subcloned into a known expression vector.
  • the screening method in the invention includes the steps of introducing an expression vector into a host cell and detecting and/or quantitating expression of a plant acetyl CoA carboxylase gene. This method of screening is useful to identify expression cassettes providing for an overexpression of a plant acetyl CoA carboxylase gene, antisense molecules that effectively inhibit acetyl CoA carboxylase synthesis, and expression of an acetyl CoA carboxylase in the chloroplast of a transformed plant cell.
  • Suitable known expression vectors include plasmids that autonomously replicate in prokaryotic and eukaryotic cells. Specific examples include plasmids such as the pBI121 or pBI221 plasmid constructed as described by Jefferson cited supra, a binary Ti vector such as PG582 as described by An cited supra, PUC119, or PBR322.
  • the preferred expression system is a pBI121 or pBI221 plasmid.
  • An expression cassette of the invention can be subcloned into an expression vector by standard methods.
  • the expression vector can then be introduced into prokaryotic or eukaryotic cells by standard methods including protoplast transformation, silicon fiber-based transformation Agrobacterium mediated transformation, electroporation, microprojectiles, e.g., preferably using gold particles of about 6 to about 10 microns in diameter although diameters that are about 100 to about 1000 times smaller may also be employed, and liposomes.
  • the expression vector can be introduced into plant cells such as tobacco, Brassica, Black Mexican sweet corn, and Arabidopsis cells.
  • the vector can also be introduced into procaryotic cells such as E. coli or Agrobacterium.
  • Transformed cells can be selected typically using a selection marker encoded on the expression vector.
  • Transient expression of a plant acetyl CoA carboxylase gene can be detected and quantitated in the transformed cells. Gene expression can be quantitated by a quantitative Western blot using antibodies specific for the cloned acetyl CoA carboxylase or by detecting an increase in specific activity of the enzyme.
  • the tissue and subcellular location of the cloned acetyl CoA carboxylase can be determined by immunochemical staining methods using antibodies specific for the cloned acetyl CoA carboxylase. Sensitivity of the cloned acetyl CoA carboxylase to herbicides can also be assessed.
  • Expression cassettes providing for overexpression of a plant acetyl CoA carboxylase or acetyl CoA carboxylase tolerant to herbicides can then be used to transform monocot and/or dicot plant tissue cells and to regenerate transformed plants and seeds.
  • the invention provides a method of conferring cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant.
  • the method includes the steps of introducing an expression cassette comprising a gene coding for a plant acetyl CoA carboxylase or a functional mutant thereof operably linked to a promoter into the cells of plant tissue and expressing the gene in an amount effective to render the cells of the plant tissue substantially tolerant to herbicides.
  • An effective amount of gene expression to render the cells of the plant tissue substantially tolerant to the herbicide depends on whether the gene codes for an unaltered acetyl CoA carboxylase gene or a mutant or altered form of the gene that is less sensitive to the herbicides.
  • acetyl CoA carboxylase gene in an effective amount is that amount that provides for a 2- to 50-fold increase in herbicide tolerance and preferably increases the amount of acetyl CoA carboxylase from at least about 2- to 20-fold over that amount of the native enzyme.
  • An altered form of the enzyme can be expressed at levels comparable to that of the native enzyme or less if the altered form of the enzyme has higher specific activity.
  • Acetyl CoA carboxylase substantially tolerant to herbicides is an enzyme that is tolerant of levels of herbicide which normally inhibit a native acetyl CoA carboxylase and preferably can function in concentrations of herbicide of about 2- to 20-fold greater than are toxic to the native enzyme.
  • Herbicide tolerance can be achieved by at least two methods including: 1) by increasing the level of gene expression of a native or unaltered acetyl CoA carboxylase gene; or 2) by introducing an altered gene coding for an acetyl CoA carboxylase that is less sensitive to herbicide inhibition.
  • the level of gene expression can be increased by either combining a plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression such as the 35S Came promoter or by introducing the gene into the cells so that multiple copies of the gene are integrated into the genome of the transformed plant cell.
  • Formation of an expression cassette comprised of a plant acetyl CoA carboxylase gene operably linked to a promoter that can be expressed in an effective amount to confer herbicide tolerance has been described previously.
  • the preferred plant cells for introducing an expression cassette of the invention to achieve herbicide tolerance for the plant cells then are monocot plants.
  • Monocot plants include corn, wheat, barley, sorghum, rice, and others.
  • An expression cassette of the invention can be introduced by methods of transformation, especially effective for monocots including biolistic transformation of Type II embryogenic suspension cells as described by W.J. Gordon-Kamm et al., Plant Cell, 2, 603-618 (1990), M.E. Fromm et al, Bio/Techno1ogy : £, 833-839 (1990) and D.A.
  • Transformed cells can be selected for the presence of a selectable marker gene.
  • Transient expression of a plant acetyl CoA carboxylase gene can be detected in the transgenic embryogenic calli using antibodies specific for the cloned plant acetyl CoA carboxylase.
  • Transformed embryogenic calli can be used to generate transgenic plants that exhibit stable inheritance of either the altered acetyl CoA carboxylase gene or overexpression of the acetyl CoA carboxylase gene.
  • Maize cell lines exhibiting satisfactory levels of tolerance to herbicide are put through a plant regeneration protocol to obtain mature maize plants and seeds expressing the tolerance traits such as described in D ⁇ afluin, cited supra., or An, cited supra.
  • the plant regeneration protocol allows the development of somatic embryos and the subsequent growth of roots and shoots.
  • regenerated plants are exposed to herbicide levels which will normally inhibit shoot and root formation and growth.
  • Mature maize plants are then obtained from maize cell lines that are known to express the trait. If possible, the regenerated plants are self- pollinated. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important inbred lines. Conversely, pollen from plants of these inbred lines is used to pollinate regenerated plants.
  • the genetics of the trait are then characterized by evaluating the segregation of the trait in the first and later generation progeny. Stable inheritance of overexpression of a plant acetyl CoA carboxylase or a functional mutant of a plant acetyl CoA carboxylase conferring herbicide tolerance to the plant is achieved if the plants maintain herbicide tolerance for at least about three to six generations.
  • Seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants. Progenies from these plants become true breeding lines which are evaluated for herbicide tolerance in the field under a range of environmental conditions. Herbicide tolerance must be sufficient to protect the monocot plants at the maximum labeled delivery rate under field conditions which cause herbicides to be most active. Appropriate herbicide concentrations and methods of application are those which are known and have been developed for the cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides in question.
  • an expression cassette comprised of a maize acetyl CoA carboxylase gene isolated from a maize cell line tolerant to sethoxydim and haloxyfop and linked to the 35S Came promoter is introduced into an herbicide sensitive monocot tissue using biolistic transformation. Transformed calli are selected and used to generate transgenic plants. Transformed calli and transgenic plants can be evaluated for tolerance to sethoxydim and haloxyfop and for stable inheritance of the tolerance trait. D. Method for Altering the Oil Content in a Plant
  • the invention also provides a method of altering the oil content in a plant.
  • the method include the steps of introducing an expression cassette comprising a gene coding for plant acetyl CoA carboxylase or functional mutant thereof operably linked to a promoter functional in a plant cell into the cells of plant tissue and expressing the gene in an amount effective to alter the oil content of the plant cell.
  • An alteration in the oil content of a plant cell can include a change in the total oil content over that normally present in that type of plant cell, or a change in the type of oil from that normally present in the plant cell.
  • Expression of the gene in an amount effective to alter the oil content of the gene depends on whether the gene codes for an unaltered acetyl CoA carboxylase or a mutant or altered form of the gene.
  • Expression of an unaltered plant acetyl CoA carboxylase gene in an effective amount is that amount that may provide a change in the oil content of the cell from about 1.2- to 20-fold over that normally present in that plant cell, and preferably increases the amount of acetyl CoA carboxylase about 2- to 20-fold over that amount of the enzyme normally present in that plant cell.
  • An altered form of the enzyme can be expressed at levels comparable to that of the native enzyme or less if the altered form of the enzyme has higher specific activity.
  • An alteration in oil content of the plant cells according to the method of the invention can be achieved in at least two ways including:
  • the level of gene expression of an unaltered plant acetyl CoA carboxylase gene can be increased by either combining an unaltered plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression, such as the 35S cauliflower mosaic virus or by introducing the expression cassette and/or selecting for plant cells having multiple copies of a plant acetyl CoA carboxylase gene integrated into the genome.
  • a decrease in expression of an unaltered acetyl CoA carboxylase can be achieved by transformation with an ACCase antisense gene containing an expression cassette.
  • acetyl CoA carboxylase gene codes for an enzyme that has an increase in specific activity, it may lead to an increase in total oil content of a plant cell even if the level of gene expression is comparable to that of the native enzyme.
  • acetyl CoA carboxylase gene codes for an enzyme having a decrease in specific activity, it may lead to a decrease in the total oil content of the plant cell compared to that normally present.
  • An expression cassette as described above can be introduced into either monocots or dicots.
  • the expression cassette is introduced into dicot plants such as soybean, canola, and sunflower.
  • An expression cassette can be introduced by standard methods including protoplast transformation, Agrobacterium-mediated transformation, microprojectiles, electroporation, and the like.
  • Transformed cells or tissues can be selected for the presence of a selectable marker gene.
  • Transient expression of a plant acetyl CoA carboxylase gene can be detected in transformed cells or tissues by immunoreactivity with antibodies specific for the cloned acetyl CoA carboxylase.
  • Overexpression of a plant acetyl CoA carboxylase can be detected by quantitative Western blots.
  • a change in specific activity of the enzyme can be detected by measuring enzyme activity in the transformed cells.
  • a change in total oil content can also be examined by standard methods, as described in Clark & Snyder, .TAGS, 66:1316 (1989).
  • Transgenic plants and seeds can be generated from transformed cells and tissues showing a change in oil content or in the amount or specific activity of a plant acetyl CoA carboxylase using standard methods. It is especially preferred that the oil content of the leaves, seeds, or fruits is increased.
  • a maize acetyl CoA carboxylase gene is combined with a 35S cauliflower mosaic virus promoter in a vector such as pBI121 or pBI221 and introduced into soybean cells using the microprojectile method.
  • Transformed soybean cells showing an increase in expression of acetyl CoA carboxylase of at least about 2-fold or at least a 1.2-fold increase in oil content are selected.
  • Transformed soybean cells exhibiting overexpression of acetyl CoA carboxylase or showing an increase in total oil content are used to generate transgenic plants and seeds.
  • the invention also provides a method of producing plant acetyl CoA carboxylase in a host cell.
  • the method includes the steps of introducing an expression cassette comprised of a gene encoding a plant acetyl CoA carboxylase or functional mutant thereof into a host cell and expressing the gene in an amount sufficient to allow for crystallization of the plant acetyl CoA carboxylase.
  • An amount sufficient to allow for crystallization of a plant acetyl CoA carboxylase is about 20- to 100-fold increase over the amount of plant acetyl CoA carboxylase that can normally be purified from plant cells, preferably about 2 to 10 mg protein.
  • Crystallized plant acetyl CoA carboxylase can be used to identify other herbicides that can bind to and inhibit acetyl CoA carboxylase function.
  • the availability of large amounts of purified enzyme provides for screening of the efficacy of such herbicides.
  • An expression cassette can include a promoter that is functional in either a eukaryotic or prokaryotic cell.
  • the expression cassette can be introduced into a prokaryotic cell such as E. coli, or a eukaryotic cell such as a plant or yeast.
  • the preferred cell is a prokaryotic cell used routinely in producing recombinant proteins such as E. co i.
  • the expression cassette can be introduced and transformed cells selected by standard methods.
  • the plant acetyl CoA carboxylase gene can be expressed in an prokaryotic cell until sufficient amount of the enzyme is produced so that it can be crystallized.
  • Plant acetyl CoA carboxylase can be isolated from bacterial cells using standard methods, including those described in Example V. The purified acetyl CoA carboxylase can then be crystallized and characterized by standard methods.
  • Buffers and cofactors were purchased from Sigma Chemical Company (St. Louis, Missouri); [2- 14 C] acetate was purchased from Research Products International; [2- 14 C]pyruvate and [ 14 C]NaHCO 3 were purchased from New England Nuclear; and [2- 1 C]malonyl coenzyme A was purchased from Amersham. Sethoxydim was a gift from BASF (Parsippany, New Jersey), and haloxyfop was provided by Dow Chemical USA (Midland, Michigan).
  • Corn (Z. mays L., 'B37 x Oh43') seeds were germinated in darkness for 96 hours in vermiculite in an incubation chamber maintained at 30°C, 80%) RH. Seedlings were then transferred to a growth chamber with a 16 hour light (25°C) and an 8 hour dark (20°C) cycle, 90% relative humidity (RH). After greening 48 hours, seedlings were returned to the dark incubation chamber for 12 hours to deplete chloroplast starch reserves. Seedlings were harvested 6 days after planting. Pea (P.
  • sativum L., 'PI 990 l-C seedlings were grown in vermiculite in a growth chamber with a 16 hour light (21°C) and 8 hour dark (16°C) cycle, 80% RH. Peas were harvested 10 to 13 days after planting.
  • Black Mexican Sweet (BMS) corn suspension cultures were maintained in a supplemented Murashige-Skoog (MS) medium (C. E. Green, Hort. Sci., 12, 7-10 (1977)), and subcultured weekly by 20-fold dilution of the suspension culture into fresh medium.
  • Chloroplasts from corn and pea seedlings were isolated at 4°C (K. Cline et al., J. Biol. Chem., 26Q, 3691-3696 (1985)). Seedlings (50 g of shoots) were homogenized in 200 ml buffer A (50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, 0.1% w/v BSA, 1 mM MgCl 2 , 1 mM MnCl 2 , 2 mM EDTA, 5 mM isoascorbate, 1.3 mM glutathione) in an omnimixer (five, 3 -second bursts at full speed).
  • buffer A 50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, 0.1% w/v BSA, 1 mM MgCl 2 , 1 mM MnCl 2 , 2 mM EDTA, 5 m
  • the homogenate was filtered through six layers of cheesecloth and two layers of miracloth, and then centrifuged at 3000 g for 3 minutes with hand- braking.
  • the pellet was gently resuspended in buffer A and layered onto a preformed linear Percoll gradient (50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, 1.9 mM isoascorbate, 1.08 mM glutathione, 0.1% w/v BSA, 50% Percoll) which was centrifuged at 3000 g for 20 minutes in a Sorvall HB-4 rotor.
  • a preformed linear Percoll gradient 50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, 1.9 mM isoascorbate, 1.08 mM glutathione, 0.1% w/v BSA, 50% Percoll
  • [ 14 C]acetate and [ 14 C]pyruvate were used as precursors to measure fatty acid biosynthesis in isolated chloroplasts (B. Liedvogel et al., Planla, 162, 481-489 (1986)).
  • [ 14 C]acetate incorporation was assayed in a 0.5 ml-volume containing: 50 mM HEPES-NaOH (pH 7.5), 330 mM sorbitol, 5 mM KH 2 PO 4 , 10 mM NaHCO 3 , 1 mM MgCl 2 , 1 mM ATP, 0.1 mM CoA, 0.15 mM [ 14 C]acetate (3.33 mCi/mmol), and chloroplasts (20 to 50 ⁇ g chlorophyll).
  • [ 14 C]pyruvate incorporation into fatty acids was assayed in the same medium except that it included 2 mM TPP, 1 mM NAD + , 0.15 mM [ 14 C]-pyruvate (1.33 mCi/mmol), but no acetate.
  • Assay suspensions were illuminated with 1400 ⁇ E/m 2 -second PAR at 25°C.
  • Assays were initiated by the addition of the labeled substrate and stopped by the addition of 0.5 ml of 40% KOH.
  • each treatment was saponified at 90°C for 30 minutes in capped vials (P. B. Hoj et al., Carl herg Res.
  • Chlorophyll D. I. Arnon, Plant Physiol.. 24, 1-15 (1949)
  • protein P. K. Smith et al., Anal. Bioche ., 150, 76-85 (1985)
  • Maize chloroplasts isolated as described above, were suspended in buffer C (0.1 M Tricine-KOH, pH 8.0; 0.3 M glycerol, and 1 mM DTT) and homogenized in a glass tissue homogenizer. The disrupted chloroplast fraction was centrifuged at 16,000 g for 15 minutes. The supernatant was desalted on a Sephadex G-25 column (1.5 x 5 cm equilibrated with 0.1 M Tricine-KOH, pH 8.0; and 0.3 M glycerol) and assayed directly. ACCase activity (B. J. Nikolau et al., Arch. Biochem. Biophys..).
  • Results are expressed as mean of two experiments ⁇ standard error.
  • a selection protocol to identify and isolate herbicide-tolerant maize cells was developed to minimize the adverse effects of high herbicide concentrations on somatic embryo development and plant regeneration capacity.
  • the procedure involved exposing tissue to gradually increasing concentrations of herbicide beginning with a sethoxydim concentration representing l/20th of lethal dose and doubling the herbicide concentration at approximately two-week intervals until the lethal dose (10 ⁇ M sethoxydim) was reached. In this way, the herbicide was allowed to take effect slowly with continuous selection pressure, thus permitting herbicide-tolerant cells to accumulate over time while not affecting the potential for plant regeneration.
  • A. Selection of a Sethoxydim-Tolerant Cell Line Many selections were carried out utilizing the selection protocol described in the preceding paragraph. The selection of one such sethoxydim- tolerant cell line that was identified and characterized is described below in detail.
  • S-2 Tolerant cell line 2167-9/2160-154 S-2
  • S-2 was characterized to evaluate: (1) the magnitude of sethoxydim tolerance; (2) cross-tolerance of haloxyfop; and (3) the biochemical basis for the tolerance.
  • ACCase activity of control tissue was 50% inhibited either by 1.5 ⁇ M sethoxydim, or by 0.25 ⁇ M haloxyfop.
  • ACCase activity of S-2 tissue was inhibited 50% either by 70 ⁇ M sethoxydim, or by 1.8 ⁇ M haloxyfop, indicating at least 40-fold and 7-fold decreases in herbicide sensitivity on concentration basis, respectively.
  • Plants surviving sethoxydim treatments of up to 0.44 kg/ha were transplanted to the genetics plot on the University of Minnesota campus, St. Paul, Minnesota. Additional S-2 plants were transplanted to the field that had not been sprayed. Sixty-five 2167-9/2160-154 control plants and ninety-five S-2 plants were grown to maturity in the field. Plants were either self-pollinated or cross-pollinated to inbred maize lines A188, A619, A641, A661, A665, B37, B73, R806, and W153R. Control seed were produced by selfmg 2167-9/2160- 154 regenerated plants, or by crossing them with the inbreds listed above. D.
  • Seeds obtained by the crossing procedure described above were viable and germinated normally. Seeds from thirty S-2 selfed plants and fifteen 2167-9/2160-154 control plants were planted in 25 x 50 cm trays of soil (28 seeds from each plant in one tray) and grown in the greenhouse. Seedlings at the 3-4 leaf stage were treated with 0.1, 0.44, and 1.1 kg/ha sethoxydim and evaluated for visual herbicide damage and shoot height. Based on visual rating of herbicide damage two weeks after treatment, selfed progeny of S-2 plants segregated approximately 1:2:1 for healthy, uninjured plants: to plants showing partial injury: to dead plants, respectively, at 0.44 and 1.1 kg/ha sethoxydim treatments.
  • Progeny of S-2 plants crossed to inbred lines and exhibiting sethoxydim tolerance may be recurrently backcrossed to the same inbreds. Progeny of each cross may be screened for sethoxydim-tolerance, and tolerant plants grown to maturity and again crossed to the recurrent parent. After six or seven cycles of backcrossing, sethoxydim-tolerant plants may be selfed and progeny screened for tolerance to produce homozygous sethoxydim tolerant maize inbreds.
  • Maize cell line 2167-9/2160-154 S-1 was selected from maize cell culture using the protocol described in detail above for the selection of Line 2167-9/2160-154 S-2. Approximately 70 plants were regenerated from Line 2167-9/2160-154 S-1, and either self-pollinated or cross-pollinated to the inbred maize lines A188, A619, A641, A661, A665, B37, B73, R806, and W153R.
  • Line 2167-9/2160-154 H-1 was selected from maize cell culture using a similar protocol described in detail above except maize callus tissue was selected using the herbicide haloxyfop. Maize callus tissue was initially plated on 0.01 ⁇ M haloxyfop. At two-week intervals, surviving tissue was subcultured onto 0.05, 0.10 and 0.20 ⁇ M haloxyfop. Approximately 50 plants were regenerated from Line 2167-9/2160-154 H-1, and were self-pollinated. C. Selection of Maize Cell Line 21 7-9/21 0-154 H-2
  • Line 2167-9/2160-154 H-2 was selected from maize cell culture using a similar protocol described in detail for line 2167-9/2160-154 H-1. No plants have been successfully regenerated from this line.
  • D Characterization of Lines 21 7-9/21 0-154 S-1. H-1 and H-2 The tolerant callus cultures were characterized to determine the magnitude of sethoxydim and haloxyfop tolerance. Callus tissue from these lines was evaluated in experiments as described above in the characterization of line 2167-9/2160-154 S-2. Table II summarizes the results of these studies.
  • Line 2167-9/2160-154 S-1 and Line 2167-9/2160-154 H-2 showed a four-fold increase in haloxyfop tolerance, while Line 2167-9/2160- 154 H- 1 exhibited approximately a 60-fold increase in haloxyfop tolerance.
  • Neither haloxyfop selected line showed a significant degree of sethoxydim tolerance, while the sethoxydim selected line S-1 exhibited approximately a 100-fold increase in sethoxydim tolerance.
  • the numbers represent the fold increase in herbicide concentration that results in a 50% reduction in growth of the selected cell lines compared to the unselected control cell line 2167-9/2160-154.
  • Acetyl Coenzyme A Carboxylase (ACCase) was extracted from cell lines S-1, H-1 and H-2 and assayed as described in detail for maize cell line
  • ACCase from line S-1 was more tolerant of both sethoxydim and haloxyfop, while the ACCase from line H-1 was more tolerant of haloxyfop, but not of sethoxydim.
  • the ACCase from line H-2 showed no difference from the unselected parent line 2167-9/2160-154 in sensitivity to either herbicide.
  • cell line H-2 exhibited approximately a five-fold higher level of ACCase activity as compared to the unselected parent line 2167-9/2160-
  • the numbers represent the fold increase in herbicide concentration that inhibits ACCase activity of the selected cell lines by 50% compared to the unselected parent cell line 2167-9/2160-154.
  • the acetyl CoA carboxylase enzyme was isolated and purified from plant tissues and characterized. The purified enzyme was used to generate antibody reagents useful in identifying cDNA clones encoding the gene or portions of the gene for ACCase.
  • ACCase was extracted from frozen shoots of 7-d-old maize (Zea Mays L. inbred A619 or B73) seedlings grown in a growth chamber (24°C, 90% RH, 16-h daylength at 210 ⁇ E m "2 s "1 ). The outermost leaf and blade were remo ved and the remainder of the shoot was frozen in liquid N 2 . Embryos and endosperm tissue from developing kernels were harvested from field-grown ears at 36 to 40 days after pollination (DAP). Black Mexican Sweet corn (BMS) maize suspension cells were obtained from cultures as previously described (W.B. Parker et al., Plant Physiol .. 91, 1220-1225 (1990)). Tissues were stored in liquid N 2 until used.
  • BMS Black Mexican Sweet corn
  • protease inhibitors (leupeptin, 2 ⁇ g mL; pepstatin A, 100 ⁇ g mL " '; benzamidine, 1 mM; ⁇ -amino-n-caproic acid, 5 mM; and soybean trypsin inhibitor, 10 ⁇ g mL " ') were included.
  • Filtered homogenates were centrifuged 20 minutes at 30,000g.
  • a portion of the crude supernatant fraction was immediately boiled 5 minutes in 1 volume of SDS sample buffer (W.B. Parker et al., Plant Physiol., 91, 1220-1225 (1990)) for SDS-PAGE analysis; the remainder was desalted on a 10-mL Sephadex G-25 column into extraction buffer minus PMSF.
  • ACCase was purified from the crude extract supernatant in four steps. This fraction was brought to 30% saturation with solid (NH 4 ) 2 SO 4 , stirred 15 minutes, and centrifuged 20 minute at 20,000g. The supernatant was then brought to 40% saturation with (NH 4 ) 2 SO 4 solution, stirred 30 minutes, and centrifuged.
  • ACCase was also analyzed from mesophyll chloroplasts and bundle sheath strands.
  • Mesophyll chloroplasts from homogenates of 7- to 8-day- old seedlings that were kept in the dark 24 hours prior to harvesting were isolated on a linear Percoll gradient according to J.D. Burton et al., Pesticide Biochemistry and Physiology, 34, 76-85 (1989), except that buffers contained 0.6 M sorbitol and centrifugation g-forces were reduced by 25%.
  • Intact chloroplasts were taken from the discrete lower green band present after Percoll gradient centrifugation (G. Morioux et al., Plant Physiol., 62, 470-473 (1981)).
  • Pelleted chloroplasts were lysed by resuspending them in ACCase extraction buffer plus PMSF and 0.01% (v/v) Triton X-100. Bundle sheath strands were obtained from the original leaf homogenate material retained on a 70- ⁇ m filter after re-homogenizing the retentate five times in a total of 2 L buffer. Triton X-treated, desalted leaf, mesophyll chloroplast, and bundle sheath strand extracts were assayed for activities of Rubisco (G. Zhu et al., Plant Physiol., 97, 1348- 1353 (1991)), NADP-dependent malate dehydrogenase (M.D.
  • Mesophyll chloroplast preparations also contained ⁇ 2.6% as much catalase, fumarase, and phosphoenolpyruvate carboxylase activities (mg " ' chlorophyll) as did whole-leaf extracts, indicating they were relatively free of peroxisomal, mitochondrial, or cytoplasmic components.
  • Avidin (10 U mL " ') was included in some assays.
  • Herbicide inhibition assays contained 1% (v/v) ethanol plus or minus 1 ⁇ M haloxyfop (2- [4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy] propanoic acid, Dow Chemical Co. analytical grade racemic mixture) or 10 ⁇ M sethoxydim (2- [1 [(ethoxylmino)butyl]-5-[2-(ethylthio)-propyl]-3-hydroxy-2-cyclohexene -1 - one, Li salt, BASF Corp. technical grade). Data are means plus standard error of three assays.
  • Protein concentrations were determined in duplicate with the Bio- Rad Coomassie blue dye-binding assay as described by the manufacturer, using BSA as the standard. Centrifuged crude extracts and proteins in purification fractions and immunoprecipitation supernatants were separated by SDS-PAGE in 6 or 7.5%o gels as previously described (W.B. Parker et al., Plant Physiol., 9_2, 1220-1225 (1990)). Purification fractions were precipitated in 10% (v/v) TCA, washed with 80% (v/v) acetone, and air-dried 10 minutes prior to electrophoresis. Proteins in gels were stained with silver (J. Heukeshoven et al., Electrophoresis, 6, 103-112 (1985)). High molecular weight protein standards for SDS-PAGE (Pharmacia) were used to estimate polypeptide masses.
  • the four-step purification procedure shown in Table LV typically yielded 30 to 190 ⁇ g of highly purified ACCase from 50 grams (fresh weight) of maize inbred A619 or B73 seedling leaves.
  • ACCase activity in the crude supernatant fraction precipitated between 30 and 40% saturation with (NH 4 ) 2 SO 4 , which appeared to increase total ACCase activity approximately 38%.
  • Crude extract components might have depressed the reaction rate shown in Table IV because the assay mixture contained 50% enzyme (v/v).
  • enzyme velocity was proportional to enzyme concentration in assay mixtures containing up to 25% (v/v) crude extract, but 50% (v/v) mixtures were not tested.
  • ACCase activity eluted from the Sephacryl S-300 gel filtration column slightly after the green void peak. Approximately 56% of the S-300 fraction ACCase activity was recovered from the Blue Sepharose column, primarily in the initial ATP-containing fractions (12.5 mL). Both 10 mM NaHCO 3 and 4.25 mM MgCl 2 (1- and 0.85-fold standard assay concentrations, respectively) were included in the Blue Sepharose buffer because they improved the total and specific ACCase activity remaining after batch absorption to Blue Sepharose beads, elution with ATP, and desalting into extraction buffer minus PMSF. Neither NaHCO 3 nor MgCl 2 improved enzyme stability of crude extracts.
  • ACCase II Mono-Q anion-exchange chromatography resulted in separation of two ACCase activity peaks which eluted at approximately 210 mM (designated ACCase II) and 250 mM KCl (designated ACCase I), as previously observed for a hybrid maize variety (J.L. Howard et al., FEBS Lett., 261, 261-264 (1990)).
  • ACCase I comprised about 85% of the total activity recovered from the column (29% of the original crude extract activity) and had high specific activity (Table IV).
  • the specific activity of ACCase II was less than 30% that of ACCase I. Both activities were inhibited >90% by avidin, as previously reported (J.L. Howard et al., FEBS Lett., 261, 261-264 (1990)).
  • the mass of native ACCase I was estimated to be approximately 490 kD by gel filtration on Superose 6.
  • Step Protein Activity Specific Fold Activity Activity Purification Yield mg units b units/mg %
  • Data are from one purification experiment starting with 50 g fresh weight of tissue and are representative of data obtained for eight purifications.
  • Antibodies are sensitive reagents that allow for the identification of gene products from cDNA and other cloned genes. Antibodies to purified ACCase were prepared and used to screen for cDNA clones encoding all or a portion of a gene for ACCase.
  • Antiserum to maize ACCase was obtained by immunizing a female New Zealand White rabbit (Egli et al., Plant Physiol ., 101 , 499 ( 1993)). An intramuscular injection of 100 ⁇ g of Mono-Q-purified, SDS-denatured ACCase I in Freund's complete adjuvant was followed by subcutaneous injections of 20 to 100 ⁇ g of gel-purified ACCase I polypeptide in acrylamide plus incomplete adjuvant every 4 to 6 weeks, for a total of six injections. Serum was stored at -20°C in 0.02% (w/v) NaN 3 .
  • ACCase and biotinylated proteins were detected with immune serum (1/10,000) plus goat anti-rabbit IgG-alkaline phosphatase conjugate or with avidin-alkaline phosphatase (W.B. Parker et al., Plant Physiol .. 91, 1220-1225 (1990)). Blots were repeated at least three times. For immunoprecipitations, equal ACCase activities (0.58 nmol min " ') in crude extracts were desalted into S-300 buffer containing 0.1 M KCl and incubated 1 hour at 25 °C with 16 ⁇ L buffer or with 16 ⁇ L serum consisting of 0 to 100% ACCase antiserum in preimmune serum.
  • Immune complexes were incubated 1 hour at 25 °C with a 2-fold (IgG binding) excess of Protein A-agarose and then micro fuged 5 minutes to obtain immunoprecipitation supernatant fractions. ACCase activity of supernatants was expressed as a percent of the 100% preimmune serum control. Data are means plus SE of three replicate assays for each of two sets of extracts.
  • Antiserum to ACCase I strongly recognized the ACCase I polypeptide in crude extracts and showed little or no recognition of ACCase II polypeptides. No bands were recognized by preimmune serum. Assuming that avidin binds similarly to ACCase I and II polypeptides, it appears that the amount of ACCase II on the Western blot was slightly less than the amount of ACCase I. However, the relative staining with antibody compared to avidin indicated that the antibody had significantly less affinity for ACCase II than ACCase I.
  • ACCase I biotinylated polypeptide of approximately 227 kD (ACCase I) that was strongly recognized by ACCase antiserum or avidin. Similar 227 kD band densities were observed when gel lanes were probed with either avidin or ACCase antiserum.
  • the 219 kD ACCase II polypeptide was readily detected in leaves only by avidin binding, but was in low abundance or not detected in extracts from other tissues.
  • Non-biotinylated proteins of 66 kD (faint) and 55 kD were also recognized by ACCase antiserum.
  • the 55 kD polypeptide was only found in leaves; it was also present in both ACCase I and II Mono Q fractions (Fig. 5) and was identified as the Rubisco large subunit based on its comigration with protein immunoprecipitated by spinach Rubisco antiserum.
  • ACCase I and II utilized propionyl-Co-A 40 to 50% as rapidly as acetyl-CoA at 50 to 500 ⁇ M substrate even though they contained no biotinylated polypeptides (Figure 5) the size of known propionyl CoA carboxylases (70 to 75 kD; see E.S. Wurtele et al., Archives of Biochemistry and Biophysics, 22&, 179-186 (1990)).
  • ACCase I and II differed significantly in their inhibition by either haloxyfop or sethoxydim (Fig. 7).
  • Acetyl-CoA or propionyl-CoA-dependent H 14 CO 3 incorporation by ACCase I was strongly inhibited (65 to 80%) by 1 ⁇ M haloxyfop or 10 ⁇ M sethoxydim, while ACCase II activity was inhibited less than 50%) for all herbicide/substrate combinations examined.
  • C. Cloning and Identification of Maize cDNA Clones Encoding ACCase Maize cDNA clones encoding a portion of the ACCase game were identified by screening a DNA library generated from maize.
  • the cDNA clones were used to identify the sequence of the ACCase gene and to identify the genomic DNA fragments encoding the gene or genes for ACCase.
  • a ⁇ gtl 1 cDNA library from maize inbred A188 seedlings was prepared by standard method for oligo-dT priming, as described for pea cDNA. (Gantt and Key, Eur. J. Biochem., 166:119-125 (1987).
  • Plaque lifts of the maize cDNA library were screened with maize ACCase antiserum (Egli et al, Plant Physiol., 101 , 499 (1993)) to identify plaques expressing ACCase- like proteins, as described by Sambrook et al., cited supra. (1989).
  • the initial screen of 800,000 plaques yielded 120 positives. Rescreening and plaque purification reduced the number of positives to 14. All 14 clones bound ACCase antibodies that, when eluted from plaque lifts (J. Hammarback et al., J. Biol.
  • Clone #15-14 contained three EcoRI fragments of 2.0, 1.2 and 0.23 kb shown in Figure 8. Southern blots showed that the 1.2 and 2.0-kb fragments of clone #15-14 each hybridized to different fragments in the other five clones, with the exception of clone #4-4 which only contained a 1.2-kb fragment.
  • the six maize cDNA clones contained EcoRI fragments that hybridized to a large transcript (ca. 7.8 kb) on Northern blots of total RNA from maize leaves, embryos and endosperm (Fig.9). BMS cell culture RNA also contained a 7.8 kb transcript, but the hybridization signal is not evident on this exposure (Fig. 9). The relative abundance of the 7.8-kb transcript in embryos was higher than the other sources which is consistent with their ACCase activity.
  • the three EcoRI fragments were subcloned from cDNA clone #15-14 into BlueScript vector and sequenced by the dideoxy chain termination method (Sequenase 2.0 USB) initially using T3 and T7 primers and then oligonucleotide primers based on insert sequence.
  • a clone #16-6 was also sequenced in a similar manner.
  • Clone #16-6 included three EcoRI fragments of 3.1 kb, 1.2 kb, and 0.23 kb and had additional sequence located upstream from that of clone #15-14. After comparing the sequence and determining that the sequence was the same, the additional 1.2 kb sequence at the 5 ' end was sequenced.
  • Clone #18-5 was sequenced in a similar manner. Clone #18-5 included 3.9 kb, 1.2 kb, and 0.23 kb EcoRI fragments and contains an additional 1.9 kb 5' sequence upstream from clone #15-14. Subclone #18-51 (3.9 kb EcoRI fragment) has been deposited with the American Type Culture Collection and given Accession No. 69236.
  • GenBank, PLR-29, and Swiss-Prot 19 data banks have been searched for amino acid homology with the corresponding amino acid sequences of the three subclones of clone #18-5.
  • Peptide sequences corresponding to the maize cDNA subclones had higher similarity to chicken, rat, and yeast ACCases than to any other peptide sequence in the data banks.
  • Figure 8 illustrates the relative organization of the 3.9, 1.2 and 0.23-kb EcoRI fragments of clone #18-5 and their co-linearity and extent of amino acid identity with chicken ACCase cDNA sequence.
  • the biotin binding site is encoded approximately 30% in from the 5' (N- terminus) end of rat, chicken and yeast ACCase genes. These functional domains are useful in mapping and further identifying other cDNA and/or genomic fragments encoding ACCase genes.
  • the cDNA clones encoding portions of the acetyl CoA carboxylase genes are useful to identify the sequence of the gene or genes and are useful as probes to locate the genomic copies of the gene or genes. Because the ACCase antibodies used to screen the ⁇ gtl 1 library recognize both the 219 and 227 kD ACCase polypeptides, it has not been determined which polypeptide is encoded by these less than full length clones. It is likely that the majority of the clones encode the 227 kD polypeptide since that polypeptide is more abundant in the leaf tissue source of the DNA library and the antibodies have a higher affinity for the 227 kD ACCase polypeptide.
  • the maize genome has been analyzed to identify copy number and location of the genomic copies of ACCase gene or genes.
  • Four distinct types of maize ACCase genomic clones have been identified, termed Al, A2, Bl and B2 (see below).
  • a maize B73 genomic library (Clontech, Palo Alto, CA) was screened with the 2 kb subclone from #15-14 and several clones of about 15 kb were identified as having homology to the ACCase cDNA. Restriction mapping and partial sequence analysis revealed two types of genomic clones (Type A and Type B) that differed in restriction sites and in their position relative to the ACCase partial cDNA sequence as shown in Figure 8.
  • the 2.5 kb EcoRI-Sall fragment (#16) from the Type A genomic clone and the 3.0 kb EcoRI-EcoRI fragment (#34) from the Type B genomic clone were shown to hybridize to the 3.9 kb probe from #18-5 and were subcloned into the Bluescript vector and sequenced.
  • Approximately 1.5 kb of DNA sequence from the genomic Type A 2.5 kb fragment were 100% identical to coding sequence from the 3.9 kb cDNA subclone #18-51 described in Example V; the remaining sequence exhibited no identity with the cDNA clone and presumably represents a noncoding intron sequence.
  • a 350 nucleotide sequence derived from the genomic Type B 3.0 kb fragment was about 95% identical to the cDNA clone indicating that its coding sequence differs from that of genomic Type A. These results also indicate that the maize genome encodes at least two different genes encoding a polypeptide having acetyl CoA carboxylase activity. To identify and clone the remainder of the gene representing the amino-terminus of maize ACCase, additional regions from the Type A genomic clone have been subcloned and partly sequenced. The 3.5 kb Hindlll-Hindlll fragment (#28) has been sequenced for about 400 nucleotides from each end.
  • the 3' end of #28 shows significant homology to the amino acid sequence from the chicken sequence located about 0.5 kb from the start of the chicken gene.
  • the complete sequence for fragment #28 can be obtained and analyzed to determine whether it contains the 5' end of the ACCase coding region.
  • the start of the transcribed region, and thus the likely start of the coding region for ACCase, can be identified by using the genomic clones in RNAse protection analysis (J. Sambrook et al., "Molecular Cloning - A Laboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)).
  • oligonucleotide primers can be constructed to synthesize cDNA molecules representing the amino terminus of the ACCase gene. These molecules can be hybridized to genomic Type A DNA fragments such as #28 and the nonhybridizing portions digested with SI nuclease. The end of the protected fragment are determined by analysis on a DNA sequencing gel. To synthesize the remaining coding region between the end of the cDNA clone #18-5 and the start of transcription, two oligonucleotide primers were synthesized.
  • Primer 1 is complementary to the DNA sequence: (SEQ ID NO:4) 5' GCCAGATTCC ACCAAAGCAT AT ATCC 3' near the 5' end of cDNA subclone #18-51 and can be used as a primer for synthesis of cDNA molecules from maize seedling, leaf or embryo RNA.
  • a primer corresponding to a DNA sequence near the transcription start site can be used in combination with primer 1 for the amplification of DNA by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Several independent clones are then sequenced and their sequences compared to the known sequence of the Type A genomic clone to determine the exact coding sequence corresponding to that maize gene for ACCase.
  • a similar strategy can be used to obtain the complete coding sequence for genomic Type B ACCase.
  • the remaining cDNA sequence was obtained by three successive rounds of RT-PCR using oligonucleotide primers based on genomic apparent exon (5') and known cDNA (3') sequences.
  • the primers used to amplify nucleotides 1-240 of the cDNA were 28sst-a5+ (SEQ ID NO. 7) and 28sst-6at3+ (SEQ ID NO: 8)
  • nucleotides 217-610 of the cDNA were 28sst-5+ (SEQ ID NO:9) and 28-2t3+ (SEQ ID NO: 10)
  • nucleotides 537-2094 of the cDNA were ACCPCR5' (SEQ ID NO:l 1) and 155 " (SEQ ID NO:30) (Table V).
  • PCR products corresponding to nucleotides 1-240, 217-610, and 537-2094 of the final sequence were cloned into PCR-script (Stratagene).
  • the 7470 bp cDNA includes a 459 nucleotide 3' untranslated region and 36 nucleotides of 5' untranslated sequences.
  • the first Met codon in the cDNA (nucleotides 37-39) was identified as the start codon based on its similarity to consensus initiation sequences (Kozak, J. Cell. Biol., ]M, 229 (1989); Lutcke et al, Embo. J.. 6, 43 (1987)).
  • An in-frame stop was found in the genomic sequence 6 nucleotides upstream of the sequenced cDNA, and RT-PCR analysis of this region suggested that the in frame stop codon was also present in the cDNA.
  • the 3' end of the coding sequence was defined by two stop codons present in the large open reading frame after nucleotide 7011.
  • the translated coding sequence predicted a polypeptide of 2325 amino acids (257 kD; SEQ ID NO:6) which was 79 to 81% identical to the multifunctional (MF) ACCases from alfalfa (Shorrosh et al., Proc. Nat'1. Acad. Sci., 9_1, 4323 (1994)) and wheat (Gornicki et al, Proc. Nat'1 Acad. Sci., 9JL, 6860 (1994)), and to a 118-amino acid predicted polypeptide of a rice expressed sequence tag (Genbank accession # D39099, T. Sasaki), but only 53 to 55%) identical to ACCase from other eukaryotes.
  • Types A and B correspond to previously published pA3 and ⁇ A4 cDNAs (Ashton et al., Plant Mol. Biol.. 24, 35 (1994)) and differ from pA3 and pA4 by ⁇ 4% in their coding sequences.
  • Type A and B genomic clones have linear sequence homology except for an insertion in an intron of the Type B genes about 1400 bp 3 ' of the A1(SEQ ID NO:5) translation start site. Analysis of the insert boundaries revealed a 3-bp target site duplication and a 6-bp direct repeat, and further sequence analysis showed the presence of two new and unique LINE elements (Long Interspersed Nuclear Elements) in Bl and B2. Mammalian LINE elements are highly abundant (10 4 to 10 5 copies), 6 to 7 kb long, and have frequent 5 '-end deletions and an A-rich 3' terminus. They are flanked by short direct repeats, and contain two ORFs, one encoding a reverse transcriptase.
  • LINE elements Three LINE elements (Cin4, 50-100 copies in maize; del2, 250,000 copies in lily; BNRl, 2-5% of genome in sugarbeet) have been described in plants (Leeton et al., Mol. Gen. Geneti., 232, 97 (1993); Schmidt et al, Chromo. Res., 3, 335 (1995); Schwarz-Sommer et al., EMBO J., 6, 3873 (1987)).
  • Maize ACCase Bl has one unique LINE element and B2 has two.
  • the two B2 LINE elements were characterized by differences in their reverse transcriptase sequence.
  • the B genomic clone inserts have characteristic LINE features including cysteine motifs and a possible polyA tail, and high abundance.
  • the LINE insert also has been found in an intron of the maize Shrunken-2 gene (Hannah et al., Plant P LysioL, ⁇ , 1214 (1992)).
  • ACCase genomic clone is shown in Figure 16 (SEQ ID NO:12).
  • the clone is a Hindlll fragment which includes nucleotides 1-931 of the cDNA in Figure 13 (SEQ ID NO:5), and the first four introns within the coding region, at positions 240 (460 nucleotides), 296 (480 nucleotides), and 872 (76 nucleotides) of SEQ ID NO:5.
  • the clone also has 1395 nucleotides 5' to the cDNA of SEQ ID NO:5 (i.e., 1431 nucleotides 5' of the translational start at nucleotide position 1432).
  • the partial nucleotide sequence (1328 nucleotide) of another Type A clone is shown in Figure 17 (SEQ YD NO: 13).
  • the partial sequence is all 5 ' untranslated sequence and contains a 7 base insert between nucleotides 279-290, but is otherwise identical to SEQ ID NO: 12.
  • the partial nucleotide sequence of six Type A2 clones is shown in Figure 18 (1565, 1168, 638, 558, 976 and 852 nucleotides; SEQ LD NOs 14, 15, 16, 17, 18 and 19, respectively).
  • the A2 genomic clone is weakly amplified with Type Al PCR primers specific for the 5' UTR if the 3' primer employed is for a conserved amino acid sequence found in all ACCases (e.g., 28sst-l 10F, ACTGTGCGTTTGAGAAGGTC, SEQ ID NO:28, and 28sst-2T3+, CCTCTACGTAATTGGTCAGC, SEQ ID NO:29).
  • the A2 amplified product is the same size as that from the Type Al genomic clone, and restriction analysis indicated a difference in sequence from Al .
  • Sequence differences in the 5 ' region should provide a means to distinguish between expression of Al and A2 ACCase genes and to determine whether A2 also encodes a CTP.
  • the partial nucleotide sequence (231, 207 and 180 nucleotides) of three Type B clones is shown in Figure 19 (SEQ ID NOs 20, 21 and 22, respectively).
  • the cDNAs corresponding to genomic clones A2, Bl and B2 are cloned and sequenced in a manner similar to that described above.
  • the derived amino acid sequences are aligned with known ACCase sequences. If putative CTP sequences are identified, functionality is tested as described below. Also if the tissue specificity and developmental timing of expression differ for different ACCase genes, the sequences of the promoter regions of the corresponding genomic clones are compared.
  • Gene-specific probes for specific ACCase genes can provide more information on their roles in lipid synthesis (plastid and cytoplasmic isoforms), secondary metabolism (cytoplasmic isoforms), and herbicide resistance (likely plastid isoforms).
  • a 3' Type Al ACCase cDNA probe mapped to chromosome 2S (Egli et al., Maize Genetics Newsletter. 6S, 92 (1994)) and to 1 OL (Caffrey et al., Mai e Gen. Coop., 62, 3 (1995)).
  • Two 5' Type Al cDNA probes which span the transit peptide mapped to chromosome 2S in the same location as the 3 ' probe (see maize genetic map, 1996 version, Maize Genomic Database).
  • PCR primers 28sst-97F (CCTTTTTATGGCACTGTGCG, SEQ ID NO:25) and 28sst-6t3+; (CATCGTAGCCTATATGAGGACG, SEQ ID NO:26) located in non-coding regions of Al that span the chloroplast transit peptide were used to amplify a B73 chromosome-specific product which segregated with the resistance trait.
  • a nearby 5' primer (28sst-a5+, SEQ ID NO:7) amplified all genotypes and functioned as a positive control.
  • Herbicide resistance due to the Accl-S3 mutation segregates (29/29 individuals to date) with production of a Type A 5 ' end-specific PCR product derived from the mutant parent while herbicide sensitive plants lack the transit sequence (15/17 progeny). Two individual plants which contained B73-specific DNA at this location died of unknown causes while grown in the presence of herbicide. Mutations in maize that confer resistance to cyclohexanedione and aryloxyphenoxypropionate herbicides by means of an altered ACCase target are found at two non-allelic loci, Accl and Acc2. Al and A2 appear to encode plastidic ACCases and correspond to the Accl and Acc2 herbicide resistance loci.
  • Acc2 has been mapped to 10 L (VanDee, M.S. Thesis, University of Minnesota (1994)).
  • Accl is the site of five allelic mutations including Accl-S2 and -S3 (Marshall et al., Theor. Appl. Genet.. S3, 435 (1992)), and has been mapped to chromosome 2. Only one plastidic ACCase polypeptide was identified by SDS-
  • the Type Al ACCase gene is probably located on chromosome 2, since (i) 5' untranslated and chloroplast transit peptide probes from Type Al hybridize to two bands (dark and light) in maize inbreds, and (ii) analysis of maize-oat addition lines carrying maize chromosomes 2 through 9 indicates the dark band is on chromosome 2 and the light band is on chromosome 1 or 10.
  • Type B ACCase genes are likely to encode cytosolic isoforms. Given that cytosolic malonyl-CoA is a precursor in the synthesis of many secondary metabolites including flavonoids (e.g. maysin, a corn silk component associated with corn earworm resistance), these cytosolic ACCases can have agronomic utility.
  • flavonoids e.g. maysin, a corn silk component associated with corn earworm resistance
  • Type A- and B- specific 32 P-CTP- labeled antisense transcripts were 780 nt long (662 nt of ACCase sequence + 118 nt of vector/promoter sequence) and were identical except for 15 base mismatches scattered along their length. Each antisense transcript was hybridized to total RNA from embryos at 16, 20, 23, and 42 DAP and digested with RNAse A/TI mixture to yield a 662-base fragment specific to the probe used. The results showed that the Type A transcript was more abundant than Type B at all tested stages, and that only Type A remained high in older embryos. Types A and B had similar expression patterns and peaked around 20-23 DAP. The ratio of Type A:B mRNA in leaves was about 2:1, similar to its relative abundance in cDNA expression libraries.
  • EXAMPLE VIII Expression of the Maize ACCase Chloroplast Transit Peptide
  • the N-terminus of the predicted maize ACCase polypeptide is longer than that of predicted cytosolic ACCase isoforms and has several characteristics typical of chloroplast transit peptides within the first approximately 73 amino acids of the predicted N-terminal sequence.
  • the CTP cleavage site motif is not found in the putative maize ACCase CTP, although only about 30% of known CTPs contain this consensus sequence (Gavel and von Hejne, FEBS Lett., 261, 455 (1990)).
  • the maize ACCase N-terminus appears to have several other properties typical of known CTPs: (1) a lack of acidic residues in amino acids 1-49, (2) high Ser + Thr content (69% within amino acid residues 23-35), (3) an R-rich region between S- and D-rich regions in amino acid residues 36-49, and (4) a predicted turn ⁇ sheet within amino acid residues 58-73 (von Hejne and Nishikawa, FEBS Lett., 278, 1 (1991)).
  • the function of the putative maize ACCase CTP is tested by inserting the first 258 coding nucleotides of maize ACCase in frame with and 5' of a GUS reporter gene in pBI221 (Clontech).
  • This construct and the pBAR plasmid are used to co-transform maize "Black Mexican Sweet" suspension cells by particle bombardment. Basta-resistant transformants are selected, and GUS activity and/or protein is assayed in surviving cultures or in plasmids isolated from transformants.
  • a partial ACCase construct consisting of nucleotides 1-833 of SEQ ID NO: 5 including the putative CTP (nucleotides 37 to 256) and the first domain within the biotin carboxylase region (identified by amino acid sequence comparison with E. coli biotin carboxylase; see Waldrop et al., Biochem., 33, 6249 (1994)) was amplified by PCR and cloned into the EcoRV site of PCR- script (Stratagene) to create the plasmid pBCNl. A corresponding plasmid lacking CTP sequences (nucleotides 278-833) was also made (-pBCNl). The protein encoded by -pBCNl begins at amino acid residue 83 (Val ⁇ Met).
  • Purified pBCNl was digested with EcoRI to linearize the plasmid at the 3' end of the BCN1 insert, electrophoresed in 1.5% agarose, and the plasmid band at approximately 3.8 kb was excised and Gene-Cleaned (BioLab 101).
  • the purified band was digested with 20 ⁇ g proteinase K to remove any residual RNAse, extracted with phenol and then chloroform under RNAse-free conditions. DNA content was estimated by ethidium bromide fluorescence in droplets, relative to ⁇ DNA standards (Sambrook et al, Molecular Cloning: A T .ahoratory Manual. 2nd ed. (1989).
  • RNA yield was estimated by determining the % inco ⁇ oration of a 32 P-ATP (Amersham) into a precipitable product, according to the Ambion kit instructions. Electrophoresis and autoradiography of 32 P-labeled product showed that it contained a single RNA band of approximately 895 nucleotides, as expected.
  • RNA transcripts were translated into 35 S-labeled polypeptides with Ambion's wheat germ IVT kit and approximately 45 ⁇ Ci 35 S-methionine (Amersham; 37 TBq/mmol) in a 50- ⁇ l reaction. Labeled proteins were held on ice 6 hours prior to their use in chloroplast import experiments. Pea (cv. "Little Marvel") and maize (inbred A188) plants were grown in a growth chamber at 25 °C, 16 hour day length. Chloroplasts were isolated from pea and maize leaves 7 days after planting, respectively, as previously described (Burton et al., Pestic Biochem.
  • Import experiments were carried out essentially as described by Cline et a!.(.T. Biol. Chem.. 26Q, 3691 (1985)). Import reactions containing 0.3 ml pea or maize chloroplast suspension, 40 ⁇ l 35 S-translation mixture, 3 mM Mg-ATP and 10 mM Met were incubated under light for 1-30 minutes at 25 °C. Unimported proteins were digested for 30 minutes with 40 ⁇ g of thermolysin, and proteo lysis was stopped with 10 mM EDTA.
  • Chloroplasts were re-isolated by centrifuging them through 1-ml 40% v/v Percoll gradients in the presence of resuspension buffer plus 3 mM Mg- ATP, 10 mM Met, and 20 mM EDTA, washed twice in the same buffer, and resuspended in 65 ⁇ l of 1 mM MgCl 2 /10 mM Tris buffer, pH 8.0.
  • Chloroplasts were lysed by three cycles of freeze-thawing in liquid N 2 , micro fuged, and aliquots of the supernatants and of the original in vz ' tro-translated proteins were analyzed by SDS-PAGE in 8-25% gradient Phast gels (Pharmacia), followed by direct detection of radiolabeled proteins in the wet gels (AMBIS) ( Figure 20).
  • SDS-PAGE As estimated by SDS-PAGE, a 30 minute import converted the original 32-kD BCNl polypeptide to a doublet of 27.2 and 27.5 kD in maize and produced an additional 30-kD band in pea ( Figure 20A). Neither maize or pea chloroplasts imported -pBCNl -derived polypeptides.
  • the cDNA and genomic clones encoding all or a portion of the ACCase gene can be subcloned into a known expression system and the gene products reactive with the antibodies specific for maize ACCase can be identified using a Western blot.
  • the ACCase cDNA clones are inserted into two transformation plasmids: (i) Glblexp which contains the embryo-specific maize Globulini (Glbl) promoter and 3' regions (Belanger et al., Genetics, 129_, 863 (1991)); and (ii) pAHC17 which contains the maize ubiquitin (Ubi-1) constitutive promoter and first exon and intron, and the NOS 3' terminator (Christensen et al., Plant Mol. Biol., 1£, 675 (1992); Toki et al., Plant
  • the 3' end of the A x cDNA has a unique Sail site just 3 ' of the stop codon which is used to ligate into a Sail site in both plasmids ahead of the construct terminator. Other cloning sites will be added as needed to the plasmids or cDNA to complete the ligation of the 5' end.
  • the gene products can also be further characterized structurally and/or enzymatically. This will ensure that the genomic and cDNA clones that encode acetyl CoA carboxylase can be screened for promoters that provide for ove ⁇ roduction of the native or herbicide tolerant ACCase enzyme in plants.
  • the 2 kb EcoRI fragment from clone #15-14 can be subcloned into a plant transformation plasmid pBI121 or pBI221 downstream from the 35 S CaMV promoter and upstream from the nopaline 3' polyadenylation signal sequence, as described in Jefferson, Plant Molec. Biol. Reptr.. 5_, 387-405 (1987).
  • This plasmid can then be used to transform plant cells such as tobacco, Brassica and Arabidopsis cells using protoplast or biolistic transformation, as described by W.J. Gordon-Kamm et al., Plant Cell, 2, 603-618 (1990); M.E.
  • the ACCase gene can be subcloned along with the 35S Came promoter into a binary Ti vector pGA482, as described in An, cited supra., which is a binary Ti vector system and can be used to transform plant cells by Agrobacterium-mediated transformation.
  • Stably transformed plants can be generated by standard methods as described in Example III, and levels of expression of ACCase genes can be determined by quantitative Western blots, as described in Harlow and Lane, Antibodies, Cold Spring Harbor Laboratories (1988). The ability to monitor expression of cloned ACCase genes will permit the identification of promoters that provide for enhanced expression of the ACCase gene.
  • the expression system can be used to screen for those promoters that enhance gene expression of the ACCase gene at least about 5 to 10-fold over the endogenous levels of ACCase produced normally in the plant cells. Because the 35S Came promoter is known as a strong promoter, it is likely this promoter will provide for at least a 5-fold increase in the expression of ACCase over that normally produced in the plant cell.
  • this expression system can be used to screen antisense
  • an antisense sequence can be obtained that is complementary to an about 0.5 kb region of the maize ACCase cDNA that has high homology with a portion of the chicken ACCase gene and contains the sequence for the presumed transcarboxylase active site domain, as shown in FIG.
  • the antisense sequence could be subcloned into a pBI121 or pBI221 expression under the control of an inducible plant promoter, such as nitrite reductase promoter (Back et al, Plant Molec. Biol., 12:9-18 (1991)).
  • an inducible plant promoter such as nitrite reductase promoter
  • the ability of the antisense sequence to inhibit expression of the native ACCase gene can be evaluated in transformed cells, for example as described in Hamilton et al, Nature, 346:284-287 (1990).
  • Herbicide resistant maize cell lines were generated as described in
  • Examples I, II, and FV These herbicide resistant cell lines have been shown to produce an ACCase enzyme that is less sensitive to inhibition by sethoxydim or haloxyfop.
  • the genes encoding the herbicide resistant forms of the ACCase will be identified and cloned using standard methods as described in Sambrook et al., Guide to Molecular Cloning: A Laboratory Manual (1989).
  • the genes encoding the herbicide resistant forms of ACCase can then be introduced into herbicide sensitive plant species by standard methods to confer herbicide resistance .
  • the ACCase enzyme in the maize cell line 2167-9/2160-154 S-1 is at least 100-fold less sensitive to sethoxydim than the wild-type.
  • DNA from the cell line or plants will be obtained and digested with EcoRI and/or other appropriate restriction enzymes, according to standard methods.
  • the restriction enzyme digest will be separated out by agarose gel electrophoresis and probed with either the 2 kb or the 3.9 kb cDNA ACCase probe described in Example V. Fragments hybridizing to the 2 kb or 3.9 kb probe will be subcloned into a Bluescript vector and portions of the gene will be sequenced, as described in Example V, to verify that the entire ACCase gene has been isolated.
  • the clone encodes the ACCase gene
  • it will be subcloned into the pBI121 or pBI221 expression vector, as described in Example VIII.
  • the ACCase gene product expressed by the clone in either Black Mexican sweet corn cells or tobacco cells will be evaluated for reactivity with ACCase specific antibodies, by enzyme activity, and/or resistance of the enzyme activity to inhibition with sethoxydim and/or haloxyfop. It is likely that the cloned gene will encode an ACCase which is resistant to inhibition by sethoxydim and haloxyfop.
  • This gene can then be introduced into an herbicide-sensitive embryogenic plant cell or an embryo, including maize cells or immature embryos, to confer herbicide resistance to that plant species upon regeneration.
  • the complete coding sequence encoding the herbicide resistant form of the ACCase enzyme will be cloned into a plant transformation vector such as pBI121 or pBI221 as described in Jefferson, Plant Molec. Biol. Reporter, 5:387-405 (1987).
  • This vector contains the 35S Came constitutive promoter, the ⁇ -glucuronidase structural gene, and the nopaline synthase 3 ' polyadenylation signals.
  • the ⁇ -glucuronidase gene is replaced with a cloned ACCase gene.
  • the cloned ACCase gene can be combined with natural or synthetically produced chloroplast transit peptide sequence from pea, as described in Keegstra & Olsen, Ann. Rev. Plant. Physiol ./Mol. Biol, 40:471-501 (1989) and/or unique restriction sites introduced so the cloned gene can be distinguished from the endogenous maize ACCase gene. Standard methods of subcloning will be utilized as described in Sambrook et al., cited supra.
  • type II calli can be transformed using biolistic transformation, as described by W.J. Gordon-Kamm et al., Plant Cell, 2, 603-618 (1980); M.E.
  • type I embryogenic calli can be transformed using electroporation after mechanically or enzymatically wounding calli, as described by D ⁇ afluin et al., The Plant Cell, 4:1495 (1992).
  • fertile transgenic maize plants can be regenerated, as described by D ⁇ afluin et al. cited supra.
  • Fertile transgenic plants can be evaluated for herbicide tolerance, as described in Example III.
  • the fertile transgenic plants having and expressing a cloned ACCase gene as an ACCase resistant to sethoxydim and/or haloxyfop will exhibit herbicide tolerance as compared to the corresponding untransformed plant.
  • CoA carboxylase can be introduced into monocot or dicot plant species, including maize, under the control of a promoter that provides for overexpression of the ACCase enzyme.
  • the overexpression of the ACCase enzyme is likely to lead to an increase in the oil content of the plants and seeds.
  • a gene encoding a genomic maize acetyl CoA carboxylase can be isolated, as described in Example V, and used to transform plant species by protoplast or biolistic transformation. If the gene is combined with a strong promoter, such as the 35S cauliflower mosaic virus promoter, overexpression of the ACCase gene is likely. Alternatively, selecting transformed cells with multiple copies of the gene can also result in transformed cells overexpressing the ACCase gene.
  • the gene can be cloned into a vector such as pBI121 or pBI221, as described by Jefferson, cited supra. This vector contains the 35S cauliflower mosaic virus promoter, the ⁇ -glucuronidase structural gene, and the nopaline synthase 3' polyadenylation signals. The cloned ACCase gene can replace the ⁇ -glucuronidase gene and then be used to transform plant cells, including maize, as described in Example VIII.
  • Transformed cells can be screened for ove ⁇ roduction of ACCase.
  • the presence of the cloned gene can be verified by identifying the unique restriction enzyme sites inco ⁇ orated into the cloned gene.
  • ACCase levels can be assessed by standard enzyme assay methods and quantitative Western blots using antibodies specific for maize ACCase. Fatty acid and lipid content in cells lines ove ⁇ roducing ACCase are likely to be elevated and can be assessed using standard methodologies, as described in Clark & Snyder, .TACS, 66:1316 (1989).
  • Transformed cell lines ove ⁇ roducing ACCase and having increased total oil content will be used to regenerate fertile transgenic plants and seeds, as described in D'Hafluin, cited supra.
  • Maize embryos are transformed with sense and antisense cDNA constructs encoding the plastidic Al ACCase. Selected transgenic cultures and regenerated transgenic plants and progeny are subjected to detailed analyses of:
  • ACCase transcript levels activity of ACCase I (plastidic) and ACCase II (presumably cytosolic) in various plant tissues; fatty acid synthesis; lipid accumulation in kernels (primarily embryos); and other plant traits.
  • the culmination of these tests indicates whether plastidic ACCase activity can be modified via transformation and whether fatty acid synthesis is affected. 1. Transformation of maize embryos and plant regeneration Immature maize embryos of the Hi-II genotype are transformed by particle bombardment according to slight modifications from previously described procedures (Fromm et al., Biotech., S, 833 (1990); Koziel et al., Biotech., 11, 194 (1993)).
  • F2 or backcross progeny are grown in the greenhouse and field and tested for Basta resistance and presence of the Al ACCase transgene to identify homozygous transgenic plants. Homozygous inbred transgenic lines are then developed. 2.
  • Plants recovered from at least 100 independent transformation events (i.e., from different bombarded embryos) for both the UBIl and GLB1 vectors are recovered. Regenerated plants are tested for the presence of the intact ACCase Al transgene and its cosegregation with the Basta-resistance marker. Homozygous and heterozygous transgenic lines are assayed for total ACCase activity in leaves for UBIl transformants and in developing embryos (22-26 DAP) for both UBIl and GLB1 transformants.
  • Sethoxydim and haloxyfop inhibition are used to quickly determine the levels of herbicide- sensitive ACCase I (plastidic) and the herbicide-insensitive ACCase II activity in both leaves and embryos.
  • Increased expression of the ACCase Al transgene contributes to plastidic ACCase activity and not to ACCase II activity.
  • Kernel fatty acid and oil content are analyzed at maturity and their relationship with ACCase I determined by methods well known to the art.
  • Transformants that differ in kernel ACCase activity and/or fatty acid and oil content are then selected for more detailed analysis of embryos throughout development (4-day intervals from 16 DAP to maturity). These analyses include RNAse protection assays to determine total Al + A2 transcript levels using a non-specific probe and to determine relative levels of endogenous Al versus transgene Al transcripts by use of antisense riboprobes spanning the 5' UTR region of the Al transgene constructs.
  • Antisense transformation vectors were constructed by blunt-end ligation of nucleotides 1-833 of SEQ ID NO:5 in reverse orientation into multicloning sites of both the GLBl and UBIl plasmids. A sense construct with the same 833-bp cDNA sequence also was made with the GLBl plasmid to serve as a transformation control. Insert orientations were verified by restriction mapping. UBIl antisense, GLBl antisense, and GLBl sense constructs were introduced into > 2100, > 2900 and > 2000 embryos, respectively, and Basta- resistant callus were selected.
  • UBIl and GLBl antisense constructs will be determined by PCR analysis for unique transgene sequences such as the Ubi-1 intron/ ACCase Al junction or Glbl 5' UTR/ ACCase Al junction, or by Southern blotting to detect unique fragments.
  • Plants and lines homozygous or heterozygous for the antisense transgene are analyzed for steady state level of the ACCase Al antisense transcripts in appropriate tissues/organs (such as leaves, tassels, ears, embryos and endosperm for UBIl; leaves and embryos for GLBl) by using ACCase Al sense riboprobes for hybridization on RNA blots or for RNAse protection assays.
  • Total ACCase activity both ACCase I and ACCase II isoforms
  • fatty acid and lipid content are determined for the antisense transgenic lines and for corresponding tissues from nontransformed control plants.
  • EXAMPLE XIII Expression of Plastidic and Cytosolic ACCases during Plant Development Intact embryos are isolated from developing kernels of field- grown inbred B73 at 2 to 4 day intervals between 16-42 DAP and frozen immediately in liquid nitrogen. Samples also are saved for fresh and dry weight determinations. Subsamples from each stage are analyzed for total lipid and fatty acid content. Seedling leaves are sampled along the leaf blade ranging from the etiolated, meristematic basal region to the fully expanded, green tip.
  • Leaves and other tissues (e.g., epidermis) of maize genotypes that accumulate anthocyanin pigments are also analyzed to assess whether a specific ACCase (such as a cytosolic ACCase) is more highly expressed in tissues in which malonyl-CoA also is required as a substrate for chalcone synthase in the flavonoid pathway leading to anthocyanin synthesis.
  • a specific ACCase such as a cytosolic ACCase
  • Gene-specific antisense riboprobes in RNAse protection assays are employed to determine Al, A2, Bl and B2 transcript levels.
  • the corresponding sense transcripts are produced in vitro and used as standards to verify specificity and quantitate the sample transcript levels. Quantisation is done on an AMBIS radioanalytic image system.
  • Herbicide inhibition of total ACCase activity provides an assessment of levels of herbicide-sensitive ACCase I (plastidic) and the herbicide-insensitive ACCase II activity in these tissues. ACCase I and II isoforms are separated by ion-exchange chromatography.
  • Total proteins are separated by SDS-PAGE and Western blots probed with avidin to detect the biotinylated 227-kD ACCase I and 219-kD ACCase II isoforms or probed with ACCase I-specific antibodies.
  • oat grain is produced for many pu ⁇ oses such as for animal feed, human food, and cosmetic and pharmaceutical additives (Webster, 1986). Seventy-eight percent of the world oat production is used for livestock feed (Schrickel, 1986).
  • oat groats have the highest lipid concentration (Morrison, 1978), but due to their high fiber content oat is lower in energy value than many other cereals when used for livestock feed.
  • Oat is also unique among the cereals because more than 50% of total seed lipid is deposited in the starchy endosperm rather than the embryo of developing caryopses (Peterson and Wood, 1997; Youngs, 1986). An increase in oil content is desirable for making oat a higher-energy feed grain. On the other hand, a decrease in oil content would make oat a more attractive commodity for use in many current human diets where oat is desired for the type and concentration of its fiber (Ripsin et al, 1992).
  • a polygenic pattern of inheritance has been indicated for groat oil content with heritability estimates ranging from 63 to 93% (Schipper and Frey, 1991a) with primarily additive gene action (Thro and Frey, 1985).
  • the average groat oil concentration in contemporary cultures is 6 to 7% but variation from 2 to 16% oil occurs in diverse germplasm (e.g., Kanota has about 11%, Ogle about 9% and Marion about 8% of oil (tryglycerides plus polar lipids) content, per dry weight).
  • Genotype by environment interaction for this trait is small (Gullard, 1986; Branson and Frey, 1989).
  • genomic regions can be identified which significantly influence seed oil content.
  • two populations the high oil by low oil population (Alrefai et al., 1995) and the high protein by low protein population (Goldman et al., 1994) were analyzed to identify genomic regions influencing kernel oil content.
  • a region on chromosome 6 was identified near the linoleic acid! locus.
  • this region explains 63% of the phenotypic variation in the ratio of oleic to linoleic (18:1 to 18:2) fatty acids and also influenced the total kernel oil concentration (Alrefai et al., 1995).
  • oat plants with an altered fatty acid composition as a result of a qualitative genetic mutation have not been identified, possibly due to the hexaploid nature of the genome.
  • graminicides cause plant cell death by inhibiting
  • ACCase and cessation of fatty acid biosynthesis (Burton et al., 1989).
  • Maize mutants have been isolated that grow in the presence of normally lethal concentrations of ACCase-inhibiting herbicides, exhibit up to 20-fold increase in wild-type ACCase activity, and synthesize fatty acids in the presence of herbicide (Parker et al., 1990).
  • Other results point to the role of ACCase in regulating seed oil deposition.
  • a herbicide-insensitive ACCase mutation, Accl exhibits increased ACCase activity in corn embryos paralleling the reporting time course of oil deposition in this tissue (Somers et al., 1993).
  • Arabidopsis cytosolic ACCase in B. napus plastids alters fatty acid composition, with the largest effect being an increase in oleic acid, and increasing total seed oil content by about 5% (Roesler et al., 1997).
  • the second population was chosen on the basis of having a common parent, Kanota, to provide a degree of
  • Groat oil content was measured by a Bran+Luebbe InfraAlyzer 500 on dehulled ground samples.
  • the Near Infrared (NIR) calibration was based on total fat as determined by acid hydrolysis.
  • the calibration set contained 348 oat samples (representing commercial and elite breeding lines from North American oat breeding programs) with fat contents that ranged from 4.95 to 14.01%.
  • the calibration equation had a correlation coefficient of 0.985, a Standard Error of Estimate of 0.299, and a F-value of 11500.
  • Acid hydrolysis extracts the total lipid content of the groat.
  • the reported values represent the sum of the neutral lipids (triglycerides) plus the polar lipid fractions (Youngs et al., 1982). Thus, these values are higher than those obtained from ether extracts which only contain the neutral lipid fraction.
  • oat groat cDNA clone was isolated based on its hybridization to a maize cDNA fragment which included portions of the less conserved mid- peptide sequence and the transcarboxylase domain (Shorrosh et al., 1994). Sequence identity indicated that the oat cDNA (3031 bp) corresponded to maize ACCase nt 3124-6169 (Egli et al., 1995). The 1010-aa predicted polypeptide was nearly identical to portions of wheat (89%) and maize (84%) plastidic multifunctional ACCases; it had lowe?
  • Oat ACCase cDNA sequence was compared to other plant ACCases with the BESTFIT program (BESTFIT, Wisconsin Package V. 9.1, Genetics Computer Group (GCG), Madison, WI).
  • BESTFIT Genetics Computer Group
  • GCG Genetics Computer Group
  • Pileup alignments of maize, wheat, barley, and rice ACCase coding sequence were used to identify conserved regions (i) for design of new primers or (ii) regions of identity to available maize ACCase oligos, to be used for PCR amplification of oat ACCase.
  • PCR2 (5'-ggcagagcaa agcttggagg; SEQ ID NO:25) and PCR1 (5'-tccaccagag aagcctctcc; SEQ ID NO:26) appeared like to amplify oat cDNA based on their identity to other monocots and previous use to amplify soybean ACCase.
  • These primers are located within the transcarboxylase domain, surrounding the acetyl-CoA binding site of ACCase.
  • the first procedure was single factor ANOVA (ANOVA and GLM procedures; SAS Institute, 1990) with the added criteria for consistency in association of genomic regions with a trait over environments and linkage distances (Kianian et al., 1998). If the association were significant when considering the average of all the environments, the association was also significant over the majority of the environments (3 of 5 in KO and 2 of 3 in KM), and closely linked markers within that region, if available, also showed significant associations.
  • a "backward elimination” process as described by Kennard and Havey (1995) was used to construct a multi-locus model containing the most important markers identified by the ANOVA procedure.
  • Marker loci were excluded one at a time from this model, based on the criterion of least significant (P ⁇ 0.05) Type-Ill sum of squares (Kennard et al., 1994).
  • MQTL software (Tinker and Mather, 1995a & b) was used to strengthen and validate the ANOVA results.
  • This software performs Simple Interval Mapping (SIM) and Simplified Composite Interval Mapping (sCIM). It also has the advantages of searching for one QTL while simultaneously accounting for the effect of others, providing separate analyses for each environment, and calculation of a threshold to control the type-I error rate. One thousand permutations were performed to estimate the threshold for a type-I error rate below 5%.
  • the KO population contained individuals with the highest and lowest groat oil concentrations, possibly due to a larger difference in parental values (3% between Kanota and Ogle, 2% between Kanota and Marion). Overall, the KM population contained more transgressive segregants than the KO population (Table VI).
  • a hexaploid oat RFLP map that contains 561 loci has been generated from 71 RILs in the KO population (O'Donoughue et al., 1995).
  • Initial quantitative analysis was performed using this subset of the KO population, the available trait data, and 360 of the 561 loci that are co- dominantly segregating and evenly distributed across the oat genome.
  • the remaining 66 RILs in this population were also mapped.
  • 153 loci have been mapped with these individuals. Detection of QTL with either the subset (360 markers and 71 individuals) or the entire data set (153 markers and 137 individuals) of the KO population did not make a major difference, except in two situations. In both cases, QTLs were detected in the subset, but were not detected in the entire population due to the absence of RFLP data for the remaining 66 RILs. Results of the entire population are presented herein except for the aforementioned differences.
  • the positive alleles (those that cause an increase in groat oil content) for all of the genomic regions influencing this trait were from the Kanota parent (Table 3). Overall, the alleles from Kanota contributed 2.8% (4.1% if linkage group 37 and Coleoptal loci were included) to the groat oil content. Regions on linkage groups 6 and 11 are considered highly significant and remained in the full model after the backward elimination process (Table 4). However, the two additional loci from linkage group 37 and Coleoptal were also included in the model if only the first 71 individuals in this population were being analyzed. However, the two additional loci from linkage groups 37 and Coleoptal were also included in the model if only the first 71 individuals in this population were being analyzed.
  • Locus Xcdo665B on linkage group 11 accounted for 36% to 48% (depending on the environment) of the phenotypic variance for this trait (P ⁇ 0.0001).
  • a substitution of Kanota alleles for their Ogle counte ⁇ arts caused an average increase of 2% in groat oil content.
  • Substitution of Kanota alleles for Ogle alleles at the locus Xcdol414B (or XumnS IB) on linkage group 37 caused an average increase of 1% in groat oil content and was the second most significant locus considering only the first 71 individuals in this population.
  • Table VIII Genomic regions significantly influencing groat oil content in oil identified by single factor analysis of variance
  • linkage group and parent contributing the positive allele are depicted in parentheses (i.e., linkage group 11 with Kanota allele being positive (11, K)).
  • the models are based on five environments in KO and three in KM.
  • the VM/VP (variance explained by the QTL(s) over the phenotypic variance) is presented as the R 2 for MQTL.
  • XaccaseA is closely linked to Xcdo665B and gives the same relative effects as Xcdo665B.
  • the markers mapped were not chosen for their association with groat oil content in the KO subset population, but for their association with other groat chemical and physical characteristics (Kianian et al., 1998). Since the level of polymo ⁇ hism in hexaploid oat is relatively high, most probes detected sequences mapping to more than one location. A total of 60 loci were mapped with the 139 RILs in the KM population.
  • locus Xcdo665B on linkage group 11 was highly significant for its effect on groat oil content (P ⁇ 0.0001), accounting for 16% to 20% (depending on the environment) of the phenotypic variance for this trait. Overall, the three significant loci explained 15% of the phenotypic variance and together accounted for a total difference of 1.7% in groat oil content.
  • oat groat cDNA was isolated based on its hybridization to a maize cDNA fragment which included portions of the less conserved mid- peptide sequence and the transcarboxylase domain (Shorrosh et al, 1994).
  • the 3031-bp oat cDNA was nearly identical to wheat (90%) and maize (84%) genes encoding plastidic ACCase (Egli et al., 1995; Gornick et al., 1997).
  • a 424 nucleotide sequence form the 3' end of the insert was translated and compared to other plant ACCase peptides.
  • the oat peptide sequence was 93% identical to plastidic isozymes from maize and wheat (Egli et al., 1995; Gornicki et al., 1997), but only 78 to 83% identical to cytosolic ACCase from maize, wheat (Gronicki et al., 1994), or alfalfa (Shorrosh et al, 1994).
  • the 3031-bp oat ACCase cDNA clone identified a single polymo ⁇ hic locus in the KO population ⁇ AccaseA) and two polymo ⁇ hic loci in the KM population ⁇ AccaseA and AccaseB).
  • the AccaseA locus maps 2.3 cM from Xcdo665B and 3.7 cM from Xisu2287 on linkage group 11.
  • the AccaseB locus maps 11 cM from Xcdo665D and 7.3 cM from Xbcdl 729B.
  • the ACCase cDNA clone from developing oat seeds hybridizes to a genomic region that is linked to a QTL locus on linkage group 11 having a major influence on the total lipid content of oat groats.
  • the influence of this locus was detected in various environments and two genetic backgrounds, thus confirming the value of biological as well as environmental replication.
  • the relative proportion of variance due to the environmental influence on this locus was much higher in the KM population than in the KO population.
  • This example illustrates the power of biological replication (i.e., having populations with a common parent) in identifying quantitative trait loci. Identification of the most significant locus does not preclude the identification of other less important loci due to the influence of genetic background and environment on the overall effect. The consistency in identification of significant regions by various quantitative analysis methods in the KM population is a good example. The regions on linkage groups 5, 11 and 22 and Xcdo395C locus are considered to make important contributions to the groat oil content in this population.
  • the Xcdo1 199C locus located near the Xacor242 locus on linkage group 5 of the published hexaploid oat RFLP map (O'Donoughue et al., 1995) has as great an effect as Xcdo665B-XaccaseA region on linkage group 11 in this population.
  • This locus was not detected in the KO population presumably because the Ogle allele had a similar influence as Kanota and in the KM population the Marion parent was carrying the allele with positive influence on the groat oil content.
  • the other two significant loci in the KM population have a relatively minor effect on this trait and would be more difficult to detect in a population segregating for regions with a larger influence on oil such as in the KO population.
  • results from other QTL studies can help identify possible pleiotropic effects of other traits such as heading date and vernalization response on groat oil concentration.
  • a region on linkage group 3 with alleles being contributed by Kanota is implicated as having a major influence on heading date and possibly yield, thus reducing its importance as having a direct effect on groat oil concentration (Siripoonwiwat et al., 1996; Holland et al., 1997).
  • the cultivar Ogle was found to have loci on linkage groups 22 and 37 that influence plant height in the KO population (Siripoonwiwat et al., 1996).
  • Loci on linkage group 11 is believed to have an influence on test weight and other groat physical characteristics, thus providing additional evidence of their role in oat groat mo ⁇ hophysiology (Siripoonwiwat et al., 1996; Cakir et al., 1996). These and other studies of quantitative traits in oat are helping identify genomic regions that influence the growth, development and physiology of grain, the primary product. Thus, genetic and molecular manipulation of the oat genome to improve grain quality and quantity can be directed at critical segments.
  • Cultivated oat is a hexaploid composed of three ancestral genomes designated A, C, and D (Rajhathy and Thomas, 1974). Recent studies have indicated that the organization of hexaploid oat genomes into discrete homoeologous groupings such as that of hexaploid wheat is difficult (Rooney et al., 1994; O'Donoughue et al., 1995; Kianian et al., 1997). However, C-genome chromosomes, due to their distinctiveness, have been used as a basis to identify homoeologous segments among the other chromosomes (Kianian et al., 1997).
  • hexaploid wheat plastidic ACCase maps to the short arm of homoeologous group 2 near the telomere (Gornicki et al., 1997). Plastidic ACCase genes in wheat have been shown to be transcriptionally active in seedling leaves but their effect on seed lipid content are unknown (Gornicki et al, 1997). ACCase in maize has been mapped to short arm of chromosome 2 near the centromere and long arm of chromosome 10 (Egli et al., 1995; Van Dee, 1994).
  • a QTL with moderate effect on oil concentration in progeny of Illinois High and Low oil maize lines was located near the ACCase locus on maize chromosome 2 (Berke and Rocheford, 1995). Additionally, there are numerous defective kernel (dek), reduced endosperm (ren), and Endosperm factor (Ef) loci influencing grain mo ⁇ hophysiology located on chromosomes 2 and 10 of maize near these synthetic segments (MaizeDB). In maize, oil is stored in embryos and embryo size is associated with oil content of whole seeds (Dudley, 1974).
  • oat grains contain significant amounts of lipid in the endosperm (Peterson and Wood, 1997), and thus QTLs affecting pathway flux could be more important than those altering seed anatomy.
  • the regions in wheat and maize agree well in terms of conserved syntony and correspond to chromosome 4 of rice, linkage group C of diploid oat and linkage group 11 of hexaploid oat map (Ahn and Tanksley, 1993; Van Deynze et al., 1995).
  • This comparative evidence from other grass species and homoeologous evidence from oat further help reinforce the validity of the association of these genomic regions with groat oil content and composition in oat.
  • Oat groats are unique among cereals for the high level and the embryo-plus-endosperm localization of lipids. Genetic manipulation of groat quality traits such as oil is desired for optimizing the value of oat in human and livestock diets. A locus having a major effect on oil content in oat ⁇ Avena sativa L.) groats was located on linkage group 11 by single factor analysis of variance, simple interval mapping and simplified composite interval mapping.
  • the major locus plus additional QTLs represent three homologous genomic segments in oat that can be followed by as few as three RFLP markers to produce a large shift in the groat oil content.
  • a maize ACCase gene was linked only to a moderate oil QTL. Unlike maize, where embryo size affects seed lipid concentration, oat stores significant amounts of lipid in the endosperm, thus QTLs affecting pathway flux may be more important than those altering seed anatomy. If an ACCase gene is indeed the major groat oil QTL, differential ACCase gene expression and/or enzyme activity may control groat oil content. Moreover, if an ACCase DNA is a QTL in oat, it may be useful as a marker for marker-assisted breeding in oat and other cereals, e.g., maize and wheat.
  • Groat oil values were averages of five (KxO) or three (KxM) environments. QTLs were identified by single factor analysis of variance.
  • Oat is unique among the cereals in regard to seed lipid deposition (Welch, 1995; Youngs, 1986). Most other cereals accumulate storage lipid primarily in the embryo of developing caryopses. Mature oat groats contain more than 50% of total seed lipid content in the starchy endosperm (Peterson and Wood, 1997). Additionally, significant amounts of oil are found in the bran fraction of processed oats. A substantial portion of the bran is in starchy endosperm and aleurone cell layers which do not separate cleanly from the bran as in wheat. Together the oat endosperm and bran fractions contain >90% of the total lipid in the groat. Thus, the endosperm is the major source of the oil in the groat.
  • the first type is typical of eukaryotic organisms where a single, nuclear gene encodes the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains within a single, multifunctional polypeptide of 210-280 kDa that, in plants, forms a dimeric active enzyme.
  • Plant lipid synthesis begins in plastids, where ACCase synthesizes the malonyl-CoA needed to initiate and elongate fatty acids of up to 18 carbons (Guenther, 1996).
  • multifunctional ACCase genes from maize, wheat and Brassica have been shown to encode functional chloroplast transit peptides.
  • Both monocots and dicots also appear to contain a cytoplasmic form of the multifunctional ACCase.
  • the cytoplasmic ACCase may be involved in the synthesis of malonyl-CoA needed for such reactions as the synthesis of very long chain fatty acids and flavonoids in plants.
  • the second, multisubunit type of ACCase consists of three functional domains formed from four separable polypeptides and is found in dicot plastids and in prokaryotic organisms, but appears not to be present in the Gramineae.
  • the biotinylated polypeptide component of this multisubunit form of ACCase is absent from the Gramineae and the plastid-localized multifunctional ACCase is believed to be responsible for fatty acid biosynthesis.
  • ACCase Complete herbicide inhibition of ACCase results in plant cell death due to cessation of fatty acid biosynthesis (Burton et al., 1989). Based on the apparent flux control coefficient for ACCase in barley and maize leaves exposed to low levels of graminicides, ACCase was shown to be the major flux-controlling enzyme for light-stimulated lipid synthesis in these leaves (Page et al., 1994). Parker et al. (1990b) isolated maize tissue cultures that were selected for growth in the presence of normally lethal concentrations of ACCase inhibiting herbicides.
  • Monocot plastid-localized, multifunctional ACCase is likely involved in regulating seed oil deposition.
  • Herbicide-resistant maize mutant plants were isolated using tissue culture selection (Parker et al., 1990a). The herbicide-resistant phenotype of these plants is conferred by mutations converting plastid-localized, multifunctional, herbicide-sensitive ACCase to herbicide-resistant ACCase (Marshall et al, 1992; Parker et al., 1990a).
  • Two mutant genes for herbicide-insensitive ACCase, Accl and Acc2 have been characterized and both are partially dominant (VanDee et al, 1992).
  • ACCase activity assayed in extracts of developing kernels harvested from herbicide- resistant plants was tested for herbicide inhibition.
  • the embryo and endosperm ACCase activities exhibited similar reductions of herbicide inhibition as ACCase extracted from the leaves of these herbicide-resistant mutants, indicating that the Accl gene was the major ACCase expressed in the embryo and endosperm of the developing corn kernel.
  • homozygous herbicide-resistant and wild-type susceptible kernels were isolated four days after pollination and cultured in vitro to maturity in the presence and absence of a range of herbicide concentrations (Somers et al, 1993).
  • Maize multifunctional ACCase isoforms are encoded by at least four genes, Al, A2, Bl, and B2 (Egli et al., 1997).
  • Type A cDNAs are > 98% identical, and are 95% identical to a type B cDNA.
  • Types A and B encode highly similar N-termini and are both likely to encode plastid-targeted isoforms (Egli and Gengenbach, 1996; Egli et al., 1997; Egli et al., 1995).
  • Herbicide resistance in corn has been mapped to the same chromosomal locations as an ACCase cDNA probe with high identity to both A and B types (Caffrey et al., 1995; Egli et al, 1994; VanDee et al., 1992).
  • Herbicide-tolerant ACCase is expressed in developing corn kernels (Somers et al., 1993) and both A- and B-type cDNAs are found in immature embryos at the time of peak ACCase activity (Egli and Gengenbach, 1996; Somers et al., 1993).
  • Hexaploid wheat In hexaploid wheat, six cDNAs encoding cytosolic ACCase were attributed either to three homoeologous sets of duplicate genes or to allelic differences in each genome (Podkowlinski et al., 1996). Hexaploid wheat also contains three transcriptionally active genes encoding plastidic ACCase with ⁇ 98% exon sequence identity. These genes are located on homoeologous chromosomes (Gornicki et al, 1997). Oat is used for both human food and animal feed. Crop improvement goals for oat involve two divergent end uses in that high oil oat is desired as an animal feed whereas a lower oil oat is desired to make low-fat oat products for human consumption.
  • Low oil in oat may be a value- added trait for the crop that may allow processors to produce low-fat oat products that contain previously identified cholesterol-lowering constituents including, but not limited to, ⁇ -glucans, thus making oats a more healthful human food.
  • Targeted antisense down-regulation of ACCase in the endosperm using an endosperm-specific promoter may avoid problems that would be caused by inhibition of ACCase in the embryo. Determination of the antisense phenotype by detection of reduced ACCase activity and by quantitation of groat oil content in transgenic lines provides direct evidence for the involvement of ACCase in oat seed oil deposition.
  • ACCase cDNA e.g., oat ACCase DNA
  • oat ACCase DNA is employed in an antisense orientation to specifically down-regulate oat endosperm ACCase activity and thus reduce groat oil content.
  • a plant having a dominantly inherited low-oil gene useful for production of low-oil varieties is prepared.
  • oat oil is primarily localized in the starchy endosperm, down-regulation of endosperm ACCase activity is less likely to cause deleterious effects on the embryo.
  • converting oil reduction to a single, dominantly inherited antisense trait is useful in rapid production of low oil varieties in this allohexaploid.
  • Seed from transgenic plants are analyzed for expression of the transgene during development and for oil content at maturity.
  • low oil oat varieties are produced.
  • Reduction of groat oil content may decrease processing problems associated with oil rancidity and can decrease caloric value resulting in a lower fat food.
  • Reduced- fat oatmeal and other oat products may increase the use of oat as a human food and create a value-enhanced crop for producers of the low-fat oat.
  • the preparation of high-energy (high oil) oat feed through use of a high-oil gene is also contemplated. Isolation and Characterization of Oat ACCase cDNAs.
  • oat groat cDNA clone was isolated based on its hybridization to a maize cDNA fragment which included portions of the less conserved mid- peptide sequence and the transcarboxylase domain (Shorrosh et al., 1994). Sequence identity indicated that the oat cDNA (3031 bp) corresponded to maize ACCase nt 3124-6169 (Egli et al., 1995).
  • the 1010-aa predicted polypeptide was nearly identical to portions of wheat (89%) and maize (84%) plastidic multifunctional ACCases; it had lower identity to cytosolic ACCases, including wheat (67.5%) and alfalfa (64.6%) (Gornicki et al, 1994; Shorrosh et al., 1994). Therefore, the oat-91 cDNA is likely to encode a portion of a plastid-targeted, multifunctional ACCase polypeptide.
  • An amplified 'Prairie' oat developing groat cDNA library R.
  • Oat-91 Five colonies per transformation reaction were rescreened (as colony lifts) with an Aflll-BbrPI fragment of maize ACCase cDNA (4281-5797; Egli et al., 1995).
  • Oat-91 was sequenced using the ABI Prizm system (Perkin-Elmer Co ⁇ .) at the Advanced Genetic Analysis Center (University of Minnesota, St. Paul, Minnesota). Sequence identity indicated the 3.05-kb EcoRI insert of oat-91 corresponded to maize ACCase nt 3124-6169 (Egli et al., 1995).
  • Oat ACCase cDNA sequence was compared to other plant ACCases with the BESTFIT program (BESTFIT, Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, Wisconsin).
  • primers are located within the transcarboxylase domain, surrounding the acetyl- CoA binding site of ACCase (50).
  • One I of the oat cDNA library was amplified with maize primers (PCR2/PCR1), using the Expand High Fidelity PCR system (BMB) in a GeneAmp PCR System 9600 thermal cycler (Perkin-Elmer) after denaturing phage heads at 94 °C for 10 minutes.
  • the single, 222-bp product was purified by Wizard PCR Plus (Promega Co ⁇ .) and sequenced in an ABI 377 (Perkin-Elmer) using PCR1 as primer. The resulting sequence appeared to be from oat since it had high identity to maize plastidic ACCase (89%; 4, 12, Egli, unpublished). Additional 5' cDNA was obtained by (i) PCR of the oat cDNA library with T3/PCR1 primers to generate a complex band mixture and (ii) reamplification of 0.1 1 products with maize ACCase primers 1324+ (5'- caatcacgtcagactg)/PCRl to obtain a 1.9-kb product with the same 3' sequence as the PCR2/PCR1 product.
  • oat ACCase cDNA clone oat-91 encodes a plastidic isoform
  • 422 nt of 3 ' insert sequence was translated and compared to predicted amino acid sequences of maize plastidic ACCases, a putative maize cytosolic ACCase, and to multifunctional ACCases from wheat, barley, alfalfa, soybean, Arabidopsis, Brassica, chicken, human, Ustilago, yeast and Cyclotella. Aligned, degapped peptides were used to construct a phylogram by the UPGMA method based on Kimura protein distances (GCG, Madison, Wisconsin).
  • the oat ACCase sequence was > 93% identical to plastidic maize and wheat ACCase sequences, but only 78-83% identical to cytosolic ACCases from maize, wheat, or dicots. The corresponding nucleotide identities were 88-92% and 73-78%, respectively. In the phylogram, monocot ACCases were separated from dicots and grouped according to their known or expected cellular locations ( Figure 25). Both sequence identity and phylogenetic grouping indicate that oat cDNA encodes a plastidic ACCase.
  • Additional 5 ' sequence is obtained by screening the cDNA library with the most 5' oat probes or by PCR of the library with near 5' (upstream) oat plus vector-specific oligonucleotide primers.
  • the 5' end of ACCase message may be obtained by 5 ' RACE PCR (Frohman, 1993).
  • Total RNA is isolated from developing groats, and digested with DNAse.
  • Complementary DNA is synthesized with Superscript II reverse transcriptase (Gibco BRL) by priming with oat ACCase primers.
  • PCR amplification of cDNA utilizes oat primers plus 5' maize primers.
  • chloroplast transit peptide sequences are not well conserved between ACCase genes of maize, wheat, and Brassica, or between different gene families of a single species, and because 5' non-coding sequences also are unlikely to be conserved, completion of the 5' sequence of oat ACCase cDNA is accomplished by 5' RACE-PCR (GIBCO- BRL; Gornicki et al., 1994). 5' RACE products and downstream sequences are sequenced to verify that a chloroplast transit peptide coding sequence is present and to identify primers that may be used to generate a complete or partial oat ACCase cDNA product by PCR.
  • Oat cDNA is reamplified using either the Expand High Fidelity or Long Template PCR kits (Boehringer Mannheim Biochemicals), depending on the length of desired product. Products are blunt- cloned into pBluescript (Stratagene) or a similar vector, or extended with dATP and cloned into pGEM-T (Promega). Adjacent sites in vector polycloning regions or unique sites introduced by PCR are utilized to move oat ACCase cDNA into expression vectors. Co-segregation of Oat ACCase cDNA and a Major Groat Oil QTL
  • a major quantitative trait locus (QTL) controlling groat oil content in oat was recently identified by the oat genome mapping program at the University of Minnesota (See Example XIV).
  • Single factor ANOVA showed that RFLP marker Xcdo665B on linkage group 11 was associated with a QTL accounting for approximately 37% of the variance in groat oil in a Kanota by Ogle mapping population (O'Donoughue et al, 1995).
  • Antisense ACCase cDNAs are constructed using standard procedures in the plasmids that are routinely used for oat transformation (Torbert et al., 1995).
  • the cDNA is ligated in antisense orientation relative to an endosperm-specific promoter and is terminated using the nopaline synthase (NOS) 3' untranslated sequence of the Agrobacterium tumefaciens NOS gene, although other termination regions may be employed.
  • NOS nopaline synthase
  • This antisense cassette is ligated into a plasmid backbone carrying a neomycin phosphotransferase II (npffl) gene under control of the cauliflower mosaic virus 35S promoter as a selectable marker (Torbert et al., 1995).
  • ACCase Complete inhibition of vegetative- or seed-expressed ACCase is likely to be lethal because the enzyme is required for general fatty acid biosynthesis, as shown by the lethality of herbicide inhibition of ACCase in susceptible monocots.
  • tissue-specific isozymes or homo logs of ACCase exist, or if they do, whether they vary significantly in nucleotide sequence. Therefore, it is important to target antisense down- regulation of ACCase to the developing oat endosperm and that the antisense gene exert a partial down-regulation of endosperm ACCase homo logs to avoid deleterious effects of possible endosperm ablation due to complete inhibition of ACCase.
  • the wheat Dy 10 high molecular weight glutenin promoter confers strong expression of GUS in oat and expression is specifically limited to the endosperm. Because of its tissue specificity and the likelihood that its expression pattern does not directly coincide with the expression of ACCase during oat endosperm development, the DylO promoter may be useful in restricting the ACCase antisense transcripts to the oat endosperm resulting in a partial inhibition of ACCase expression and the normal development of endosperm cells. Oat Transformation
  • Elite cultivars of oat can be transformed and on average one transgenic tissue culture is produced that regenerates fertile, transgenic plants per two microprojectile bombardments. Generally, from 15 to 30 transgenic plants are produced from the microprojectile bombardments conducted during one experiment.
  • the oat ACCase antisense plasmid is introduced into regenerable oat tissue cultures of oat genotypes that exhibit high oil content using microprojectile bombardment as described by Torbert et al. (1995). Tissue cultures are selected for resistance to paromomycin conferred by the nptll selectable marker gene.
  • Paromomycin-resistant tissue cultures are regenerated and plants analyzed for the nptll protein using an NPTII ELISA kit (3 ' > 5 ' Inc., Boulder, CO). Plants that express the nptll protein are grown to maturity.
  • the progeny from at least 100 independent transgenic events are then evaluated. Characterization of the ACCase Antisense Phenotype Homozygous T 2 seed are divided for maintenance, advancement to produce greater quantities of seed for more detailed analyses, and for preliminary analysis. Single oat plants may produce up to 100 seed. Therefore, sufficient seed should be available to determine if a transgene phenotype is detectable. Dried mature seed of antisense homozygotes and nontransgenic sister lines are analyzed for oil content initially using nondestructive Near Infrared Reflectance (NIR) spectroscopy. This method requires only 1 g of dehulled groats and is inexpensive. NIR is employed to rapidly identify transgenic lines that exhibit lowered oil content.
  • NIR nondestructive Near Infrared Reflectance
  • Selected low-oil lines are advanced an additional generation. Seed is isolated at different stages of development and stored under conditions to preserve ACCase activity and mRNA for Northern blot analysis. ACCase activity levels and sense and antisense transcript levels are determined to further characterize the antisense phenotype of these plants.
  • oat While oat is considered a beneficial alternative crop with multiple uses, production acreage continues to shrink due to poor profitability and international competition. Yet oat is used as a high quality animal feed and for production of nutritious human food. Numerous positive human health qualities are attributed to consumption of oatmeal. Foremost among these is the cholesterol-lowering property of oats. A major problem confronting oat processors is that current oat varieties have elevated oil content that precludes the production of a low- fat oat product. Thus, in the production of oat products such as oatmeal, the high oil content offsets the benefit of the cholesterol- lowering constituents of the product. Oat oil content is relatively easy to manipulate using conventional plant breeding. However, the trend has been to increase oil content of oats for use as animal feed and only recently has low-oil oat been recognized as important for food products.
  • oat varieties with reduced oil content may be prepared using genetic engineering.
  • a single dominant gene system can vastly simplify conversion of existing agronomically adapted oat varieties to low oil as well as being useful in the production of new low-oil varieties.
  • Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets. Proc. Natl. Acad. Sci. U.S.A., 24:14179-14184 (1997).
  • Robertson, D. S. A possible technique for isolating genie DNA for quantitative traits in plants. J. Theor. Biol., 112:1-10 (1985).
  • Multifunctional acetyl-CoA carboxylase from Brassica napus is encoded by a multi- gene family: Indication for plastidic localization of at least one isoform. Proc. Natl. Acad. Sci. U.S.A.. 24:3465-3470 (1997).
  • IQTJL http ://probe.nalusda.gov: 8000/otherdocs/j qtl j qtl 1995-01 ) ( 1995 a).
  • IQTL http://probe.nalusda.gov:8000/otherdocs/jqtl jqtll995-02) (1995b).

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Abstract

L'invention concerne la totalité de la séquence d'ADNc d'acétyle CoA carboxylase du maïs, ainsi que des procédés servant à conférer une tolérance aux herbicides et/ou à modifier la teneur en huile de plantes par introduction et expression d'un gène d'acétyle CoA carboxylase dans les cellules de ces plantes. Le procédé permettant d'obtenir une tolérance aux herbicides consiste à introduire une cassette d'expression codant une acétyle CoA carboxylase de plante ou une séquence d'ADN antisens complémentaire de la séquence d'un gène d'acétyle CoA carboxylase de plante lié à un promoteur agissant dans des cellules de plantes, dans les cellules du tissu de la plante et à exprimer le gène d'acétyle CoA carboxylase selon un niveau suffisant pour rendre soit acétyle CoA carboxylase, soit les cellules de la plante tolérantes aux herbicides. Le procédé servant à modifier la teneur en huile d'une plante consiste à introduire une cassette d'expression dans des cellules de plantes et à exprimer le gène d'acétyle CoA carboxylase selon un niveau suffisant pour modifier la teneur en huile des cellules. On peut également introduire cette cassette d'expression dans d'autres cellules hôtes afin d'augmenter la productivité d'acétyle CoA carboxylase, de façon à pouvoir utiliser l'enzyme cristallisé, de manière à cribler et à identifier d'autres herbicides se fixant à cet enzyme et l'inhibant. L'invention concerne également une séquence partielle d'ADN codant acétyle CoA carboxylase de l'avoine.
PCT/US1999/014022 1998-06-22 1999-06-22 ADN CODANT ACETYLE CoA CARBOXYLASE DE L'AVOINE WO1999067367A1 (fr)

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AU47044/99A AU4704499A (en) 1998-06-22 1999-06-22 Dna encoding oat acetyl coa carboxylase

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EP1571901A2 (fr) * 2002-12-18 2005-09-14 Agrinomics, LLC Production de plantes dont la teneur en graisse est modifiee
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WO2008015692A2 (fr) * 2006-05-09 2008-02-07 Reliance Life Sciences Pvt Ltd CLONAGE MOLÉCULAIRE ET SÉQUENÇAGE DU GÈNE ACÉTYL CoA CARBOXYLASE (ACCase) PROVENANT DE JATROPHA CURCAS
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