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WO1998005758A1 - STRUCTURE ET EXPRESSION DE LA SOUS-UNITE ALPHA-CAROBXYLTRANSFERASE DE L'ACETYL-CoA CARBOXYLASE HETEROMERE - Google Patents

STRUCTURE ET EXPRESSION DE LA SOUS-UNITE ALPHA-CAROBXYLTRANSFERASE DE L'ACETYL-CoA CARBOXYLASE HETEROMERE Download PDF

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WO1998005758A1
WO1998005758A1 PCT/US1997/013532 US9713532W WO9805758A1 WO 1998005758 A1 WO1998005758 A1 WO 1998005758A1 US 9713532 W US9713532 W US 9713532W WO 9805758 A1 WO9805758 A1 WO 9805758A1
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John B. Ohlrogge
Basil S. Shorrosh
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Board Of Trustees Operating Michigan State University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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

Definitions

  • the present invention relates generally to the ⁇ -carboxyltransferase subunit of acetyl-CoA carboxylase and more particularly, the ⁇ -carboxyltransferase subunit of heterome ⁇ c acetyl-CoA carboxylase from pea (Pis ⁇ m sativ ⁇ m L.) and its use in controlling the carboxylation of acetyl-CoA in plants
  • Acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis leading to the synthesis of malonyl-CoA from acetyl-CoA. Examination of this reaction in vitro and in vivo has implicated it as a key regulatory step for plastidial fatty acid biosynthesis in spinach (Post-Beittenmilier, D., et al. J. Biol. Chem. 266.1858-1865 (1991) and Post-Beittenmiller, D , et al Plant Physiol. 100.923-930 (1992)), barley and maize leaves (Page, R A , et al Biochem Biophys Acta.
  • Cytosolic ACCase has a molecular mass of more than 200 kd and contains the biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and ⁇ - carboxyltransferase (S-CT) subunits as functional domains (Egin-Buhler, B., et al., Eur. J. Biochem. 133:335-339 (1983); Egli, .A., et al., Plant Physiol. 101 :499-506 (1993); Slabas, A.R., et al., Plant Sci.
  • BCCP biotin carboxyl carrier protein
  • BC biotin carboxylase
  • S-CT ⁇ - carboxyltransferase
  • ACCase The plastidial form of ACCase in most plants is similar to prokaryotic ACCase (Alix, J-H. DNA, 8:779-789 (1989); Kondo, H , et al , Proc. NatI Acad. Sci. USA, 88:9730-9733 (1991); Li, S-J., et al., J. Biol Chem 267 855-863 (1992); and Li, S-J., et al., J. Biol. Chem. 267:16841-16847 (1992)) tn that it is a heteromeric enzyme composed of dissociable subunits of different sizes, and it is thus referred to as the multi-subunit (MS) form.
  • MS multi-subunit
  • a purified and isolated nucleic acid sequence encoding the ⁇ - carboxyltransferase ( ⁇ -CT) subunit of heteromeric ACCase is provided.
  • Vectors comprising the nucleic acid sequence, plant cells transformed with the vectors, as well as plants transformed with the nucleic acid sequence and seeds of the transgenic plants, are also provided.
  • the ⁇ -CT nucleic acid sequence may be used to control carboxylation of acetyl-CoA to produce malonyl-CoA.
  • carboxylation of acetyl-CoA to produce malonyl-CoA may be increased or decreased.
  • fatty acid synthesis and elongation in plants and seeds which is dependent on malonyl-CoA may also be increased or decreased.
  • Secondary metabolite production in plants which is also dependent on acetyl-CoA and malonyl-CoA may also be controlled.
  • long-term control of the carboxylation of acetyl-CoA to produce malonyl-CoA may be obtained by genetically altering plants with the ⁇ -CT nucleotide sequence.
  • IEP96 pea chloroplast cDNA of unknown function
  • Figure 1 sets forth the results of the fractionation of pea chloroplast proteins by gel permeation chromatography:
  • Figure 1A shows ACCase and CT activities and protein profiles
  • Figure 1 B shows the determination of CT activity
  • Figure 1C is a Western blot analysis of gel permeation fractions using BC, biotin (BCCP), ?-CT, and ⁇ -CT antibodies;
  • Figures 2A, 2B and 2C show the immunoprecipitation of pea chloroplast ACCase and CT activities by ?-CT and ⁇ -CT antibodies;
  • Figures 3A and 3B show DEAE analysis of gel permeation purified chloroplast ACCase
  • Figure 4 shows two dimensional analysis of gel permeation purified chloroplast ACCase
  • Figure 5 is a photograph of the Western blot analysis of pea leaf proteins.
  • nucleic acid sequence IEP96 encodes the ⁇ -CT subunit of chloroplast heteromeric ACCase.
  • the cDNA and deduced amino acid sequence of IEP96 are set forth in SEQ ID Nos. 1 and 2, respectively. Sequences of the present invention may thus be used to increase or decrease the carboxylation of acetyl-CoA to produce malonyl-CoA in the plastid of plants, thereby increasing or decreasing fatty acid synthesis.
  • a method of controlling carboxylation of acetyl-CoA to produce malonyl-CoA and controlling fatty acid synthesis is thus provided by the present invention.
  • Vectors containing the ⁇ -CT nucleic acid sequence, plant cells transformed with the vectors of the present invention as well as plants containing the sequences of the present invention and seeds produced thereby, are also within the scope of the present invention.
  • the methods of the present invention generally comprise the step of introducing in sense or antisense orientation the ⁇ -CT gene described herein into a plant cell and growing the cell into a plant.
  • genes both sense and antisense orientation
  • genes may also be introduced into the plant cell, e.g., genes encoding the other known subunits of the heteromeric form of ACCase, BCCP (Choi, J-K., et al., Plant Physiol. 109:619-625 (1995)), 0-CT (Sasaki., Y., et al., J. Biol. Chem. 268:25118-255123 (1993)), and BC (Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995)).
  • the ⁇ -CT gene in sense or antisense orientation may be fused to a gene or fragment thereof which allows the ⁇ -CT gene to be transported and expressed in a plant cell.
  • the ⁇ -CT gene in sense or anti-sense orientation in combination with the gene or gene fragment is referred to as a "construct" herein.
  • the constructs of the present invention may contain any regulatory elements necessary and known to those skilled in the art for expression of the ⁇ -CT gene in either orientation.
  • constructs prepared with either seed-specific promoters such as the napin seed storage protein promoter of rapeseed, or with a constitutive promoter such as the cauliflower mosaic virus 35 S (CaMV35S) promoter, are contemplated by the present invention.
  • seed-specific promoters may be more desirable and effective in altering seed oil amounts or composition by avoiding possible deleterious effects in the plant.
  • the constitutive promoter may be more effective in, for example, engineering general herbicide resistance in the whole plant. Plants of the Gramineae family are extremely sensitive to certain "grass-selective" herbicides. This sensitivity is known to result from the inhibition of the homomeric ACCase. Certain grass biotypes which are partially resistant to the herbicides have been shown to have altered ACCase. The heteromeric ACCase is completely resistant to the "grass-selective" herbicides.
  • Gramineae plants such as Gramineae grasses (e.g., corn, wheat, barley, oats, etc.), can provide resistance in these species to such herbicides.
  • genes encoding the other subunits of the heteromeric ACCase may also be introduced in addition to or in combination with the gene of the present invention.
  • a constitutive promoter such as CaMV35S would be used in the construct to create such transgenic plants.
  • the present invention also provides a method of controlling plant and seed fatty acid synthesis and elongation.
  • Increasing seed fatty acid synthesis by overexpressing the ⁇ -CT gene is useful in increasing oil content of rapeseed, soybean, or other oilseed crops.
  • overexpression of the homomeric ACCase increases the malonyl-CoA/acetyl-CoA ratio and increases the amount of oil stored in the seeds. Because the homomeric ACCase is a 250 kDa protein, achieving high levels of expression and successfully targeting to the plastid at sufficiently high expression levels, may be more difficult than the overexpression of heteromeric ACCase.
  • Increasing oil content of oil seeds by overexpressing the ⁇ -CT gene is therefore advantageous.
  • the heteromeric ACCase gene described herein encodes a plastid protein and, since fatty acid synthesis takes place primarily in the plastid, a construct which includes the gene described herein does not require a plant plastid transit peptide.
  • An effective increase in ACCase activity in the plastid thus results when the plastid ACCase gene of the present invention is overexpressed. It should be appreciated that decreasing seed fatty acid synthesis by decreasing gene expression is also useful in producing "low-fat" seeds such as low-fat peanuts.
  • acetyl-CoA and malonyl-CoA are precursors of various plant secondary metabolites. Decreasing expression of ⁇ -CT may therefore decrease fatty acid synthesis in the plastid and subsequent long chain fatty acid synthesis in the cytosol This increases the amount of cytosolic malonyl-CoA available for synthesis offlavonoids, isoflavonoids, and other secondary metabolites Furthermore, decreasing expression of the ⁇ -CT gene set forth herein may ultimately decrease the amount of malonyl-CoA present and increase the amount of acetyl-CoA present Thus, altering expression of the ⁇ -CT gene may favorably alter the amount of acetyl-CoA or malonyl-CoA available for production of secondary plant products, many of which have value in plant protection against pathogens or for medicinal or other uses Furthermore, it is not necessary that these products be naturally present in plants.
  • bacterial genes may be introduced into plants to produce polyhydroxybutyrate which can be used to synthesize biodegradable plastics Poirier Y et al , Science 256 520-524 (1992) Production of polyhydroxybutyrate or other acetyl-CoA derived products in a plant will require adequate supply of cellular and plastidial acetyl-CoA If the fatty acid synthesis pathway is drawing on this acetyl-CoA supply for oil storage, the amount available for alternative, higher-value products will be less Therefore, inhibition of the fatty acid synthesis pathway may be desirable to allow diversion of more carbon into products other than fatty acids, e.g., increasing the acetyl-CoA to malonyl-CoA ratio by decreasing ACCase gene expression may allow more carbon flux into polyhydroxybutyrate production thereby resulting in higher yields of polyhydroxybutyrate or other acetyl-CoA derived products
  • the methods of the present invention further include introducing the constructs of the present invention including the sense or antisense orientation of the ⁇ -CT gene into a plant cell, and growing the cell into a callus and then into a plant; or, alternatively, breeding a transgenic plant produced from the above method with a second plant to form an F1 or higher hybrid (e.g., F2)
  • constructs containing the nucleotide sequences of the present invention may be introduced into plants by cocultivation with Agrobactenum containing the construct Transgenic plants are therefore produced by the methods of the present invention and are also contemplated by the present invention .
  • nucleic acid is intended to mean natural and synthetic linear and sequential arrays of nucleotides and nucleosides, e.g., cDNA, genomic DNA (gDNA), mRNA, and RNA, oligonucleotides, oligonucleosides, and derivatives thereof.
  • cDNA genomic DNA
  • gDNA genomic DNA
  • mRNA mRNA
  • RNA oligonucleotides
  • oligonucleosides oligonucleosides
  • sense orientation refers to the orientation of a gene such that its RNA transcript, following removal of introns, is translatable into the polypeptide product of the gene.
  • antisense orientation is used to mean the opposite orientation of a gene such that its transcript is complementary to the normal transcript of the gene when in sense orientation.
  • encoding is intended to mean that the subject nucleic acid may be transcribed and translated into either the desired polypeptide or the subject protein in an appropriate expression system, e.g., when the subject nucleic acid is linked to appropriate control sequences such as promoters, operators, regulators, and the like, in a suitable vector (e.g., an expression vector) and when the vector is introduced into an appropriate system or cell.
  • polypeptides includes not only full length protein molecules but also fragments thereof which, by themselves or with other fragments, generate substantially similar physiological activity as the full length protein. It will further be appreciated that synthetic polypeptides of the protein of the present invention are also within the scope of the invention and can be manufactured according to standard synthetic methods.
  • oilseed plant and “oilseed crop” are used interchangeably herein and refer to those plants and crops known to those skilled in the art as part of the oilseed variety, including but not limited to rapeseed, soybean, Crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed and sunflower.
  • the term “capable of hybridizing under stringent conditions” is used to mean annealing a first nucleic acid to a second nucleic acid under stringent conditions (defined below).
  • the first nucleic acid may be a test sample
  • the second nucleic acid may be a portion of the nucleic acid sequence set forth in SEQ ID No. 1.
  • Hybridization of the first and second nucleic acids is conducted under stringent conditions, from low stringency to high stringency, e.g., at a temperature and/or salt content, which tend to disfavor hybridization of noncomplementary nucleotide sequences.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.
  • SSC sodium chloride/sodium citrate
  • OX SSC at 50° C are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C to high stringency conditions, at about 65° C. It will be appreciated, however, that although reference herein is made to nucleic acids capable of hybridizing under stringent conditions, hybridization in the practice of the present invention need not actually be conducted under such conditions. The following Specific Example further describes the present invention.
  • the formation of the product is completely dependent on the presence of biotin methyl ester as the carboxyl group acceptor, and is linear with time and enzyme.
  • the CT activity of gel- permeation purified ACCase preparations are 1-5 % of ACCase activity. This relatively low activity can most likely be attributed to the use of biotin methyl ester rather than BCCP as acceptor and because the back rather than forward reaction is measured.
  • a pea cDNA, IEP96 was obtained which has sequence similarity to the ⁇ -CT subunit of E. co// ' ACCase. Hirsh, S., et al., Plant Mol. Biol. 27:1 173-1181 (1995).
  • the first 300 amino acids of the proposed mature IEP96 protein is 47% identical and 68% similar to the 35 kDa ⁇ -CT subunit of E. coli ACCase. Li, S.J. et al. J. Biol. Chem. 267:16841-16847 (1992).
  • IEP96 is unrelated to other ACCase sequences and instead resembles several cytoskeleton proteins such as integrin, myosin heavy chain and US01 , a cytoskeleton component in yeast involved in intracellular protein trafficking from the ER to the Golgi. Nakajima, H., et al., J. Cell Biol. 113:245-260 (1991). Moreover, because there are numerous carboxylation reactions in plants which are catalyzed by proteins with related sequences, IEP96 may have represented a subunit of any of a number of enzymes.
  • IEP96 may be a component of the heteromeric-ACCase enzyme. While both the ⁇ -Cl and IEP96 protein levels gradually decreased after fraction 32, the BC and BCCP protein levels dropped at a faster rate. These results correlate with the detection of CT activity, but not ACCase activity, in fractions 32 to 44 and may indicate that the ACCase subunits are undergoing partial dissociation during chromatography.
  • the gel permeation elution patterns described above were reproducible throughout at least six independent pea chloroplast isolations.
  • FIG. 2 shows the immunoprecipitation of pea chloroplast ACCase and CT activities by ⁇ -Cl and IEP96 ( ⁇ -CT) antibodies.
  • Figures 2A and 2B show the percent of maximum ACCase or CT activities remaining after treatment with antibodies to IEP96 ( ⁇ -CT), ⁇ -Cl or pre-immune sera (Pre ⁇ - CT).
  • Figure 2B shows the resolution of the proteins immunoprecipitated with antibodies to IEP96 ( ⁇ -CT), ⁇ -Cl, and pre-immune sera (Pre ⁇ -CT) by 7.5% SDS-
  • Figure 3 provides the DEAE analysis of gel permeation purified chloroplast ACCase, with Figure 3A showing CT and ACCase activities in fractions 6 and 9 after mixing, and Figure 3B showing that individual DEAE column fractions were resolved by 10% SDS-PAGE, blotted to nitrocellulose and probed with BC, biotin (BCCP) and IEP96 ( ⁇ -CT) antibodies.
  • BCCP biotin
  • IEP96 ⁇ -CT
  • the ⁇ - CT and IEP96 proteins migrated together in the first dimension, entering about 2 cm into the gel, between MW markers thyroglobulin (669 kd) and ferritin (440 kd).
  • the mature IEP96 has a calculated pl (from its primary sequence) of 9.7 and therefore, at pH 8.5-8.7 (running buffer) this protein is positively charged and would not be expected to enter the native gel.
  • 35 S-labeled IEP96 did not enter the first dimension (data not shown).
  • Pea chloroplast ACCase dissociates into two complexes. These data demonstrate that pea chloroplast-ACCase dissociates into two complexes ( ⁇ - CT/IEP96 and BC/BCCP) that can be resolved by either DEAE chromatography or native PAGE. These results extend the observation of Alban, C, et al., Biochem J. 300:557-565 (1994), that pea ACCase could be dissociated into two inactive fractions by differential (NH 4 ) 2 S0 4 precipitation and that the activity could be restored by simple recombination of the two fractions. Thus, the accumulated data suggest that, unlike in E.
  • a complex of all four subunits of pea ACCase remains associated during gel filtration and can be at least partially reconstituted from components separated by salt or anion-exchange fractionation.
  • the BC/BCCP complex and the ⁇ -Cll ⁇ -Cl complexes are more stable than the entire 600-700-kd ACCase complex.
  • the ⁇ -Cll ⁇ -Cl complex is clearly the most stable, and this is perhaps related to the strikingly different pl values.
  • the calculated pl of the pea ⁇ -Cl subunit is 4.1 whereas the ⁇ -CT subunit pl is 9.7. Based on their dissociation by salt, anion-exchange chromatography and PAGE, the BC/BCCP complex most likely interacts with the ⁇ -Cll ⁇ -Cl complex through ionic interactions.
  • BC activity was purified from pea chloroplasts. Alban, C, et al., Plant Physiol. 109:927-935 (1995). It was reported that the structure of the active BC corresponded to a complex made up of BCCP with a MW of 38 kd and a BC polypeptide of 31 kd. The 31 -kd polypeptide is substantially different in size from the 50-kd polypeptide previously reported to be the BC subunit of the pea ACCase. Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995) and Roesler, K.R., et al., Planta, 198, in press (1995).
  • pea Native structure of pea ACCase. To date, four subunits of the heteromeric ACCase have been characterized and cloned.
  • the deduced mature molecular weights of ⁇ -Cl (pea), BC (tobacco), BCCP (Arabidopsis), and ⁇ -CT (IEP96) (pea) are 67, 51 , 21 , and 91 kd, respectively, whereas the apparent molecular weights determined by SDS-PAGE are 86 (pea), 51 (pea), 38 (pea), and 91 kd (pea), respectively.
  • the plant heteromeric ACCase would have a native holoenzyme mass of 230 kd (based on the deduced molecular weights) or 266 kd (based on the apparent molecular weights). If plant heteromeric ACCase has the same organization of subunits as found in E. coli (two of each subunit) then the native complex would have a mass in the range of 460-532 kd. This is somewhat lower than the MW of 600-700 kd determined by gel permeation chromatography. Alternatively, if three of each subunit make up the native ACCase then the native MW could be 690 to 798 kd.
  • chloroplast ACCase elutes anomalously during gel filtration chromatography, has other unidentified subunits, or its molar subunit stoichiometry is different than that of E. coli ACCase.
  • the deduced IEP96 protein is approximately twice the size of the E. coli ⁇ -CT and a putative red algal ⁇ -CT (Genbank Accession number Z33874).
  • the first 300 amino acids of the proposed mature IEP96 protein share 47% identity and 68 - 71 % similarity with E. coli and red algal ⁇ -CT protein sequences.
  • the remaining amino acid sequence (positions 346-873) of IEP96 is unrelated to other ACCase sequences and instead resembles several cytoskeleton proteins such as integrin, myosin heavy chain and USO1 , a cytoskeleton component in yeast involved in intracellular protein trafficking from the ER to the Golgi.
  • Such proteins are characterized by coiled-coil helical structures, and heptapeptide repeats having hydrophobic amino acids in every fourth and seventh position.
  • GCG motif a structural motif program identified in IEP96 a motif (IVIGEGGSGGALAIGC) similar to a prokaryotic membrane lipoprotein lipid attachment site.
  • membrane lipoproteins are synthesized with a precursor signal peptide, which is cleaved by a specific lipoprotein signal peptidase (Signal Peptidase II).
  • the peptidase recognizes the consensus sequence (D,E,R,K,) ⁇ 6 residues ⁇ (L,A) ⁇ 2 residues ⁇ (I) (G) C and cleaves upstream of the cysteine residue to which a glyceride-fatty acid lipid is attached.
  • ACCase assay is based on the acetyl-CoA dependent formation of acid-stable radioactive malonyl-CoA from H 1 C0 3 " and acetyl-CoA. Sauer, A., et al., Naturforsch 39C:268-275 (1984); Alban, C., et al., Biochem. J. 300:557-565 (1994).
  • [2- 14 Cjmalonyl-CoA (50 //Ci/ ⁇ mol) was prepared from [1- 14 C]acetate using pea chloroplast extracts as described by Roughan, G., Biochem. J. 300:355-358 (1994).
  • Carboxyl transfer from [2- 14 C]malonyl-CoA to free d-biotin methyl ester was determined in a 20//L reaction containing 5 mM biotin methyl ester, 50 mM Tris pH 8.1 , 12500 d.p.m. of [2- 14 C]malonyl-CoA, and 6 ⁇ l enzyme. After 15 min at 30° C, 5 ⁇ of 2 M neutral hydroxylamine was added.
  • a disadvantage of the assay is that the TLC application and separation of the modified substrate and product require approximately 6 h. Also, the modified product (acetylhydroxamate) evaporates with very long storage at room temperature. Fractionation of pea chloroplast proteins by gel permeation chromatography. Chloroplasts were isolated from pea [Pisum sativum cv little admire (Burpee)] seedlings as described by Roughan, P.G., Methods Enzymol. 148:327-337 (1987).
  • Chloroplasts were lysed in 50 mM Hepes (pH 8.0), 0.1 % Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine hydrochloride, and 5 mM e-amino-n-caproic acid. Complete lysis was achieved by freezing the resuspended chloroplasts at -80° C.
  • the stromal fraction recovered after centrifugation at 37000 g for 30 min, was loaded onto a HiPrep 16/60 Sephacryl S-300 HR column (Pharmacia, Uppsala, Sweden) and equilibrated with lysis buffer minus Triton X-100 (Solution A). Fractions were collected at 1.5 ml/fraction and assayed for ACCase and CT activities, and used for immunoprecipitations and immunoblots. Protein concentrations were determined by the Bradford, M.M., Anal. Biochem. 72:248-254 (1976) method using bovine gamma globulin as a standard.
  • Immunoglobulin G was prepared from both immune and preimmune sera using diethylaminoethyl (DEAE)-cellulose chromatography.
  • the IgG immune and preimmune sera (200 ⁇ g of each) were lyophilized and resuspended in solution B [solution A (described above) containing 0.25% (w/v) gelatin], 1 % (w/v) casein, and 2% (w/v) BSA] and incubated at 4° C for 1 h to overnight.
  • IgG-sorb with binding capacity of 2 mg/mL IgG (The Enzyme Center, Maiden, MA, USA) was mixed with an equal volume of solution B and incubated at 4° C for 1 h to overnight. Subsequently, the blocked IgG-sorb was aliqated into fractions that can bind at least 60 ⁇ g of IgG, and pelleted down by centrifugation. The blocked immune or pre-immune IgG (10, 30, and 60 ⁇ g) was incubated with the enzyme (40 ⁇ L gel permeation fraction) in a total volume of 64 ⁇ L (made up in solution B) for 1 h on ice.
  • the antigen-antibody complexes were mixed with the IgG-sorb pellets and stored on ice for 30 min.
  • the antigen-antibody complexes were precipitated by centrifugation at 14000 g and the supernatant was assayed for ACCase and CT activities.
  • the protein pellets were boiled in SDS sample buffer (Laemmli, U.K. Nature, 227:680-685 (1970)), resolved by SDS-PAGE, and blotted to nitrocellulose. Two dimensional gel electrophoresis and blotting procedures.
  • Chloroplast proteins resolved by gel permeation chromatography (1 mL each of fractions 27 to 31 ) were pooled and loaded on a 1 mL RESOURCE Q column (Pharmacia) equilibrated with solution C (20 mM Tris pH 8.5 / 10% glycerol). Protein fractions (20 fractions) were collected at 1.0 mL/fraction using a buffer gradient formed by mixing solution C with the elution buffer D (solution C plus 0.5 M NaCI). At the end of the gradient, solution C containing 1 M NaCI was used to elute any remaining proteins bound to the column. An aliquot (38 ⁇ L each) from collected fractions was resolved by SDS-PAGE and immunoblotted.
  • ORGANISM Pisum sativum
  • I ORGANELLE Chloroplast
  • i x FEATURE :
  • GCA GAT GGC A ⁇ ATA CCG GAG CCC CTG GCT GGT GCA CAT ACT GAT CCA 1347 Ala Asp Gly He He Pro Glu Pro Leu Ala Gly Ala His Thr Asp Pro 350 355 360 AGT TGG ATG TCT CAA CAG ATT AAA A ⁇ GCA ATC AAT GAA GCT ATG GAT 1395 Ser Trp Met Ser Gin Gin He Lys He Ala He Asn Glu Ala Met Asp 365 370 375

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Abstract

La présente invention concerne la séquence nucléotidique codant pour la sous-unité α-carboxiltransférase (α-CT) de l'acétyl-CoA carboxylase et de sa séquence d'aminoacides déduite. L'invention concerne aussi des vecteurs comprenant la séquence nucléotidique, des cellules végétales transformées au moyen des vecteurs, ainsi que des plantes transformées au moyen de la séquence nucléotidique et des graines de la plante transgénique. En régulant l'expression du gène α-CT, on peut commander la carboxylation de l'acétyl-CoA. Ainsi, en introduisant des constructions du gène de la présente invention selon une orientation sens ou antisens, on peut augmenter ou diminuer la carboxylation de l'acétyl-CoA pour produire du malonyl-CoA et réguler la synthèse des acides gras et l'élongation dans les plantes et les graines qui dépendent du malonyl-CoA. La figure illustre les activités de ACCase et CT ainsi que les profils des protéines.
PCT/US1997/013532 1996-08-02 1997-08-01 STRUCTURE ET EXPRESSION DE LA SOUS-UNITE ALPHA-CAROBXYLTRANSFERASE DE L'ACETYL-CoA CARBOXYLASE HETEROMERE WO1998005758A1 (fr)

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AU41462/97A AU4146297A (en) 1996-08-02 1997-08-01 Structure and expression of the alpha-carboxyltransferase subunit of heteromeric-acetyl-coa carboxylase

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US60/023,384 1996-08-02

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2000001828A1 (fr) * 1998-07-01 2000-01-13 Iowa State University Research Foundation, Inc. Agent promoteur du cac1, cac2 ou cac3 de l'arabidopsis thaliana modifie, element suppresseur du cac1, cac2 ou cac3 de l'arabidopsis thaliana, et leurs procedes d'utilisation
EP1241262A3 (fr) * 2001-03-13 2002-12-04 Nara Institute of Science and Technology Procédé pout augmenter la synthèse d'acides gras dans une plante
US6566584B1 (en) 1998-08-20 2003-05-20 Pioneer Hi-Bred International, Inc. Compositions and methods for altering an acetyl-CoA metabolic pathway of a plant
WO2018009626A3 (fr) * 2016-07-07 2018-02-15 The Curators Of The University Of Missouri Augmentation de la teneur en huile végétale par amélioration de l'activité de l'acétyl-coa carboxylase
US10883113B2 (en) 2015-08-28 2021-01-05 The Curators Of The University Of Missouri Increasing plant oil content by altering a negative regulator of acetyl-coa carboxylase
US12286649B2 (en) * 2018-05-30 2025-04-29 The Curators Of The University Of Missouri Methods of altering seed weight and seed oil content by manipulating alpha-carboxyl-transferase (a-CT) activity via carboxyl-transferase interaction (CTI) protein expression

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WO1996032484A2 (fr) * 1995-04-14 1996-10-17 Arch Development Corporation COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001828A1 (fr) * 1998-07-01 2000-01-13 Iowa State University Research Foundation, Inc. Agent promoteur du cac1, cac2 ou cac3 de l'arabidopsis thaliana modifie, element suppresseur du cac1, cac2 ou cac3 de l'arabidopsis thaliana, et leurs procedes d'utilisation
US6566584B1 (en) 1998-08-20 2003-05-20 Pioneer Hi-Bred International, Inc. Compositions and methods for altering an acetyl-CoA metabolic pathway of a plant
EP1241262A3 (fr) * 2001-03-13 2002-12-04 Nara Institute of Science and Technology Procédé pout augmenter la synthèse d'acides gras dans une plante
US10883113B2 (en) 2015-08-28 2021-01-05 The Curators Of The University Of Missouri Increasing plant oil content by altering a negative regulator of acetyl-coa carboxylase
US11959087B2 (en) 2015-08-28 2024-04-16 The Curators Of The University Of Missouri Increasing plant oil content by altering a negative regulator of acetyl-CoA carboxylase
WO2018009626A3 (fr) * 2016-07-07 2018-02-15 The Curators Of The University Of Missouri Augmentation de la teneur en huile végétale par amélioration de l'activité de l'acétyl-coa carboxylase
US11802286B2 (en) 2016-07-07 2023-10-31 The Curators Of The University Of Missouri Increasing plant oil content by improving activity of acetyl-CoA carboxylase
US20230416760A1 (en) * 2016-07-07 2023-12-28 The Curators Of The University Of Missouri Increasing plant oil content by improving activity of acetyl coa carboxylase
US12286649B2 (en) * 2018-05-30 2025-04-29 The Curators Of The University Of Missouri Methods of altering seed weight and seed oil content by manipulating alpha-carboxyl-transferase (a-CT) activity via carboxyl-transferase interaction (CTI) protein expression

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