WO1994023027A2 - Gene vegetal specifiant la carboxylase du coenzyme a d'acetyle, et plantes transformees contenant ce gene - Google Patents

Gene vegetal specifiant la carboxylase du coenzyme a d'acetyle, et plantes transformees contenant ce gene Download PDF

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Publication number
WO1994023027A2
WO1994023027A2 PCT/GB1994/000653 GB9400653W WO9423027A2 WO 1994023027 A2 WO1994023027 A2 WO 1994023027A2 GB 9400653 W GB9400653 W GB 9400653W WO 9423027 A2 WO9423027 A2 WO 9423027A2
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accase
plant
dna
rape
cdna
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PCT/GB1994/000653
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WO1994023027A3 (fr
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Anthony Rysard Slabas
Keiran Michael Elborough
Simon William Jonathan Bright
Philip Antony Fentem
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Zeneca Limited
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Priority to AU64331/94A priority Critical patent/AU6433194A/en
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Publication of WO1994023027A3 publication Critical patent/WO1994023027A3/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

Definitions

  • This invention relates to a plant gene specifying the enzyme acetyl Coenzyme A carboxylase (ACCase) and to plant genomes genetically transformed with the said gene.
  • ACCase acetyl Coenzyme A carboxylase
  • the invention relates to ACCase genes from plants of the Brassica species, especially Brassica napus (oilseed rape) and control of expression of the gene by Brassica plants which are genetically transformed with the gene or its antisense configuration.
  • Acetyl Coenzyme A carboxylase is one of the genes involved in the synthesis of oil by oil-producing crops such as oilseed rape. Variation of the expression of that gene leads to alteration in the quantity and/or quality of the oil produced.
  • An object of the invention is to provide a gene specifying ACCase in plants.
  • partial cDNAs specifying ACCase, isolated from seed of Brassica napus. having the nucleotide sequences set forth in Figures 6 and 12, and variations thereof permitted by the degeneracy of the genetic code.
  • the invention further provides the partial cDNA, isolated from wheat germ, having the nucleotide sequence set forth in Figure 4, and variants thereof permitted by the degeneracy of the genetic code.
  • Also provided by this invention is the full length genomic DNA specifying ACCase from Arabidopsis thaliana having the nucleotide sequence set forth in Figure 8, and variants thereof permitted by the degeneracy of the genetic code.
  • the invention further provides the following clones,
  • the present invention also provides genetically transformed plants, plant cells and plant parts, containing a DNA of the invention or fragment thereof in sense orientation or a complete or partial sense or antisense variant thereof * 0 It is preferred that the plant be of a species which produces substantial quantities of oil, rather than starch. Such plant species are well known and are simply referred to as "oil-seed” crops and include, oilseed rape, canola, soya and sunflower. Methods for the 5 genetic transformation of many oil crops are known; for example, transformation by Aqrobacterium tumefaciens methods are suitable for most. Such methods are well-described in the literature and well-known and extensively practised in the art.
  • fatty acids which are the building blocks of plant oils utilise the substrate acetyl Coenzyme A which is the same substrate required by 10 the polyhydroxyalkanoate genes.
  • Methods for the regulation of gene expression are well-known in the art. Two principal methods are commonly employed, these being referred to loosely as “sense” and "antisense” regulation. In antisense regulation a gene construct is assembled which,, when : 0 inserted into a plant cell, results in expression of a messenger RNA which is of complementary sequence to the messenger produced by a target gene.
  • the theory is that the complementary RNA sequences form a duplex thereby inhibiting translation to protein.
  • the complementary 5 sequence may be equivalent in length to the whole sequence of the target gene but a fragment is usually sufficient and is more cnvenient to handle.
  • sense regulation a copy of the target gene is inserted into the plant genome. Again this may be a full length or partial 0 sequence.
  • a range of phenotypes is obtained from which individuals in which the expression of the protein encoded by the target gene is inhibited may be identified and isolated as may individuals where expression of the gene product is increased.
  • Sense regulation using partial sequences tends to favour inhibition. The mechanism is not well understood. Reference is made to
  • the clones of the invention may be used to probe
  • genomic or cDNA libraries 10 plant DNA ( genomic or cDNA libraries) to obtain homologous sequences. These may be truncated or full length cDNAs or genomic DNAs for ACCase genes from, for example, wheat, or oil crops such as rape, canola, soya, sunflower, maize, oil palm and coconut.
  • Partial cDNAs of rape seed ACCase may be used in conjunction with a plant-recognised promoter to create an expression cassette (partial sense or antisense) for use in transforming rape plants to down-regulate production of the ACCase enzyme. This will give plants 0 with a lower oil content or oil of altered quality.
  • the same cassette can be used to down-regulate the production of ACCase enzyme in other plants of the Brassica species.
  • cDNAs isolated from other crops can be used to create expression cassettes (partial, sense or antisense) for use 5 in transformation of these crops in order to modify the oil content.
  • Down-regulation of oil synthesis can be used to divert the substrate, acetyl Coenzyme A, into synthesis of alternative storage 0 materials such as starch, protein, or novel polymers introduced by genetic modification, for example polyhydroxyalkanoates.
  • Full length clones of rape or Arabidopsis ACCase DNA can be used to create expression cassettes, either with powerful promoters, or by inserting extra gene copies, to promote over-expression of ACCase in rape or other oil crops, leading to plants with enhanced oil content in the seed.
  • the ACCase DNA may also be put under 5 the control of a seed-specific promoter such as the napin promoter, which has a different window of expression from the ACCase promoter during seed development. In this way the period over which ACCase is expressed in the developing seed is extended, and the oil content of the 10 seeds increased.
  • Genomic DNAs of rape ACCase can be used to recover the promoter of the ACCase gene.
  • This promoter can be used to generate RNA in a tissue-specific and developmentally regulated fashion.
  • the promoter so 15 generated may promote the expression of ACCase, or it may control the expression of a gene construct placed after it (for example the structural gene of a different enzyme) which will then be expressed specifically in the developing seed.
  • the full length cDNA and genomic DNA of rape or Arabidopsis ACCase contain a sequence between the translation start site and the N-terminal sequence of the mature protein, known as a "transit peptide" sequence. This directs the gene product to the plastids and is 5 cleaved off during import of the protein into the plastids. This transit peptide sequence may be used in gene fusions to direct different gene products to the plastids.
  • Monocotyledonous plants such as wheat, barley, 0 maize and rice, are normally sensitive to the aryloxyphenoxy- propionate and alkylketone herbicides to which the dicotyledonous plants are normally resistant.
  • Monocots with resistance to these herbicides may be created by: (a) transforming ACCase from a dicotyledonous species such as rape and Arabidopsis, into the monocot genome;
  • Partial cDNAs of rape seed ACCase of this invention may be used in conjunctipon with a plant-recognised promoter to create an expression cassette (partial sense or antisense) for use in transforming plants to down-regulate production of the cytosolic ACCase. This will alter oil quality by inhibiting production of long chain fatty acids) chain length greater than about C18) .
  • a second plastid form of ACCase has been identified in plants.
  • This ACCase is composed of dissociable sub-units for transcarboxylase, biotin carrier protein (BCP) and biotin carboxylase (BC) .
  • the transcarboxylase gene is encoded by the chloroplast genome; BCP and BC are nuclear encoded. Sequence homology between the cDNAs of the invention and the BCP and BC may be used to isolate BCP and BC. Sense and antisense constructs may be raised against BCP and BC in order to effect down-regulation of these genes. 9.
  • the cDNAs of the invention may themselves have sufficient homology with the BCP and BC genes to be used directly for the down-regulation of these genes.
  • the deduced amino acid sequence for wheat ACCase cDNA shows complete homology in four regions of sequence to the amino acid sequences obtained from four peptides isolated from the ACCase enzyme purified from wheat embryo.
  • the deduced amino acid sequence shows high homology with both" the rat and chicken ACCase genes. High homology at the amino acid level with maize leaf ACCase was found, with two sections of 48 amino acids completely conserved.
  • the deduced amino acid sequence from the rape seed partial cDNA (pRSl) sequence shows high homology to the sequences of the maize leaf cDNA and the chicken, rat, yeast and algal ACCase genes.
  • the deduced amino acid sequence from the Arabidopsis genomic DNA shows high homology with the rat, chicken and yeats ACCase genes.
  • High homology with the amino acid sequence of the rape seed ACCase partial cDNA (pRSl) was found, with one section of 48 amino acids almost completely conserved.
  • Figure 1 shows the elution profiles of wheat embryo ACCase from Q-Sepharose ( Figure 1A) and Blue-Sepharose ( Figure IB) during purification of the enzyme.
  • the dotted line represents the sodium chloride gradient concentration and the activity of ACCase, represented by the boxes, was measured as described hereinbelow;
  • Figure 2k shows an SDS PAGE gel of wheat embryo ACCaseshowing the alteration in mobility caused by the binding of streptavidin.
  • Lane 1 contains 500ng myosin (200kDa) ;
  • lane 2 contains lO ⁇ l Post Blue-sepharose material without Streptavidin; and
  • lane 3 contains 10 ⁇ lPost Blue-sepharose material with streptavidin.
  • ACCase is indicated by asterisks (*) at its normal migration and that of the ACCase/streptavidin complex respectively.
  • Figure 2B shows an SDS PAGE gel of purified wheat embryo ACCase, with the 220kd band taken for sequencing indicated.
  • Lane 1 contains l ⁇ l Post Blue-sepharose material and lane 2 contains 10 ⁇ l Post Blue-sepharose material;
  • Figure 3 shows a comparison of four sections of amino acid sequence deduced from the pKlll wheat ACCase cDNA with the amino acid sequences obtained from four peptides isolated fr_om the purified wheat embryo ACCase enzyme;
  • Figure 4 shows the sequence of the sense strand of the wheat embryo ACCase clone pKlll, with three-phase translation shown. The sequences homologous with the peptide amino acid sequences are underlined;
  • Figure 5 shows a dot matrix plot of the deduced amino acid sequence of wheat ACCase clone pKlll against that of the maize ACCase clone pA3;
  • Figure 6 shows the derived amino acid sequence from the rape cDNA encoding the transcarboxylase domain of ACCase.
  • the amino acid sequence is translated from the first open reading frame shown pictorially.
  • the full vertical lines represent stop codons and the half vertical lines ATG sequences.
  • Figure 7 shows the rape transcarboxylase domain comparison with known ACCase sequences.
  • the Dot Matrix 5 (DNA Strider, Stringency 9 Window 21) of derived rape
  • ACCase amino acid sequence (transcarboxylase domain) is compared against rat, yeast and algal (Chlorellal ACCase.
  • Figure 8 shows the 5' sequence from the sense strand of the Arabidopsis genomic subclone pKLU81, with three 10 phase translation shown.
  • Figure 9 shows the 3' sequence from the sense strand of the Arabidopsis genomic subclone pKLU ⁇ l, with three- phase translation shown.
  • Figure 10 shows a comparison of the Arabidopsis pKLU81 15 5' translated open reading frame with the sequences of rat and chicken ACCase genes obtained from SWISSPROT database.
  • Figure 11 shows the assignment of domain order to higher plant ACCase.
  • Figure 11A is a schematic diagram 0 showing the yeast ACCase domain orders relative to the sequenced regions (hatched boxes) of the Arabidopsis genomic clone. The areas of sequenced genomic clone are named A-F for easy identification in the text.
  • Figure llBiii shows a DNA sequence comparison by dot matrix (DNA Strider, Stringency 15 Window 23) of the rape transcarboxylase domain of ACCase and areas E/F from the Arabidopsis genomic clone.
  • Figure 12 shows the rape ACCase biotin binding domain sequence.
  • Figure 12Ai shows the derived amino acid sequence
  • Figure 12Aii shows the direct comparison of the biotin binding site with that of the corresponding
  • Figure 12B shows the dot matrix comparison (DNA Strider, Stringency 9 Window 21) of derived rape ACCase amino acid sequence (biotin binding domain) against yeast
  • Figure 13 shows ACCase Southern blot analyses of rape and Arabidopsis genomic DNA. Restrriction endonuclease digested DNA was hybridised to the Arabidopsis ACCase genomic clone by Southern blot. Hybridisation and washing 0 conditions were carried out as described in materials and methods. The blot shown was exposed for 5 days, further exposure provided no extra information. Both 1 Hindlll and OX 174 Haelll DNA markers (indicated on the left hand side) were run on the same 1% gel and viewed by ethidium 5 bromide staining/UV.
  • Figure 14 shows Northern blot analysis of rape ACCase.
  • the graph shows the oil content as total fatty acid (mg/seed) in relation to the stage of rape e bryogenesis. Details of the analysis method are 0 presented in materials and methods.
  • the three Northern blots shown, relating to the different stages of embryogenesis, are all derived from the same blot after successive stripping.
  • the probes used were as indicated in the text and the amount of polyA+ RNA was 5 ⁇ g for each stage. Hybridisation and washing conditions were as in materials and methods. Exposure was for 7 days.
  • Acetyl CoA Carboxylase activity was assayed by incorporation of radioactivity from 14 C- bicarbonate into 15 non-volatile malonyl CoA (Hellyer et al 1986) .
  • Escherichia coli cells XLl-Blue and KW251 cells were grown overnight in 50 ml LB media/0.2% Maltose/50 ⁇ g/ml Tetracycline/10 mM MgS0 .
  • the cells were spun down at 3000 g for 10 mins and the cell pellet taken up in 2.5 ml 0 10 mM MgS0 4 and stored at 4°C. Cells were used fresh for primary screening and no older than one week for subsequent screening.
  • 2.2 cDNA Libraries 2.2.1 Wheat The cDNA library used (gift of Dr Charles Ainsworth, Wye College, London) was generated using the pooled RNA from whole developing grain of Chinese Spring harvested at 3,5,7,10,15,25,30 and 35 days post anthesis. The cDNA was cloned into the EcoRI/XhoI site of 1-ZAP II (Stratagene) and the host bacteria used was XL-lBlue (see 2.1 for preparations of cells) . 2.2.2 Rape
  • the Arabidopsis thaliana library used (a gift from Dr John Cowl, John Innes Institute, Norwich) was derived from leaf total DNA in 1 FIX II and the host bacteria used was E.coli KW251 (see 2.1 for preparation of cells). 2.4 Probe preparation and labelling
  • Plasmid DNA from pA3/DH5 ⁇ (ICI derived) and pRSl/DH5 ⁇ (see results for a description of pRSl) was prepared by the Quagen tip method.
  • Probe for the screening of Wheat and Rape cDNA libraries was generated by the digestion of lO ⁇ g pA3 with 20 U EcoRI or Hind III (New England Biolabs) . The fragment isolated from the probe was 2.7 and 1.54 kb in length respectively.
  • Probe for the screening of the Arabidopsis genomic library was generated by a Xho I/Pst I (10 U of each) double digest of lO ⁇ g pRSl to give an isolated fragment size of 1.2 kb.
  • the probes (200-300 ng) were radio-labelled with - p 32 ⁇ ;dCTP using the Megaprime kit as recommended by the manufacturers (Amersham International) to a level of 5 x IO 9 dpm/ ⁇ g. Un-incorporated label was removed using Biospin chromatography columns (Biorad) .
  • the filters were incubated in pre-hybridisation buffer (50 mis X6 SSC, XI Dendhart's, 0.5% SDS, 0.05% sodium pyrophosphate, SO ⁇ g/m 1 herring sperm DNA with constant 0 mixing for 3 hours at 65°C at which point the buffer was discarded.
  • the radio-labelled probe (see 2.4) was added to 10 ml hybridisation buffer (50 mis X6 SSC, XI Dendhart's, 0.5% SDS, 0.05% Sodium Pyrophosphate, ImM EDTA) previously equilibrated to 65°C.
  • the filters were 5 incubated with constant mixing for 14 hours at 65°C and the hybridisation buffer/probe removed but retained at -20°C for the subsequent screens.
  • the filters were washed 4 times with XI SSC, 0.1% SDS for 30 minutes at 65°C. 0 Filters were air dried and exposed to film overnight. Positive plaques were located and pulled out from the plate using the wide end of a 1 ml gilson tip. Only plaques that showed up positive on both lifts (30 seconds and 2 minute lifts) were used.
  • the plug was placed into 500 ⁇ l SM buffer with lO ⁇ l chloroform and incubated at room temperature for 2 hours with occasional mixing. The suspension was spun for 5 minutes on a bench top centrifuge and the supernatant containing the pfu's 5 retained.
  • Pre-hybridisation and hybridisation was carried out in 0 the same way as that in the primary screen (see 2.5), using the same probe/hybridisation buffer boiled for 5 minutes before use.
  • the positive plaques were removed as a plug using the wide end of a 200 ⁇ l Gilson tip, placed into 500 ⁇ l SM buffer with lO ⁇ l chloroform and incubated at room temperature for 2 hours with occasional mixing. The 0 suspension was spun for 5 minutes on a bench top centrifuge and the supernatant containing the pfu's retained.
  • One positive plaque was removed from a plate of the positive pfu's from the tertiary screen and incubated with 500 ⁇ l fresh KW 251 cells (see 2.1 for method of cell preparation) at 37°C for 20 minutes.
  • Pre-warmed LB media 50 ml at 37°C
  • 500 ⁇ l 1 M MgS0 was added in addition to 500 ⁇ l 1 M MgS0 and incubated with mild shaking at 37°C for 5-7 hours.
  • 250 ⁇ l Chloroform was added to the culture and incubated for a further 15 minutes at 37°C.
  • Cell debris was spun out at 10,000 g and DNase/RNase added to the supernatant to a final concentration of 1 ⁇ g ml -1 and further incubation at 37°C for 30 minutes.
  • 5 g Polyethylene Glycol 8000/3.2 g NaCl was added slowly to the supernatant at 4°C overnight with constant stirring.
  • the resultant suspension was pelleted at 10,000 g (4°C) and taken up in 5 ml 20 mM Tris-HCl pH 7.4/100 mM NaCl/10 mM MgS0 4 .
  • the solution was then subjected to 3-5 chloroform extraction's and 3-5 1:1 Phenol:Chloroform extraction.
  • To precipitate the DNA an equal volume of isopropanol (-20°C) was added and left on ice for 30 minutes.
  • the precipitated DNA was pelleted at 10,000 g and washed in 70% Ethanol (-20°C) before being pelleted again.
  • the DNA was resuspended in 300 ⁇ l T 10 E i buffer. Subcloning was carried out according to the method used by Sa brook et al (1989) . 2.11 Sequencing of DNA clones
  • Northern blot analysis Poly A+ mRNA was prepared from either 5g young leaf or 5g embryos harvested at 15, 22,29, 36, 42 and 49 days post anthesis using the recommended procedure (Pharmacia mRNA purification kit) . 1-5 ug was loaded on to a 1% formamide/formaldehyde agarose gel for electrophoresis. The Northern blot procedure was as described previously ( Elborough et al 1994) . 4. Southern blot analysis
  • Powdered (NH 4 ) S0 was added to a final saturation of 60% and stirred for 1 hour. After spinning at 20,000g the pellets were resuspended in 100 ml 100 mM Tris-HCl pH 7.5/100m M NaCl. The supernatant was dialysed for 1 hour against 5 litres 100 mM Tris-HCl pH 7.5/100 mM NaCl and subsequently overnight with fresh buffer (5 litres) . Powdered (NH 4 ) 2 S0 4 was added to a final saturation of 25% and stirred for 1 hour spun at 20,000 g and the supernatant brought up to 70% saturation.
  • the resulting pellet was resuspended in 50 ml 20 mM Tris-HCl pH 7.5, 20 mM NaCl and dialysed with 3 x 1 hour changes against 5 litres 20 mM Tris-HCl pH 7.5, 20 mM NaCl/20% glycerol.
  • the resultant suspension was diluted to a conductivity of ⁇ 4.3 x 10"" 3 cm -1 and stirred slowly with 150 ml of pre-equilibrated Q-sepharose (in 20 mM Tris-Hcl pH 7.5 20 mM NaCl/20% glycerol) for 2 hours.
  • the unbound protein was removed using a sintered glass funnel and the matrix washed with 10 volumes of 20 mM Tris-HCl pH 7.5, 20 mM NaCl/20% glycerol. The slurry was packed into a 10 cm diameter Pharmacia column. Protein was eluted from the column using a gradient of 60-500 mM
  • the washed matrix was packed into a 10 cm diameter
  • 25 material was identified as ACCase by both its ability to change mobility during SDS PAGE in the presence of streptavidin and its estimated molecular weight (Egin-Buhler et al. (1980) .
  • SDS PAGE X5 loading buffer (5 ⁇ l) was added to 20ul post Blue-sepharose material, 0 boiled at 100°C for 2 mins. and l ⁇ l of a 5mM Steptavidin stock added immediately. The solution was incubated at 650°C for 5 mins. and loaded onto an SDS PAGE gel- next to*, myosin (Mr 200kDa) and untreated post Blue-sepharose material sample for comparison (see Fig 2A) . Streptavidin clearly reduced the mobility of the 220kDa band, indicating that it is biotin containing. The only known biotin enzyme with a MW of 220kDa is ACCase.
  • Chromaphor green Promega was added at 1:1000 dilution to the upper tank during electrophoresis to allow the visualisation of protein.
  • the ACCase protein band at approx. 220 kDa was cut out of the gel, frozen and stored at -20°C overnight.
  • the gel slices were trimmed of excess acrylamide and loaded on to one well of a 3mm thick large Biorad Protean gel.
  • the gel slices once loaded were overlaid with Endoproteinase LysC (Promega) at 6.5% protein concentration in 50% glycerol/0.125M Tris pH 6.8,/0.1%. 0 SDS/3% B-mercaptoethanol/0.005% Bromophenol Blue.
  • the gel was run until the protein was at the stacker interface at which point electrophoresis was stopped for lhr at room temperature. Electrophoresis was resumed until the dye front reached the bottom of the gel.
  • Peptides were 5 semi-dry blotted into ProBlot (Applied Biosystems Inc.) according to manufacturers instructions.
  • Rapid Coomassie staining of the blot identified peptide fragments which were excised from the membrane and loaded onto an ABI 477A pulse liquid protein 0 sequencer. Sequence data was obtained at an amino acid level of 10-20pM (see Fig 3) .
  • a wheat cDNA library was probed with a 2.7 kb EcoRl fragment, and a 1.54 kb Hindlll fragment of the maize partial cDNA clone pA3 which contains 4.5 kb of the 3' maize ACCase. This yielded a 1.85 kb clone inserted between the Eco Rl and Xhol site in the multi cloning cassette of pSK.
  • the DNA was recovered by plasmid rescue in the host strain DH5 . This clone was denoted pKlll.
  • the nucleotide sequence data of this partial cDNA, with the derived amino acid sequence from the three reading frames is shown in Figure 4.
  • Figure 4 also shows that sections of pKlll show complete homology with the amino acid sequence of the 4 peptides isolated from the purified wheat germ enzyme, providing good evidennce that the cDNA does indeed code for wheat embryo ACCase.
  • 1AYE4 and 1AYE8 hybridised strongly to the pRSl ACCase probe. These were denoted 1AYE4 and 1AYE8. 1AYE8 was subcloned to produce two plasmids : pKLU81, a 5.3 kb subclone in the EcoRl site of pGEM 3ZF+ ; and pKLS2, which was excised from the 1 clone by a partial Sail digest and subcloned into pSK+.
  • the pKLU81 subclone considered to be a partial length genomic clone, was partially sequenced from the 5' and 3' ends. Therefore two sets of data are presented for the 5' and 3' sequences from the same clone.
  • 15 pKLU 81 subclone was a partial length genomic clone corresponding to a portion of the sequence of pKLS2.
  • the blotrs contained 5 ug rape poly A+ m RNA prepared from a set of staged embryos taken from Brassica napus Jet Neuf at 15, 22, 29, 35,42 and 49 days post-anthesis. Embryos taken from the same seed set were also analysed for oil content to monitor development. The oil content data is presented (expressed as fatty acid/mg seed) graphically in Figure 14A.
  • the three probes used were embryo derived cDNAs for enoyl reductase (1.15 kb) , ⁇ keto reductase (1.185 kb) and ACCase (2.5 kb) . All three cDNAs were highly expressed in seed with maximum expression
  • reductase and ⁇ ketoreductase This may be in part due to the successive stripping of the blot and degradation of the large 7.5 kb message during handling.

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Abstract

Des séquences d'ADN d'une carboxylase du coenzyme A d'acétyle ACCase issue de plantes sont insérées dans le génome de plantes avec une orientation sens ou antisens afin d'inhiber l'expression du produit génique du gène ACCase endogène, entraînant une réduction de la conversion du substrat de l'enzyme, le coenzyme A d'acétyle, en synthèse d'acide gras, laissant le substrat disponible pour une diversion dans d'autres processus de biosynthèse. On peut effectuer une telle diversion en munissant le génome d'une plante de gènes spécifiant la synthèse de polymères de polyhydroxyalcanoate.
PCT/GB1994/000653 1993-03-29 1994-03-29 Gene vegetal specifiant la carboxylase du coenzyme a d'acetyle, et plantes transformees contenant ce gene WO1994023027A2 (fr)

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007357A2 (fr) * 1993-09-04 1995-03-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Promoteurs
WO1995029246A1 (fr) * 1994-04-21 1995-11-02 Zeneca Limited Gene vegetal specifiant l'acetyl-coenzyme a-carboxylase et plantes transformees contenant ce gene
WO1996031609A2 (fr) * 1995-04-05 1996-10-10 Regents Of The University Of Minnesota PLANTES TRANSGENIQUES EXPRIMANT LE GENE D'ACETYLE CoA CARBOXYLASE
WO1996032484A2 (fr) * 1995-04-14 1996-10-17 Arch Development Corporation COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION
US5610041A (en) * 1991-07-19 1997-03-11 Board Of Trustees Operating Michigan State University Processes for producing polyhydroxybutyrate and related polyhydroxyalkanoates in the plastids of higher plants
US5650555A (en) * 1991-07-19 1997-07-22 Board Of Trustees Operating Michigan State University Transgenic plants producing polyhydroxyalkanoates
US5792627A (en) * 1992-10-02 1998-08-11 Arch Development Corporation Cyanobacterial and plant acetyl-CoA carboxylase
US5925805A (en) * 1994-05-24 1999-07-20 Board Of Trustees Operating Michigan State University Methods of increasing oil content of seeds
WO1999045122A1 (fr) * 1998-03-06 1999-09-10 Metabolix, Inc. Modification du metabolisme des acides gras dans des plantes
US5962767A (en) * 1994-05-24 1999-10-05 Board Of Trustees Operating Michigan State University Structure and expression of an arabidopsis acetyl-coenzyme A carboxylase gene
US6083729A (en) * 1995-10-26 2000-07-04 Metabolix, Inc. Methods for isolating polyhydroxyalkanoates from plants
US6222099B1 (en) 1993-02-05 2001-04-24 Regents Of The University Of Minnesota Transgenic plants expressing maize acetyl COA carboxylase gene and method of altering oil content
US6306636B1 (en) 1997-09-19 2001-10-23 Arch Development Corporation Nucleic acid segments encoding wheat acetyl-CoA carboxylase
US6414222B1 (en) 1993-02-05 2002-07-02 Regents Of The University Of Minnesota Gene combinations for herbicide tolerance in corn
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US6586658B1 (en) 1998-03-06 2003-07-01 Metabolix, Inc. Modification of fatty acid metabolism in plants
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

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US7109007B2 (en) 1987-06-29 2006-09-19 Massachusetts Institute Of Technology Polyhydroxybutyrate polymerase
US6881560B2 (en) 1987-06-29 2005-04-19 Massachusetts Institute Of Technology Polyhydroxybutyrate polymerase
US6528706B1 (en) 1987-06-29 2003-03-04 Massachusetts Institute Of Technology Polyhydroxybutyrate polymerase
US5650555A (en) * 1991-07-19 1997-07-22 Board Of Trustees Operating Michigan State University Transgenic plants producing polyhydroxyalkanoates
US5610041A (en) * 1991-07-19 1997-03-11 Board Of Trustees Operating Michigan State University Processes for producing polyhydroxybutyrate and related polyhydroxyalkanoates in the plastids of higher plants
US5910626A (en) * 1992-10-02 1999-06-08 Arch Development Corporation Acetyl-CoA carboxylase compositions and methods of use
US5792627A (en) * 1992-10-02 1998-08-11 Arch Development Corporation Cyanobacterial and plant acetyl-CoA carboxylase
US6414222B1 (en) 1993-02-05 2002-07-02 Regents Of The University Of Minnesota Gene combinations for herbicide tolerance in corn
US6268550B1 (en) 1993-02-05 2001-07-31 Regents Of The University Of Minnesota Methods and a maize acetyl CoA carboxylase gene for altering the oil content of plants
US6222099B1 (en) 1993-02-05 2001-04-24 Regents Of The University Of Minnesota Transgenic plants expressing maize acetyl COA carboxylase gene and method of altering oil content
US6069298A (en) * 1993-02-05 2000-05-30 Regents Of The University Of Minnesota Methods and an acetyl CoA carboxylase gene for conferring herbicide tolerance and an alteration in oil content of plants
US6146867A (en) * 1993-02-05 2000-11-14 Regents Of The University Of Minnesota Methods for expressing a maize acetyl CoA carboxylase gene in host cells and encoded protein produced thereby
US6133506A (en) * 1993-09-04 2000-10-17 Max-Planck-Gesellschaft Zur Forerung Der Wissenschaft E.V. Keto-acyl-(ACP) reductase promoter from cuphea lanceolata
WO1995007357A3 (fr) * 1993-09-04 1995-07-13 Max Planck Gesellschaft Promoteurs
WO1995007357A2 (fr) * 1993-09-04 1995-03-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Promoteurs
WO1995029246A1 (fr) * 1994-04-21 1995-11-02 Zeneca Limited Gene vegetal specifiant l'acetyl-coenzyme a-carboxylase et plantes transformees contenant ce gene
US5962767A (en) * 1994-05-24 1999-10-05 Board Of Trustees Operating Michigan State University Structure and expression of an arabidopsis acetyl-coenzyme A carboxylase gene
US5925805A (en) * 1994-05-24 1999-07-20 Board Of Trustees Operating Michigan State University Methods of increasing oil content of seeds
WO1996031609A3 (fr) * 1995-04-05 1996-11-07 Univ Minnesota PLANTES TRANSGENIQUES EXPRIMANT LE GENE D'ACETYLE CoA CARBOXYLASE
WO1996031609A2 (fr) * 1995-04-05 1996-10-10 Regents Of The University Of Minnesota PLANTES TRANSGENIQUES EXPRIMANT LE GENE D'ACETYLE CoA CARBOXYLASE
WO1996032484A3 (fr) * 1995-04-14 1997-05-01 Arch Dev Corp COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION
WO1996032484A2 (fr) * 1995-04-14 1996-10-17 Arch Development Corporation COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION
US6709848B1 (en) 1995-10-26 2004-03-23 Metabolix, Inc. Methods for isolating polyhydroxyalkanoates from plants
US6083729A (en) * 1995-10-26 2000-07-04 Metabolix, Inc. Methods for isolating polyhydroxyalkanoates from plants
US6306636B1 (en) 1997-09-19 2001-10-23 Arch Development Corporation Nucleic acid segments encoding wheat acetyl-CoA carboxylase
US6586658B1 (en) 1998-03-06 2003-07-01 Metabolix, Inc. Modification of fatty acid metabolism in plants
WO1999045122A1 (fr) * 1998-03-06 1999-09-10 Metabolix, Inc. Modification du metabolisme des acides gras dans des plantes
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

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