WO2005014833A1 - Biosynthese 1 d'acides gras - Google Patents

Biosynthese 1 d'acides gras Download PDF

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WO2005014833A1
WO2005014833A1 PCT/GB2003/003352 GB0303352W WO2005014833A1 WO 2005014833 A1 WO2005014833 A1 WO 2005014833A1 GB 0303352 W GB0303352 W GB 0303352W WO 2005014833 A1 WO2005014833 A1 WO 2005014833A1
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nucleic acid
acid molecule
plant
acid sequence
cell according
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PCT/GB2003/003352
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English (en)
Inventor
Yves Poirier
Enea Rezzonico
Laurence Moire
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The Universtiy Of York
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Priority to AU2003255742A priority Critical patent/AU2003255742A1/en
Priority to PCT/GB2003/003352 priority patent/WO2005014833A1/fr
Publication of WO2005014833A1 publication Critical patent/WO2005014833A1/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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)

Definitions

  • the invention relates to transgenic plant cells expressing nucleic acid molecules which encode polypeptides with expoxygenase or acetylenase activity wherein said cells are deficient in omega 3 fatty acid desaturase activity and contain increased levels of linoleic acid and including plants comprising said cells.
  • Oils comprising fatty acids are used in a vast array of industrial processes. For example oils are used in the manufacture of surface coatings, glues and resins, plastics, surfactants and lubricants. It is often the case that these oils are extracted from petrochemical sources and some of the fatty acids are not a major component. This presents a problem of both extraction and purification. The provision of a reusable source of rare or unusual fatty acids would be desirable and the engineering of plants by genetic means to produce these fatty acid is an alternative and attractive solution to this problem.
  • Membranes of plant cells are composed primarily of five "common” fatty acids, namely stearic, palmitic, cis-7,10,13-hexadecatrienoic acid (16:3), oleic, linoleic, and linolenic acids.
  • TAG triacylglycerides
  • fatty acids vary in a number of features, including the length of the acyl chains, the number, position, and nature of unsaturated bonds, as well as the presence of functional groups, such as hydroxy, epoxy, and acetylenic groups. These fatty acids are often referred as "unusual" fatty acids since their structure is different from the common fatty acids found in membranes.
  • ricinoleic acid (12-hydroxy-octadec-ct.s'-9-enoic acid) or vernolic acid (12,13-epoxy- octadec-ct-s-P-enoic acid) can be found in paints, lubricants, nylons, plastics and cosmetics.
  • lauroyl-acyl carrier protein (ACP) thioesterase under the constitutive CaMN35S promoter led to accumulation of lauric acid in seeds but not in leaves, despite the demonstration that lauric acid could be produced from isolated leaf chloroplasts (Eccleston et al., 1996).
  • a futile cycle has been demonstrated whereby medium-chain fatty acids synthesized in leaves are rapidly degraded by the peroxisomal ⁇ -oxidation cycle (Eccleston and Ohlrogge, 1998; Poirier et al., 1999).
  • failure to accumulate unusual fatty acids may not only be due to poor synthesis but also by their lack of stable incorporation into lipids such as TAG leading to their rapid degradation through the ⁇ -oxidation cycle.
  • the napin-C. palaestina epoxygenase was transformed into wild type (Columbia) plants as well as into a transgenic plant expressing the PHA synthase into the peroxisome. Expression of the PHA synthase should have no effect on the accumulation of linoleic acid or vernolic acid since it does not affect the fatty acid biosynthetic pathway. Both were thus used as controls.
  • the same napin-C. palaestina epoxygenase construct was transformed into the fadS/fad7-l/fad8 triple mutant having a very low level of omega-3 desaturase activity resulting in a high level of linoleic acid.
  • the seeds from all primary transformants were harvested and analyzed for the amount of epoxygenated fatty acids (vernolic acid + 12-epoxy-9, 15- octadecadienoic acid) .
  • the results of the analysis with the accompanying statistical analysis is provided in Figure 6. The conclusion is that expression of the epoxygenase in a plant accumulating 60% more linoleic acid then wild type resulted in a 50% increase in the accumulation of epoxygenated fatty acids in mature seeds.
  • a napin-C. alpina acetylenase gene construct was transformed into wild type plants, transgenic plants expressing the PHA synthase into the peroxisome, and into the fad3/fad7-l/fad8 triple mutant having a very low level of omega-3 desaturase activity resulting in a high level of linoleic acid.
  • the seeds from all primary transformants were harvested and analysed for the amount of 9-octadecen-12-ynoic acid.
  • transgenic plant cell wherein the genome of said cell is modified which modification is the fransfection of at least one nucleic acid molecule which encodes a polypeptide with epoxygenase activity or acetylenase activity and additionally wherein said cell is deficient omega 3 fatty acid desaturase activity.
  • said nucleic acid molecule encodes a polypeptide selected from the group consisting of: i) a polypeptide encoded by a nucleic acid molecule consisting of a nucleic acid sequence as represented in Figure la or 2a; ii) a polypeptide encoded by a nucleic acid molecule which hybridises to the nucleic acid molecule as represented by Figure la and which has acetylenase activity; iii) a polypeptide encoded by a nucleic acid molecule which hybridises to the nucleic acid molecule as represented by Figure 2a and which has epoxygenase activity; iv) a polypeptide encoded by a nucleic acid molecule consisting of a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i), (ii) or (iii).
  • said cell is deficient in at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of: i) a polypeptide encoded by a nucleic acid molecule consisting of a nucleic acid sequence as represented in Figures 3 a, 4a or 5 a; ii) a polypeptide encoded by a nucleic acid molecule which hybridises to the nucleic acid molecule in (i) and which has omega 3 fatty acid desaturase activity; iii) a polypeptide encoded by a nucleic acid molecule consisting of a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) and (ii).
  • said nucleic acid molecule comprises a nucleic acid sequence which hybridises under stringent hybridisation conditions to a nucleic acid molecule as represented by the nucleic acid sequence as represented in Figure la or 2
  • said nucleic acid molecule comprises a nucleic acid sequence which hybridises under stringent hybridisation conditions to a nucleic acid molecule as represented by the nucleic acid sequence as represented in Figure 3a, 4a or 5a.
  • Stringent hybridisation/washing conditions are well known in the art. For example, nucleic acid hybrids that are stable after washing in O.lx SSC,0.1% SDS at 60°C. It is well known in the art that optimal hybridisation conditions can be calculated if the sequence of the nucleic acid is known. For example, hybridisation conditions can be determined by the GC content of the nucleic acid subject to hybridisation. A common formula for calculating the stringency conditions required to achieve hybridisation between nucleic acid molecules of a specified homology is:
  • T m 81.5° C + 16.6 Log [Na + ] + 0.41 [ % G + C] -0.63 (%formamide).
  • hybridisation conditions uses 4 - 6 x SSPE (20x SSPE contains 175.3g NaCl, 88.2g NaH 2 PO 4 H 2 O and 7.4g EDTA dissolved to 1 litre and the pH adjusted to 7.4); 5-1 Ox Denhardts solution (50x Denhardts solution contains 5g Ficoll (type 400, Pharmacia), 5g polyvinylpyrrolidone abd 5g bovine serum albumen; lOO ⁇ g- l.Omg/ml sonicated salmon/herring DNA; 0.1-1.0% sodium dodecyl sulphate; optionally 40-60% deionised formamide.
  • 5-1 Ox Denhardts solution 50x Denhardts solution contains 5g Ficoll (type 400, Pharmacia), 5g polyvinylpyrrolidone abd 5g bovine serum albumen; lOO ⁇ g- l.Omg/ml sonicated salmon/herring DNA; 0.1-1.0% sodium dodecyl sulphate
  • Hybridisation temperature will vary depending on the GC content of the nucleic acid target sequence but will typically be between 42°- 65° C
  • said nucleic acid molecule comprises a nucleic acid sequence as represented in Figure la or 2a.
  • said nucleic acid molecule consists of a nucleic acid sequence as represented in Figure la or 2a.
  • said nucleic acid molecule comprises a nucleic acid sequence as represented in Figure 3a, 4a, or 5a.
  • said nucleic acid molecule consists of a nucleic acid sequence as represented in Figure 3 a, 4a, or 5 a.
  • said cell over-expresses said nucleic acid molecule encoding said epoxygenase or acetylenase polypeptide.
  • said over-expression is at least 2-fold higher when compared to a non-transformed reference cell of the same species.
  • said over-expression is: at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, or at least 10-fold when compared to a non-transformed reference cell of the same species.
  • omega 3 fatty acid desaturase activity is reduced to at least 10% when compared to a non-transgenic reference cell of the same species.
  • said activity is reduced by between 10%-99%.
  • said activity is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 99% when compared to a non-transgenic reference cell of the same species.
  • said transgenic cell is null for a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as represented in Figures 3 a and/or, 4a and/or, and/or 5 a; ii) nucleic acid molecules consisting of nucleic acid sequences which hybridise to the sequences of (i) above and which have omega 3 fatty acid desaturase activity; and iii) nucleic acid molecules consisting of nucleic acid sequences that are degenerate as a result of the genetic code to the sequences defined in (i) and (ii) above.
  • Null refers to a cell which includes a non-functional copy of the nucleic acid sequence described above. Methods to provide such a cell are well known in the art and include the use insertional mutagenesis using T-DNA. Furthermore, the use of antisense genes and double stranded inhibitory RNA (RNAi) to regulate the expression of specific targets enables the relative abundance of mRNA to be modulated.
  • RNAi double stranded inhibitory RNA
  • said cell is transformed with a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence which encodes at least part of a omega 3 fatty acid desaturase wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette.
  • RNAi expression cassettes can be provided which are adapted such that individual desaturase genes are silenced, pairs of desaturase genes are silenced, or all genes are silenced by RNAi. Comparison of the sequences of FAD 3, 7 and 8 allows the skilled artisan to design such RNAi expression cassettes.
  • said cassette is provided with at least two promoters adapted to transcribe sense and antisense strands of said nucleic acid molecule.
  • said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts.
  • said first and second parts are linked by at least one nucleotide base, hi a further preferred embodiment of the invention said first and second parts are linked by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide bases. In a yet further preferred embodiment of the invention said linker is at least 10 nucleotide bases.
  • the length of the RNA molecule is between 10 nucleotide bases (nb) and lOOOnb.
  • RNA molecule is lOOnb; 200nb; 300nb; 400nb; 500nb; 600nb; 700nb; 800nb; 900nb; or lOOOnb in length. More preferably still said RNA molecule is at least lOOOnb in length.
  • the length of the RNA molecule is at least lOnb; 20nb; 30nb; 40nb; 50nb; 60nb; 70nb; 80nb; or 90nb in length. More preferably still said RNA molecule is 21nb in length.
  • said expression cassette is part of a vector.
  • the nucleic acid in the vector is operably linked to an appropriate promoter or other regulatory elements for transcription in a host plant cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the example of nucleic acids according to the invention this may contain its native promoter or other regulatory elements .
  • promoter is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription.
  • Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design.
  • Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.
  • Constitutive promoters include, for example CaMN 35S promoter (Odell et al (1985) Nature 313, 9810-812); rice actin (McElroy et al (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al . (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al
  • ALS promoter U.S. Application Seriel No. 08/409,297
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid.
  • promoters of interest include steroid- responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellie et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline- repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.
  • tissue-specific promoters can be utilised.
  • Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al (1996) Plant Physiol. 112(2): 525-535; Canevascni et al (1996) Plant Physiol.
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • the promoter is an inducible promoter or a developmentally regulated promoter.
  • said developmentally regulated promoter is a promoter active in the tissues accumulating reserve oils in developing seeds. Promoters of this type are known in the art, for example, the napin promoter is a strong promoter which has the requisite expression pattern, see US Patent No. 5,608, 152.
  • vectors are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors, h : Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148). Suitable vectors may include plant viral-derived vectors (see e.g. EP-A- 194809).
  • Vectors may also include selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. phosphinotricin, chlorsulfuron, methotrexate imidazolinones and glyphosate) and antibiotics (kanamycin, hygromycin, gentamycin, spectinomycin).
  • selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. phosphinotricin, chlorsulfuron, methotrexate imidazolinones and glyphosate) and antibiotics (kanamycin, hygromycin, gentamycin, spectinomycin).
  • a plant comprising a cell according to the invention.
  • a plant selected from the group consisting of: corn ⁇ Zea mays), canola ⁇ Brassica napus, Brassica rapa ssp.), flax ⁇ Linum usitatissimum), alfalfa ⁇ Medicago sativa), rice ⁇ Oryza sativa), rye ⁇ Secale cerate), sorghum ⁇ Sorghum bicolor, Sorghum vulgare), sunflower ⁇ Helianthus annus), wheat ⁇ Tritium aestivum), soybean ⁇ Glycine max), tobacco ⁇ Nicotiana tabacum), potato ⁇ Solanum tuberosum), peanuts ⁇ Arachis hypogaea), cotton ⁇ Gossypium hirsutum), sweet potato ⁇ Iopmoea batatus), cassava ⁇ Manihot esculenta), coffee (Cofea spp.), coconut ⁇ Cocos nucifera), pineapple ⁇ Cocos nucifera), pineapple
  • plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea), and other root, tuber or seed crops.
  • Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybeamsorghum, and flax (linseed).
  • Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower.
  • the present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper.
  • Grain plants that provide seeds of interest include oil-seed plants and leguminous plants.
  • Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc.
  • Oil-seed plants include cotton, soybean, safflower, sunflower,
  • Leguminous plants include beans and peas.
  • Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava been, lentils, chickpea, etc.
  • a seed comprising a cell according to the invention.
  • a method to modulate the fatty acid content of a plant cell comprising the steps: i) providing a cell according to the invention; ii) cultivating said cell into a plant; and optionally iii) analysing the fatty acid content of said plant.
  • said plant is an oil-seed plant.
  • said fatty acid is an epoxygenated fatty acid, for example vernolic acid or 12-epoxy-9, 15-octadecadienoic acid.
  • said fatty acid is an acetylenated fatty acid, for example 9-octadecen-12-ynoic acid fatty acids.
  • Figure la is the nucleic acid sequence of Crepis alpina delta 12 fatty acid acetylenase DNA
  • Figure lb is the amino acid sequence of Crepis alpina delta 12 fatty acid acetylenase protein
  • Figure 2a is the nucleic acid sequence of Crepis palaestina delta 12 fatty acid epoxygenase DNA
  • Figure 2b is the amino acid sequence of Crepis palaestina delta 12 fatty acid epoxygenase protein
  • Figure 3a is the nucleic acid sequence of FAD 3 omega-3 fatty acid desaturase DNA
  • Figure 3b is the amino acid sequence of FAD 3 omega-3 fatty acid desaturase protein
  • Figure 4a is the nucleic acid sequence of FAD 7 omega-3 fatty acid desaturase DNA
  • Figure 4b is the amino acid sequence of FAD 7 omega-3 fatty acid desaturase protein
  • Figure 5a is the nucleic acid sequence of FAD 8 omega-3 fatty acid desaturase DNA
  • Figure 5b is the amino acid sequence of FAD 8 omega-3 fatty acid desaturase protein
  • Figure 6. Analysis of epoxy fatty acids produced in wild type A. thaliana and a mutant showing accumulation of linoleic acid.
  • the napin-epoxygenase construct was transformed into either wild type A. thaliana (WTNE), wild type expressing a peroxisomal PHA synthase (N10.3NE) or in the fad3/fad7-l/fad8 mutant (fadNE).
  • FIG. 7 -Analysis of acetylenic fatty acids produced in wild type A. thaliana and a mutant showing accumulation of linoleic acid.
  • the napin-acetylenase construct was transformed into either wild type A. thaliana expressing a peroxisomal PHA synthase (N10.3NA) or in the fad3/fad7-l/fad8 mutant (fadNA).
  • the amount (in mol%) of acetylenic fatty acids (crepenynic acid) produced in the mature seeds of each transformed plant is indicated by a circle.
  • Statistical analysis revealed significant differences between WTNA and N10.3NA (P ⁇ 0.05) ;
  • Figure 8 is a diagrammatic representation of vector construct pART27-napin- epoxygenase
  • Figure 9 is a diagrammatic representation of vector construct pART27-napin- acetylenase.
  • the cDNAs with the upstream and downstream regulatory elements were cloned as Sacl-Pstl fragments in the pCAMBIA 1300 vector (www.cambia.org.au) containing the hygromycin resistance gene under the control of CaMN35S promoter. All binary vectors were transferred into Agrobacterium tumefaciens pGN3101 by electroporation.
  • Tl transformants were isolated by plating seeds on medium containing half-strength Murashige and Skoog salts, 1% (w/v) Sue, 0.7% (w/v) agar and 30 ⁇ g/ml hygromycin. Hygromycin resistant plants were subsequently transferred to soil and grown under continuous fluorescent light at 19°C. PHA and fatty acid analysis
  • Seed fatty acid methyl-esters were prepared by acid-catalysed (IN HCL in methanol, 2h, 80°C) or base-catalysed (0.1M NaOH in methanol, lh, 60°C) transesterification. After reaction, the fatty acid methyl-esters were extracted with hexane and water and the organic phase was transferred to vials. Analysis was performed using a gas chromatograph equipped with a glass capillary column (model SP230, Supelco, Bellefonte, PA) and a flame ionization detector.
  • the napin-C. palaestina epoxygenase was transformed into wild type (Columbia) plants as well as into a transgenic plant expressing the PHA synthase into the peroxisome. Expression of the PHA synthase should have no effect on the accumulation of linoleic acid or vernolic acid since it does not affect the fatty acid biosynthetic pathway. Both were thus used as controls.
  • the same napin-C. palaestina epoxygenase construct was transformed into the fad3/fad7-l/fad8 triple mutant having a very low level of omega-3 desaturase activity resulting in a high level of linoleic acid.
  • the results of the analysis with the accompanying statistical analysis is provided in annex 1. The conclusion is that expression of the epoxygenase in a plant accumulating 60% more linoleic acid then wild type resulted in a 50% increase in the accumulation of epoxygenated fatty acids in mature seeds, see Figure 6. Similarly, a napin-C.
  • alpina acetylenase gene construct was transformed into wild type plants, transgenic plants expressing the PHA synthase into the peroxisome, and into the fad3/fad7-l/fad8 triple mutant having a very low level of omega-3 desaturase activity resulting in a high level of linoleic acid.
  • the seeds from all primary transformants were harvested and analysed for the amount of 9-octadecen-12-ynoic acid.
  • Phospholipid:diacylglycerolacyltransferase an enzyme that catalyzes the acyl-CoA independent formation of triacylglycerol in yeast and plants.Eroc. Natl. Acad. Sci. USA, 91, 6487-6492. de Boer, G.J., Kater, M.M., Fa cett, T., Slabas, A.R., Nijkamp, H.J.J. and Stuitje, A.J.
  • Eccleston, V.S., Cranmer, A.M., Voelker, T.A. and Ohlrogge, J.B. (1996) Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase. Planta 198, 46-53. Eccleston, V.S. and Ohlrogge, J.B. (1998) Expression of lauroyl-acyl carrier protein thioesterase in Brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation. Plant Cell 10, 613-621.
  • Ruuska S.A., Girke, T., Benning, C. and Ohlrogge, J.B. (2002) Contrapuntal networks of gene expression during Arabidopsis seed filling. Plant Cell 14, 1191- 1206.

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Abstract

La présente invention concerne des cellules végétales transgéniques exprimant des molécules d'acides nucléiques qui codent pour des polypeptides dotés d'une activité d'époxygénase ou d'acétylénase, lesdites cellules présentant une activité déficiente en acides gras oméga-3-désaturase. L'invention se rapporte également à des végétaux comportant de telles cellules.
PCT/GB2003/003352 2003-07-31 2003-07-31 Biosynthese 1 d'acides gras WO2005014833A1 (fr)

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