WO1999011805A1 - Molecules d'adn recombinees et procede pour augmenter la teneur en huile dans des plantes - Google Patents

Molecules d'adn recombinees et procede pour augmenter la teneur en huile dans des plantes Download PDF

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WO1999011805A1
WO1999011805A1 PCT/EP1998/005461 EP9805461W WO9911805A1 WO 1999011805 A1 WO1999011805 A1 WO 1999011805A1 EP 9805461 W EP9805461 W EP 9805461W WO 9911805 A1 WO9911805 A1 WO 9911805A1
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plants
recombinant dna
transgenic plant
dna molecule
agpase
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PCT/EP1998/005461
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German (de)
English (en)
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Norbert Martini
Ekkehard Neuhaus
Jozef Schell
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU97396/98A priority Critical patent/AU9739698A/en
Publication of WO1999011805A1 publication Critical patent/WO1999011805A1/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
    • 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 to recombinant DNA molecules and methods for increasing the oil content in plant cells and plants, in particular recombinant DNA molecules which contain regulatory sequences of an embryo-specific promoter in plants, a DNA sequence which is functionally linked to this regulatory sequence Plants can be expressed and their gene product inhibits a protein with the enzymatic activity of an ADP-glucose pyrophosphorylase (AGPase).
  • AGPase ADP-glucose pyrophosphorylase
  • This invention further relates to vectors, host cells and kits which contain such recombinant DNA molecules.
  • the present invention further relates to methods for producing transgenic plant cells, plant tissue and plants which, owing to the introduction of the recombinant DNA molecules or vectors described above, have an increased fatty acid synthesis and / or an increased oil content.
  • the present invention relates to transgenic plant cells, plant tissues and plants which contain recombinant DNA molecules or vectors according to the invention or which can be obtained by the process described
  • Oils and fats which are chemically glycerol esters and fatty acids (triacylglycerols (TAGs)), play an important role due to their high energy content Role in human nutrition.
  • TAGs triacylglycerols
  • Ninety percent of the vegetable oils produced are used for human consumption, mainly in marguerine, shortening, salad oils and roasting oils (Röbbelen, Proceedings of the World Conference on Biotechnology for the Fats and Oils Industry (1988), 78).
  • the focus has always been on improving oils that are used for these purposes.
  • the plants selected showed significant changes in the composition of the seed oil, suggesting that plants can tolerate larger fluctuations in the fatty acid composition of the storage lipids (Lühs, Designer Oil Crops (1993), 5; Kinney, Curr. Opin.
  • the present invention is therefore based on the object of providing recombinant DNA molecules and methods which can be used to increase the oil content in plants. This object is achieved by the provision of the embodiments characterized in the patent claims.
  • the present invention relates to a recombinant DNA molecule comprising
  • a protein with the enzymatic activity of an ADP-glucose pyrophosphorylase is understood to be a protein which activates glucose as a key enzyme in starch biosynthesis.
  • ADP-glucose pyrophoshorylase is also referred to, inter alia, by the term “ADPGPP”, which is also used interchangeably and interchangeably with the terms “AGPase” or “AGP” for the gene encoding the AGPase in the present application.
  • An embryo-specific promoter is understood to mean a promoter which controls the transcription of a functionally linked gene sequence in cells of embryos, preferably in their storage organs in plant seeds. It is known to the person skilled in the art that the regulatory sequences of an embryo-specific promoter in plants or the promoter itself need not necessarily come from plants. For example, they can also be synthetic and / or chimeric regulatory sequences or promoters which are derived from plant promoters, but are not identical to them.
  • a regulatory sequence, which can serve as a transcription termination sequence in plants, is able to mediate the termination of the transcription and, if appropriate, the addition of a poly-A tail to the transcript, which is believed to have a function in stabilizing the transcripts.
  • Such elements for example the terminator of the octopine synthase gene from agrobacteria, have been described in the literature (cf. Gielen, EMBO J. 8 (1989), 23- 29) and are interchangeable.
  • Further examples of suitable terminators are 3 ' transcribed, non-translated regions of the 35S rna gene of the cauliflower mosaic virus (CaMV) and of plant genes such as the 3 ' region of the CIFatB4 gene from C. lanceolata.
  • the terminators of, for example, t-RNA genes can also be used.
  • the present invention is based on the surprising finding that by suppressing the accumulation of transitory starch, fatty acid production in ripening seeds of oil plants such as e.g. Rapeseed can be increased significantly.
  • both carbon skeletons and energy equivalents are introduced into the heterotrophic plastids.
  • a number of different metabolites that differ in the molecular nature of their carbon skeletons have been identified as efficient precursors to fatty acid biosynthesis. These include, for example, acetate (Kleppinger-Sparace, Plant Physiol. 98 (1991), 723-727; Möhlmann, Planta 194 (1994), 492-497), pyruvate (Kang, Plant J. 6 (1994), 795-805) , Malat (Smith, Plant Physiol. 98 (1992), 1233-1238) or glucose-6-phosphate (Kang, Planta 199 (1996), 321-327).
  • fatty acid and starch biosynthesis in the plastids compete for carbon and energy. It is known that the inhibition of starch biosynthesis in developing embryo tissues (including rapeseed) causes a significant increase in the content of structural and storage lipids.
  • the starch biosynthesis for example in developing rapeseed, is reduced with the aid of molecular biological methods, so that carbon and energy are increasingly directed into the synthesis of lipids.
  • transgenic plants are generated, in their storage organ, e.g. Seed, the activity of the enzyme that limits the starch biosynthesis, ADP-glucose pyrophosphorylase (AGPase), is specifically reduced.
  • AGPase ADP-glucose pyrophosphorylase
  • the DNA sequence, the gene product of which inhibits a protein with the enzymatic activity of an ADP-glucose pyrophosphorylase is a DNA sequence which comprises a peptide, polypeptide, sense RNA, antisense RNA and / or ribozyme coded.
  • AGPase ADP-glucose pyrophosphorylase
  • Embryo-specific modulation of AGPase activity can e.g. by antibodies or antibody fragments such as single-chain Fv antibodies (scFv), which consist of the variable domains of the heavy and light immunoglobulin chains. Connected to each other via a flexible linker peptide, scFv can be encoded by a single gene.
  • a cytoplasmic scFv antibody was used to modulate the activity of the phytochrome A protein in genetically modified tobacco plants (Owen, Bio / Technology 10 (1992), 790-4; review: Franken, E, Teuschel, U. and Hain, R. , Current Opinion in Biotechnology 8, (1997), 411-416; Whitelam, Trends Plant Sei.
  • a scFv anti-AGPase antibody provided with a transit peptide for plastid localization could bring about a reduction in the starch synthesis rate in plastids of embryos of oily plant seeds.
  • the DNA sequence encoding a non-functional derivative of a protein with the enzymatic activity of an AGPase can be isolated from natural sources are, preferably plants, or can be synthesized by known methods. Using common molecular biological techniques, it is possible (see, for example, Sambrook, 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) to make various types of mutations in the DNA sequence that a protein contains encoding the enzymatic activity of an AGPase, in the recombinant DNA molecules according to the invention, which results in the synthesis of proteins with altered properties.
  • deletion mutants in which the synthesis of correspondingly shortened proteins can be achieved by progressive deletions from the 5 'or from the 3' end of the coding DNA sequence. It is also possible to produce proteins in a targeted manner, which are localized in certain compartments of the plant cell by adding corresponding signal sequences. Such sequences are known (see for example Braun, EMBO J. 11 (1992), 3219-3227; Wolter, Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald, Plant J. 1 (1991) , 95-106).
  • mutants can be produced which have an altered substrate or product specificity. Furthermore, mutants can be produced which have a changed activity-temperature profile.
  • the recombinant DNA molecules according to the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or a sequence change by recombining DNA sequences.
  • base exchanges can be carried out made or natural or synthetic sequences are added.
  • adapters or linkers can be attached to the fragments.
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis and other biochemical-molecular biological methods are generally carried out as the analysis method.
  • the sense RNA is a non-translatable mRNA of a protein which has the enzymatic activity of an AGPase.
  • the DNA sequences which represent a non-translatable mRNA of an AGPase are usually variations of these sequences which do not code for a functional protein, e.g. DNA sequences whose coding region is interrupted early by a stop codon.
  • These can be both naturally occurring variations, for example sequences from other organisms, or mutations, wherein these mutations can have occurred naturally or have been introduced by targeted mutagenesis, for example by deletion, substitution, insertion and / or recombination.
  • the variations can be synthetically produced sequences.
  • the allelic variants can be both naturally occurring variants and also synthetically produced variants or those produced by recombinant DNA techniques.
  • the antisense RNA and / or the ribozyme is directed against a nucleotide sequence which encodes a protein with the enzymatic activity of an ADPGPP or a subunit thereof.
  • a DNA sequence of homologous origin with respect to the plants to be transformed.
  • DNA sequences can also be used which have a high degree of homology to endogenously present AGPase genes, in particular homologies higher than 80%, preferably homologies between 90% and 100% and particularly preferably homologies over 95%. Sequences up to a minimum length of 15 bp can be used.
  • sequences are used which are shorter than 5000 base pairs, preferably sequences which are shorter than 2500 base pairs.
  • ribozymes for reducing the activity of certain enzymes in cells is also known to the person skilled in the art and is described, for example, in EP-A1 0 291 533, EP-A1 0 321 201 and EP-A1 0 360 257.
  • the expression of ribozymes in plants For example, cells were described in Steinecke, EMBO J. 11 (1992), 1525-1530 and Feyter, Mol. Gen. Genet. 250: 329-338 (1996).
  • Suitable target sequences and ribozymes can e.g. as described in Steinecke (Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460), can be determined by secondary structure calculations of ribozyme and target RNA and their interaction.
  • the protein with the enzymatic activity of an AGPase comes from plants; preferably Arabidopsis thaliana or Brassica napus.
  • promoters which ensure transcription in plant cells.
  • the promoter is chosen so that the expression is only in a specific tissue, preferably in the embryo or seed and / or at a specific point in time of plant development or at an external level Influences determined point in time.
  • the promoter can be homologous or heterologous to the plant.
  • Useful promoters are also, for example, chemically inducible promoters such as the Tet system (Gatz (1991), Mol. Gen. Genet. 227, 229-237).
  • the embryo-specific promoter is from plants, preferably from Cuphea lanceolata, Brassica rapa or Brassica napus.
  • the first two aforementioned promoters are promoters of the CIFatB3 or CIFatB4 genes which have already been successfully used in transgenic oilseed rape for the biosynthesis of medium-chain fatty acids and therefore have a suitable “expression window” for solving the present problem.
  • the invention further relates to vectors which contain recombinant DNA molecules according to the invention.
  • vectors which contain recombinant DNA molecules according to the invention.
  • These are preferably plasmids, cosmids, viruses, bacteriophages and other vectors customary in genetic engineering.
  • a large number of cloning vectors are available to prepare the introduction of foreign genes into higher plants, which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the desired sequence can be introduced into the vector at a suitable restriction site.
  • the plasmid obtained is used for the transformation of E. coli cells. Transformed E. coli cells are grown in a suitable medium, then harvested and lysed.
  • the plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.
  • the vector according to the invention contains a selectable marker gene.
  • Suitable marker genes are known to the person skilled in the art from the prior art, for example antimetabolite resistance as a selection basis for dhfr, which transmits resistance to methotrexate (Reiss, Plant Physiol. (Life Sei. Adv.) 13 (1994), 142-149); npt, conferring resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J.
  • hygro conferring resistance to hygromycin (Marsh, Gene 32 (1984), 481-485 ) and Sul, which confer resistance to sulfadiazine (Guerineau, Plant Mol. Biol. 15 (1990), 127-136).
  • Additional selectable genes have been described, namely trpB, which enables cells to use indole instead of tryptophan; hisD, which enables cells to use histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci.
  • Mannose-6-phosphate isomerase which enables cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase), which transmits resistance to the ornithine decarboxylase inhibitor, 2- (diflouromethyl) -DL-omithin, DFMO ( McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed.) Or Deaminase from Aspergillus terreus, which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338) .
  • Suitable detectable markers are known to the person skilled in the art and are commercially available.
  • This marker is preferably a gene which contains luciferase (Giacomin, PL Sc. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996) , 44-47) or ⁇ -glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907).
  • the marker gene is not necessarily in the vector which contains the DNA Molecule carries, must be present, but can also be co-transformed with this (Lyznik (1989) Plant Mol. Biol. 13, 151-161; Peng (1995) Plant Mol. Biol. 27, 91-104).
  • This option is useful, for example, if no physical coupling of the marker gene and the recombinant DNA molecule is desired.
  • the marker gene and the recombinant DNA molecule according to the invention can independently segregate in subsequent crossings.
  • Another strategy to generate marker-free transgenic plants is the use of sequence-specific recombinases.
  • two strategies can be used, for example: (i) re-transforming a starting line expressing recombinase and crossing out the recombinase after removal of the selection marker which was associated with the desired gene. (ii) Co-transformation followed by outcrossing. Prerequisites for this recombinase strategy are (i) flanking the selectin marker with recognition sequences for the recombinase and (ii) a recombinase that is active in plants and does not use any plant-specific sequences for recombination.
  • RecA Reiss (1996) Proc. Natl. Acad. Sei.
  • the invention relates to host cells which contain the recombinant DNA molecules or vectors according to the invention transiently or stably.
  • a host cell is understood to mean an organism which is able to take up recombinant DNA in vitro and, if appropriate, to express the DNA sequence contained in the recombinant DNA molecules according to the invention.
  • the invention relates to plant cells which contain the recombinant DNA molecules or vector systems or derivatives or parts thereof according to the invention.
  • the cells according to the invention are preferably characterized in that the introduced recombinant DNA molecule according to the invention is either heterologous with respect to the transformed cell, ie does not naturally occur in these cells, or is located at a different location in the genome than the corresponding naturally occurring sequence.
  • kits which contain a recombinant DNA molecule according to the invention or a vector according to the invention and optionally further recombinant DNA molecules which e.g. Contain DNA sequences that encode enzymes that are involved in fatty acid change.
  • a recombinant DNA molecule according to the invention or a vector according to the invention and optionally further recombinant DNA molecules which e.g. Contain DNA sequences that encode enzymes that are involved in fatty acid change.
  • the kit according to the invention can furthermore contain, for example, agents for the detection of plants transformed with the recombinant DNA molecules or vectors according to the invention, for example suitable PCR primers, probes, components for AGPase enzyme test, selection agents for plants etc.
  • kits according to the invention can be used in a variety of ways. Exemplary areas of application such as the production of transgenic plant cells and plants are given below.
  • the present invention thus also relates to a method for producing transgenic plant cells, plant tissue and plants, which comprises the following steps:
  • a variety of techniques are available for introducing DNA into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and other possibilities.
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent
  • the fusion of protoplasts the injection
  • the electroporation of DNA the introduction of DNA using the biolistic method and other possibilities.
  • Simple plasmids such as e.g. pUC derivatives can be used.
  • a selectable marker is present.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right boundary, but frequently the right and left boundary of the Ti and Ri plasmid T-DNA as the flank region, must be linked to the genes to be introduced.
  • the DNA to be introduced must be cloned into special plasmids, either in an intermediate vector or in a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination.
  • Intermediate vectors cannot be found in agrobacteria replicate. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation).
  • Binary vectors can replicate in both E. coli and agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters, Mol. Gen. Genet. 163 (1978), 181-187).
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region.
  • the vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
  • the agrobacterium transformed in this way is used to transform plant cells.
  • the use of T-DNA for the transformation of plant cells has been intensively investigated and described in EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46 and An, EMBO J. 4 (1985), 277-287.
  • plants or plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Whole plants can then be regenerated in a suitable medium from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells).
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • Other ways of introducing foreign DNA using the biolistic method or by protoplast transformation are known (see, for example, Christou (1996) Trends in Plant Science 1, 423-431; Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi- Volume Comprehensive Treatise (HJ. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).
  • the recombinant DNA molecule according to the invention or the vector according to the invention is introduced in the method according to the invention by means of Agrobacterium tumefaciens, particle bombardment, electroporation, microinjection, liposomes or polyethylene glycol.
  • the present invention also relates to transgenic plant cells which contain a recombinant DNA molecule or a vector according to the invention or which have been obtained by the method according to the invention and to transgenic plant cells which originate from plant cells transformed in this way.
  • Such cells can be distinguished from naturally occurring plant cells in that they contain at least one recombinant DNA molecule according to the invention which does not naturally occur in these cells or in that such a molecule is located at one location in the genome of the cell is integrated in which it does not occur naturally, ie in a different genomic environment.
  • the plant cell according to the invention contains at least one further foreign gene.
  • the crossing combination with e.g. Plants that are overexpressing acetyl-CoA carboxylase, preferably rapeseed lines, ensure that the metabolic intermediates obtained can preferably flow off into the fatty acid biosynthesis.
  • the foreign gene encodes acetyl-CoA carboxylase or another enzyme determining the fatty acid and / or lipid biosynthesis rate such as e.g. ⁇ -ketoacyl synthase or diacylglycerol acyltransferase.
  • the transgenic plant cells can be regenerated to plant tissue and whole plants using techniques known to those skilled in the art.
  • the plant tissues and plants obtainable by regeneration of the transgenic plant cells according to the invention are likewise the subject of the present invention.
  • the invention furthermore relates to plants and plant tissues which contain the transgenic plant cells described above.
  • the promoter chosen eg 35S CaMV
  • the adult plants are also resistant to the selection agent and can be selected by administration of this compound.
  • tissue-specific promoters this possibility is eliminated and the person skilled in the art can use molecular biological methods such as PCR, for example, to identify these plants.
  • the person skilled in the art can, of course, for example after self-breeding or backcrossing against the parent, lay out seeds of such plants on media contained in the selection medium and infer them based on the germination capacity of these seeds or survival of the plants in a later stage of development (depending on the chosen promoter) whether the plants are transgenic or not.
  • the transgenic plants can in principle be plants of any plant species, ie both monocot and dicot plants. It is preferably useful plants such as wheat, barley, rice, rapeseed, pea, corn, sugar beet, sugar cane or potato.
  • Oil plants are particularly preferred, in particular the six most important oil plants (soybean, oil palm, oilseed rape, sunflower, cotton and peanut), from which 84% of the vegetable oils are produced worldwide (Lühs, (1993), supra), which mainly consist of palmitin, Stearic, oleic, linoleic and linolenic acid exist.
  • the invention also relates to propagation material and harvest products of the plants according to the invention, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings etc.
  • the present invention provides recombinant DNA molecules and vectors which can be used for the production of transgenic plant cells, plant tissue and / or plants in which the fatty acid synthesis and / or the oil content is preferably increased.
  • the recombinant DNA molecules, methods and plant cells or plants according to the invention can of course also be used in connection with the production of oils with modified fatty acid profiles (Töpfer, Science 268 (1995), 681-686).
  • oils with modified fatty acid profiles Töpfer, Science 268 (1995), 681-686.
  • Currently only 10% of the edible oil produced is used in the non-food sector, such as in lubricants, hydraulic oils, alternative fuels or in oil-containing chemicals for coatings, plasticizers, soaps and detergents (Lühs, (1993), supra).
  • the economic requirements for industrially used raw materials differ considerably from the requirements for the production of cooking oil.
  • the ideal oil for an oleochemical application would consist of a certain type of fatty acid, which could be constantly supplied at a competitive price compared to raw materials from mineral oil products.
  • such a fatty acid should also contain a reactive group and thus an additional target for chemical modifications. It it is expected that such fatty acids can be produced in plants (Kishore, Curr. Opin. Biotechnol. 4 (1993), 152). It is therefore also to be expected that the recombinant DNA molecules, vectors, plant cells and plants or their seeds according to the invention can be used for the production of, for example, biodiesel and other fuels or for plastics. With the help of the recombinant DNA molecules according to the invention, it is now possible to control the photosynthesis flow and thus significantly increase the yield of oils in agriculture by returning the carbon flow to the synthesis of oils with the desired fatty acid composition.
  • Figure 1 Schematic map of the cassette for embryo-specific gene expression pTE200. EcoRI, Smal, BamHI, Xhol, Notl, Xbal, Sacl, Kpnl, Apal, Sall and Sfil mark recognition sites for restriction endonucleases. For practical reasons, Sfil (A) and Sfil (B) differ in the variable nucleotide sequence within the recognition sequence.
  • FIG. 1 Schematic representations of the AGP antisense
  • Expression cassettes fusions of the complete cDNA ⁇ AGP) of the small subunit of AGPase in antisense orientation in plasmid pTE209b with the CIFatB4 promoter (pCIFatB4) and terminator ⁇ CiFatB4); in plasmid pTE109b with the CIFatB3 promoter ⁇ pCIFatB3) and CIFatB4 terminator ( ⁇ CIFatB4) and in plasmid pG009b with the GPDH promoter CIGPDH) and the terminator (t35S) of the 35S rna gene from CaMV. EcoRI, Notl, Sall, and Xhol mark recognition sites for restriction endonucleases.
  • Figure 3 Schematic map of the AGP sense expression cassette pTE209a: This derivative of the vector pTE200 ( Figure 1) carries an incomplete cDNA for the small subunit of the AGPase ( Figure 6) in sense orientation between the recognition sites for the EcoRI and Xhol Restriction endonucleases.
  • Figure 4 Schematic map of the AGP antisense expression cassette pTE209b: This derivative of the vector pTE200 ( Figure 1) carries one incomplete cDNA for the small subunit of AGPase ( Figure 6) in antisense orientation between the two recognition sites for the EcoRI restriction endonuclease.
  • FIG. 5 Schematic map of the binary vector pMH000-0.
  • Sfil (A) and Sfil (B) differ in the variable nucleotide sequence of their recognition sequence as indicated. After Sfil cleavage, recircularization of the starting plasmid is prevented and a directed insertion of the expression cassette from the pTE200 derivative is possible.
  • RB, LB right and left border region
  • t35S termination signal of the 35S rna gene from CaMV
  • pat phosphinotricin acetyltransferase gene
  • p35S promoter of the 35S rna gene from CaMV
  • p35S (min) minimal promoter of the 35S rna Gene from CaMV
  • tp-su / sulfonamide resistance gene with transit peptides
  • ⁇ nos termination signal of the nopaline synthase gene
  • Sm / Sp bacterial resistance to streptomycin and spectinomycin
  • parA, parB and parR plasmid propagation functions from the plasmid PVS1 with a large host range and others for Agrobacterium tumefaciens and Escherichia coli.
  • Figure 6 Schematic representation of the 1365 base pair (bp) long, incomplete cDNA for the small subunit of the AGPase from rapeseed (Brassica napus, Bnagpl).
  • the black bars on both sides represent 14 bp adapters for the recognition sites of the restriction endonucleases EcoRI and Notl.
  • Cross-streaked bars with a pointed triangle represent coding regions in sense orientation, the white bar, on the other hand, represents the 3 ' untranslated area.
  • the heterologous cDNA section is for the small one Subunit of pea AGPase (Pisum sativum, PsagpSI) is shown with the base positions of the overlapping sections.
  • Figure 7 Results from the starch measurement based on the fresh weight (FG) of maturing embryos of control plants (WT) and transgenic rape plants (pMH0209a). The regression line from the control values is shown as a reference.
  • Figure 8 Results from the activity measurement of the AGPase based on the fresh weight (FG) of maturing embryos of control plants (WT) and transgenic rape plants (pMH0209a). The solid line averages the control values. It roughly reflects the course of AGPase activity in early embryonic stages described in the literature (da Silva, Planta 203 (1997), 480-487).
  • FIG 10 Contents of oil (%) and free glucose ( ⁇ mol) in mature seeds of transgenic oilseed rape plants with an average grain moisture of 8 to 9% according to near infrared spectroscopy (NIRS).
  • NIRS near infrared spectroscopy
  • the results obtained with the AGPase construct (pMH0209a) in sense orientation for individual oilseed rape plants are compared graphically with those from 11 control plants (pMH000-0).
  • the dashed line marks the mean oil content of the controls at 38.7%, while the solid line refers to the mean free glucose of the controls at 21.1 ⁇ mol.
  • Figure 11 Schematic representations of the ADPGPP antisense
  • Expression cassettes fusions of the cDNA of the small subunit of the ADPGPP in antisense orientation in the plasmid pTE209 with the CIFatB4 promoter (pCIFatB4) and terminator (CIFatB4- ⁇ er); in plasmid pTE109 with the CIFatB3 promoter (pCIFatB3) and CIFatB4 terminator ⁇ CIFatB4-te ⁇ as well as in plasmid pG009 with the GPDH promoter ⁇ pCIGPD i) and the terminator (35S-ter) of the 35S rna gene from CaMV. EcoRI, Notl, Sall, and Xhol mark recognition sites for restriction endonucleases.
  • Figure 12 Schematic map of the binary vector pRE1.
  • the expression cassette pTE200 (FIG. 1) carries the pBluescript® II SK (-) (Stratagene®, Genbank® # 52324) derivative the embryo-specific promoter pCIFatB4 and the corresponding terminator sequence from Cuphea lanceolata, which is derived from the genomic clone CITEg16 (WO 95 / 07357).
  • the approx. 1600 bp long cDNA for the small subunit of the AGPase from rapeseed is used as an Xhol - EcoRI fragment corresponding to the restriction sites in pBluescript® II SK (-) in antisense orientation after modification of the sites in the Smal site of the expression cassette pTE200 (FIG 1) introduced.
  • the plasmid pTE209 results (FIG. 11).
  • the Sall-Notl fragment from pTE209 is inserted into the Sall-Smal polylinker interfaces of the binary vector pRE1 after modification of the Notl interface (FIG. 12).
  • the 1175 bp long partial cDNA (AGP) for the small subunit of the AGPase from rapeseed was inserted as EcoRI-Xhol fragment into the corresponding interfaces within the pTE200 expression cassette (FIG. 1).
  • the plasmid pTE209a resulted from this (FIG. 3).
  • the 1360 bp long EcoRI fragment was inserted in the antisense orientation into the EcoRI site within the expression cassette pTE200 (FIG. 2).
  • the plasmid pTE209b resulted (FIG. 4).
  • the Sfil fragment from pTE209a or pTE209b was inserted into the Sfil polylinker interfaces of the binary vector pMHOOO-0 (FIG. 5).
  • the transformation vectors pMH0209a and pMH0209b resulted. 2.
  • Cloning methods such as: restriction cleavage, modification of restriction sites, DNA isolation, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria
  • Sequence analysis of recombinant DNA, Southern blot, Northemblot, Western blot analyzes were carried out according to Sambrook (Cold Spring Harbor Laboratory Press (1989); ISBN 0-87969-309-6).
  • the transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Willmitzer (Nucl. Acid Res. 16 (1988), 9877).
  • Agrobacteria were grown in YEB medium (Vervliet, Gen. Virol. (1975) 26, 33).
  • AGPase cDNA bank was used which was produced by conventional methods (Sambrook 1989, supra).
  • a full-length cDNA which codes for the small subunit of AGPase from the Arabidopsis plant, which is closely related to oilseed rape, served as the probe.
  • This cDNA was identified by the Arabidopsis genome project (EST-Kion 38E3T7) (Newman, Plant Physiol. 106 (1994), 1241-1255).
  • the "screening" was carried out using a redio-labeled probe as described (Kampfenkel, FEBS Letters 374 (1995), 351-355). 12 positive clones were isolated.
  • the longest clone according to restriction analysis was sequenced and used for further cloning steps.
  • the longest incomplete cDNA fragment has a similarity of 80.5% (using the best fit of the Wisconsin GCG computer program, Devereux, Nucl. Acid Res. 12 (1984), 387-395) to the AGP sequence from pea (Pisum sativum, Genbank, Accession X96764, Bases 1 to 1954).
  • the heterologous rapeseed sequence sweeps over that of the pea from base position 541 to 1637, excluding the 3'-untranslated region, which as a rule differs (FIG. 6).
  • Suitable oil plants such as oilseed rape, for example the DRAKKAR variety, were transformed using Agrobacterium tumefaciens according to the protocol from De Block, Plant Physiol. 91: 694-701 (1989) starting from hypocotyl pieces.
  • the agrobacterial strain GV3101 C58C1 Rifr (Van Larebeke, Nature 252 (1974), 169-170) with the Ti plasmid pMP90RK (Koncz, Mol. Gen. Genet. 204 (1986), 383-396) and the binary vector pRE1 are used .
  • kanamycin resistance is made with 50 ⁇ g / ml later with 15 ⁇ g / ml kanamycin (monosulfate, Sigma K-4000) per milliliter of medium.
  • the transformation rate is usually 10% based on the number of Hypocotyl pieces laid out. It is based on the verification of the transformation by means of Southern blot (Sambrook (1989), supra) or PCR (Edwards, Nucl. Acids Res. 19 (1991), 1349).
  • the agrobacterial strain GV3101 C58C1 Rifr with the plasmid pMP90RK and the binary vector pMHOOO-0 was used.
  • the selection for sulfonamide resistance was carried out with 15 mg / l during the regeneration of sprouts and later for rooting them with 10 mg / l sulfadiazine (99% purity, Sigma S-8626) per liter of nutrient medium.
  • the plants were then subjected to an acclimatization phase of 8-14 days in the climatic chamber before they were cultivated in the greenhouse until they were ripe.
  • the transformation rate is usually 10% based on the number of Hypocotyl pieces laid out.
  • Glufosinatammonium per liter in a 1: 100 dilution on one leaf per rape plant and the herbicide tolerance determined visually.
  • the frozen emryons were freed from the seed coat and sorted according to size (J, M, A). The number and fresh weight were determined.
  • the embryos were then frozen in liquid nitrogen.
  • the embryos were then ground in the frozen state in the Eppendorf vessel. Buffer was added to the samples (400 microliters / 100 mg FG). Buffer: 50 mM pug (pH 7.7), 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 0.1 mM Pefablock (protease inhibitors). 40 microliters were taken from this crude extract for protein determination.
  • the sample was centrifuged for 10 min at 4 ° C 10000g. The supernatant was removed, aliquoted and frozen in liquid nitrogen (for AGPase measurement). The precipitate was used to determine the starch.
  • the enzyme test includes: 75 mM Hepes pH 7.9; 10mM MgCl2; 0.75 mM NAD; 1mM ADPG (ADP glucose); 5 U G6PDH (glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides); 2 U PGM (phosphoglucomutase) and 0.75 mM Na pyrophosphate. 15 microliters of sample were used. Measurements were taken in a double-beam photometer at 334 nm (Uvikon 810) with a reference cuvette. The test was linear and linear to the amount of sample used for at least 5 min. Starch determination: The precipitate was extracted 4 times with 80% ethanol at 90 ° C. (10 min) and centrifuged 14000 g each time for 10 min, and the supernatants discarded. Then 300 microliters of water were added and the
  • the enzyme test contains 75 mM Hepes pH 7.9; 10mM MgCl2; 2mM NADP; 5 mM ATP; 1 U
  • Example 1 Oilseed rape with increased oil content in seeds by expression of an AGPase-sense RNA
  • Transgenic oilseed rape plants which carry an incomplete cDNA of the small subunit of the AGPase in sense orientation connected to the embryo-specific C / Fafß4 promoter-terminator cassette (pTE209a). Based on the observation that an incomplete transcript of a tomato gene in sense orientation was able to inhibit the expression of the endogenous gene in transgenic plants (Smith, Mol Gen Genet. 224 (1990), 477-481), a similar one became here Approach followed.
  • glycose lysis enzymes have been shown to be present in rapeseed embryonic plastids, the excess free glucose in pMH0209a lines 1D, 67D, 97D and 69 could possibly be due to a combination of these with rapeseed lines which provide increased embryonic gene expression for acetyl-CoA carboxylase (ACCase) have, ensure a further carbon flow in the fatty acid and storage lipid synthesis.
  • ACCase acetyl-CoA carboxylase
  • Example 2 Oilseed rape with increased oil content in seeds by expression of an AGPase antisense RNA
  • Transgenic oilseed rape plants which carry an incomplete cDNA of the small subunit of the AGPase in antisense orientation linked to the embryo-specific C / FafB4 promoter terminator cassette (pTE209b).
  • pTE209b embryo-specific C / FafB4 promoter terminator cassette
  • Example 3 Oilseed rape with increased oil content in seeds by expression of an ACCase and an AGPase-sense RNA
  • Transgenic rapeseed plants which contain an oilseed rape ACCase gene (WO 94/17188) under the control of the embryo-specific C / Fa.B3 promoter (WO 95/06733) and an incomplete cDNA of the small subunit of the AGPase in Wear sense orientation combined with the embryo-specific C / Fré4 promoter-terminator cassette (pTE209a).
  • Example 4 Oilseed rape with increased oil content in seeds by expression of an ACCase and an AGPase antisense RNA
  • Transgenic oilseed rape plants which contain an oilseed rape ACCase gene (WO 94/17188) under the control of the embryo-specific C / Fafß3 promoter (WO 95/06733) and an incomplete cDNA of the small subunit of the AGPase in Wear an antisense orientation linked to the embryo-specific C / Fa / S4 promoter-terminator cassette (pTE209b).
  • transgene for ACCase Since the starting material was a cleaving population, the presence of the transgene for ACCase was examined by PCR. Out of 89 lines examined, 69 contained one or more transgenes for ACCase. 67 individuals were doubly positive. In addition there were five PCR negative but bastatolerant lines for control purposes. Different developmental stages of maturing embryos were harvested from the bastatolerant plants and stored at very low temperatures. The material is examined as listed in Example 1.
  • Example 5 Further and complementary approaches to the production of rapeseed with an increased oil content in seeds
  • the expression of the cDNA or parts thereof for the subunit A and / or B of the AGPase from Arabidopsis thaliana or from Brassica napus in "antisense” or “sense” orientation at the target location of the starch and storage lipid synthesis in oil plants the resulting cDNA fused with regulatory sequences suitable for plants (promoters and terminators).
  • promoters for example, embryo-specific promoters from Cuphea lanceolata
  • pCIFatB3 promoter region of the gene on the genomic clone CITEgl (plasmid pNBM99 - TEg1 DSM 8477, deposited on August 27, 1993 with the German Culture Collection of Microorganisms and Cell Cultures GmbH in Braunschweig, Germany)
  • pCIFatB4 promoter region of the gene on the genomic clone CITEg16 (plasmid pNBM99 - TEg16 DSM 8478, deposited on August 27, 1993 with the culture collection German Collection of Microorganisms and Cell Cultures GmbH in Braunschweig, Germany)
  • pCIGPDH promoter region of the gene on the genomic clone CIGPDHg9 (pCIFatB3, pCIFatB4, WO 95/07357, pCIGPDH; WO 95/06733) used for directed expression in rape embryos and in front of the cDNA (in "antisense” or "sense") switched. Furthermore, the expression of the AGPase cDNA with the alternative promoters, in particular pCIGPDH, pNapin or pOleosin is achieved with the aim of preventing starch formation even in later embryonic stages than that described in Example 1 and of diverting the flow of available carbon into the storage lipids. The corresponding terminator areas are cloned behind it.
  • the 3 ' untranslated region for transcription termination (terminator) of chimeric plant genes normally contains a polyadenylation signal. This causes the polymerization of a poly (A) sequence at the 3 ' end of the RNA.
  • suitable terminators are 3 ' transcribed, non-translated regions of the 35S rna gene of the cauliflower mosaic virus (CaMV) and of plant genes such as the 3 ' region of the CIFatB4 gene from C. lanceolata which is preferred here.
  • the resulting vectors are transferred to rape in an Agrobacterium -mediated transformation.
  • Regenerated plants in particular developing embryos in the seeds of these plants, are examined in Northern blot analyzes for reduced "steady state" transcripts of the AGP, and analyzed in the Western blot method for correspondingly reduced amounts of enzyme.
  • the results are correlated with a throttled AGPase activity, which is measured photometrically.
  • the embryos and ripe seeds are examined for changes in the oil, starch and, as a control, also for the protein content.
  • Table 1 Characterization of wild type and transgenic lines with regard to starch content and AGPase activity.

Abstract

L'invention concerne des moyens et procédés pour augmenter la teneur en huile dans des plantes. Elle concerne également des molécules d'ADN recombinées qui contiennent les séquences de régulation d'un promoteur embryospécifique dans des plantes. Ces séquences de régulation sont reliées fonctionnellement à une séquence d'ADN dont le produit génique inhibe une protéine avec l'activité enzymatique d'une ADP-glucose-pyrophosphorylase (AGPase). Cette séquence d'ADN est reliée de façon fonctionnelle à une séquence de régulation qui peut servir de séquence terminale de transcription dans des plantes. L'invention concerne en outre des vecteurs et des cellules hôtes qui contiennent les molécules d'ADN recombinées de l'invention. L'invention concerne également des procédés pour produire des cellules et tissus végétaux et des plantes transgéniques, ainsi que des produits de récolte et des matériaux de reproduction qui contiennent les molécules d'ADN recombinées de l'invention ou qui peuvent être produits au moyen du procédé de l'invention et qui présentent une teneur élevée en huile.
PCT/EP1998/005461 1997-08-29 1998-08-28 Molecules d'adn recombinees et procede pour augmenter la teneur en huile dans des plantes WO1999011805A1 (fr)

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US6232529B1 (en) 1996-11-20 2001-05-15 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
WO2005063988A1 (fr) * 2003-12-23 2005-07-14 Pioneer Hi-Bred International, Inc. Modification de proprietes huileuses chez des vegetaux
EP1639100A2 (fr) * 2003-06-27 2006-03-29 Monsanto Technology LLC Elevation des niveaux d'huile dans les plantes

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DE19914792A1 (de) * 1999-03-31 2000-10-05 Max Planck Gesellschaft Metabolische Selektionsmarker für Pflanzen

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WO1994017188A2 (fr) * 1993-01-22 1994-08-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Gene acetyl-coa-carboxylase
EP0634491A1 (fr) * 1993-07-12 1995-01-18 Monsanto Company Semences à contenu en huile modifiée
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WO1994017188A2 (fr) * 1993-01-22 1994-08-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Gene acetyl-coa-carboxylase
EP0634491A1 (fr) * 1993-07-12 1995-01-18 Monsanto Company Semences à contenu en huile modifiée
WO1996002652A2 (fr) * 1994-07-20 1996-02-01 Zeneca Limited GENES DE β-CETOACYL-ACP-REDUCTASE PROVENANT DE BRASSICA NAPUS
WO1998022604A1 (fr) * 1996-11-20 1998-05-28 Pioneer Hi-Bred International, Inc. Procedes de production de graine a haute teneur en huile par modification des teneurs en amidon

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6232529B1 (en) 1996-11-20 2001-05-15 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
EP1639100A2 (fr) * 2003-06-27 2006-03-29 Monsanto Technology LLC Elevation des niveaux d'huile dans les plantes
EP1639100A4 (fr) * 2003-06-27 2007-12-26 Monsanto Technology Llc Elevation des niveaux d'huile dans les plantes
AU2004254373B2 (en) * 2003-06-27 2009-11-19 Monsanto Technology, Llc Elevation of oil levels in plants
WO2005063988A1 (fr) * 2003-12-23 2005-07-14 Pioneer Hi-Bred International, Inc. Modification de proprietes huileuses chez des vegetaux

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