WO2003077643A2 - Procedes pour augmenter la teneur en huile de plantes - Google Patents

Procedes pour augmenter la teneur en huile de plantes Download PDF

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WO2003077643A2
WO2003077643A2 PCT/EP2003/002733 EP0302733W WO03077643A2 WO 2003077643 A2 WO2003077643 A2 WO 2003077643A2 EP 0302733 W EP0302733 W EP 0302733W WO 03077643 A2 WO03077643 A2 WO 03077643A2
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nucleic acid
seq
storage protein
acid sequence
protein
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PCT/EP2003/002733
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WO2003077643A3 (fr
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Jörg BAUER
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Basf Plant Science Gmbh
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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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/286—Carthamus (distaff thistle)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/52—Juglandaceae (Walnut family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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 invention relates to methods for increasing the oil content in plants by reducing one or more storage proteins.
  • the invention further relates to the use of plants with a reduced storage protein content for the production of food, animal feed, seeds, pharmaceuticals or fine chemicals, in particular for the production of oils.
  • the fatty acids obtainable from vegetable oils are also of particular interest. They are used, for example, as raw materials for plasticizers, lubricants, surfactants, cosmetics etc. or are used as valuable raw materials in the food and feed industry.
  • the provision of rapeseed oils with fatty acids of medium chain length is of particular interest, since these are particularly sought after in the manufacture of surfactants.
  • plant metabolism can be advantageously changed in a way that would be difficult or impossible to achieve with traditional breeding methods.
  • unusual fatty acids for example certain polyunsaturated fatty acids, are only synthesized in certain plants or ⁇ not at all in plants and can therefore only be produced in transgenic plants by expression of the corresponding enzyme (e.g. Millar et al. (2000) Trends Plant Sei 5 : 95-101).
  • Lipids are synthesized from fatty acids and their synthesis can be divided into two sub-mechanisms, a quasi "prokaryotic” and a quasi “eukaryotic”'(Browse et al. (1986) Biochemical J 235: 25-31; Ohlrogge & Browse (1995) Plant Cell 7: 957-970).
  • the prokaryotic mechanism is in the Plastids located and comprises the biosynthesis of free fatty acids are exported to the cytosol where they are received as i 'ettklad Acidacyl CoA esters in the eukaryotic mechanism and -stert having glycerol-3-phosphate to phosphatidic acid (PA) comparable.
  • PA phosphatidic acid
  • PA is the starting point for the synthesis of leutral and polar lipids.
  • the neutral lipids are synthesized using the Kennedy route (Voelker (1996) Genetic Engineering ed .: Setlow 18: 111-113; Shankline & Cahoon (1998) -nnu Rev Plant Physiol Plant Mol Biol 49: 611-649; Frentzen ( 1998) jipids 100: 161-166).
  • Barnen are particularly dependent on storing energy and basic building blocks, e.g. to ensure later germination.
  • Storage takes place in the form of storage lipids, storage proteins or starch (storage carbohydrate).
  • the relationships between the three storage molecules vary. Rapeseed varieties contain an average of around 48% storage lipids, 19% starch and 21% storage proteins, while soybean contains 22% lipids, 12% starch and 37% proteins (each based on dry matter) (Biochemistry & Molecular Biology of the Plant ed. Buchanan, Gruissem, Jones 2000, American Society of Plant Physiologists).
  • the storage molecules are accumulated during the embryo development of the semen.
  • SP Storage proteins
  • SP in the embryo are used to store carbon, nitrogen and sulfur, which are required for the rapid heterotrophic growth when seeds or pollen germinate. They usually have no enzymatic activity. These proteins are only synthesized in the embryo during seed development. SP accumulate in protein storage vacuoles (PSV) of differentiated cells in the embryo or endosperm. As a further form, they can also be present as protein bodies associated with the endoplasmic reticulum (ER) (Her an & Larkins (1999) Plant Gell 11: 601-613). All storage proteins are originally synthesized on the rough ER (Bollini & Chrispeels (1979) Planta 146: 487-501).
  • Prolamines only occur specifically in the endosperm of grasses (Poaceae), where they are the main storage proteins (exceptions are rice and oats, where glutelin-like and globulins predominate). In contrast, globulins dominate in dicotyledons.
  • a total of four large gene families for storage proteins can be assigned based on their sequences: 2S-albumins (similar to napin), 7S-globulins (similar to phaseolin), HS / 12S-globulins (similar to legumin / cruciferin) and the zein prolamines.
  • 2S albumins are widely used in seeds of dicotyledons, including important commercial plant families such as Fabaceae (e.g. soybean), Brassicaceae (e.g. rapeseed), Euphorbiaceae (e.g. castor bean) or Asteraceae (e.g. sunflower). 2S albumins are compact globular proteins with conserved cysteine residues that often form heterodimers.
  • 7S globulins are in trimeric form and contain no cysteine residues. After their synthesis, like the 2S albumins, they are split into smaller fragments and glycosylated. Despite differences in the size of the polypeptides, the different 7S globulins are highly conserved and presumably, like the 2S albumins, are based on a common precursor protein. The 7S globulins are only present in small amounts in monocots. In dicotyledons their proportion is smaller and smaller compared to the HS / 12S globulins.
  • HS / 12S globulins represent the main fraction of the storage proteins in dicotyledons.
  • the high similarity of the different HS globulins from different plant genera in turn suggests a common precursor protein in evolution.
  • sucrose is the primary source of carbon and energy which is transported from the leaves to the developing seeds.
  • sucrose is converted to glucose-6-phosphate and pyruvate, which is transported into the plastids and used there for the synthesis of acetyl-CoA, which is the starting product for the synthesis of the fatty acids.
  • glucose-6-phosphate and pyruvate which is transported into the plastids and used there for the synthesis of acetyl-CoA, which is the starting product for the synthesis of the fatty acids.
  • EP-A 0 591 530 describes the reduction in the expression of a storage protein in seeds, in particular the storage protein glutelin in rice, with the aim of optimizing the usability of rice in fermentation processes for producing alcoholic beverages. Proteins as such are a hindrance in these processes. An effect of the reduction on the content of other herbal ingredients is not described.
  • WO 87/47731 describes the reduction of one or more storage proteins in the seed of soybeans by means of antisense technology.
  • a method is also described in which, in addition to the storage protein, the expression of a gene of fatty acid biosynthesis (microsomal ⁇ -12 desaturase [Fad 2-1] genes) is reduced. In example 2 (p.25 / Z.4-9) this is done by cosuppression.
  • the result is transgenic so bean plants with a reduced content of a storage protein and a content of oleic acid in the total amount of fatty acids, which is relatively higher in relation to other fatty acids than in non-transgenic soybean plants.
  • the change in the fatty acid profile is due to the suppression of the Fad2-1 gene and not the storage protein. No change in the total content of fatty acids is described.
  • WO 97/35023 describes storage proteins and methods for increasing the content of certain amino acids in plants by expressing said storage proteins. A description of the effects on other plant metabolites is not disclosed. WO 97/41239 describes similar methods based on sulfur-rich storage proteins.
  • WO 98/26064 describes methods for reducing one or more storage proteins, preferably in maize. Plants with an increased or changed content of amino acids or starch are also described. An impact on the oil content is not described.
  • WO 99/15004 describes the modification of the content of metabolites in the food organs of plants by expression of sulfur-rich proteins with more than 10% sulfur-containing amino acids, in particular the sunflower seed albumin (SSA; sunflower seed albumin).
  • SSA sunflower seed albumin
  • the expression in lupine an increase in the oil content (Example 1; p.28 / Z.4-5, 17).
  • the expression of the same gene in pea causes a decrease (Example 2; p.32 / Z.21).
  • EP-A 0 620 281 describes a change in the lipid composition (fatty acid pattern) in oilseed rape by reducing the expression of a storage protein (napin) by means of antisense technology.
  • Example 6 (S.ll / Z.12-15) describes that only the ratio of the individual fatty acids changed so that the content of oleic acid decreased and the content of linoleic and linolenic acid increased. It is explicitly indicated that the total fatty acid content remained unchanged.
  • Corresponding data are also disclosed in the corresponding publication by the inventors (Kohno-Murase J et al. (1994) Plant Mol Biol 26 (4): 1115-1124).
  • WO 01/81604 describes transgenic plants which contain a cytosolic acetyl-CoA carboxylase Express (ACCase).
  • a first object of the invention comprises a method for increasing the total oil content in plant organisms, characterized in that subsequent work steps are included
  • Plant organism or cells derived therefrom generally means any cell, tissue, part or reproductive material (such as seeds or fruits) of an organism which is capable of photosynthesis. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Annual, perennial, monocot and dicot plants are preferred.
  • Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
  • Plant in the context of the invention means all genera and species of higher and lower plants in the plant kingdom. Included under the term are the mature plants, seeds, sprouts and seedlings, as well as parts derived therefrom, propagation material, plant organs, tissues, protoplasts, callus and other cultures, for example cell cultures, and all other types of groupings of plant cells into functional or structural units , Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
  • Plant includes all annual and perennial, monocotyledonous and dicotyledonous plants and includes, by way of example but not by way of limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hesisocallis, Nesis gonium, panieum, pennisetum, ranunculus, senecio, salpiglossis, cucu is, browaalia, glycine, pisum
  • Plants from the following plant families are preferred: Amarantheae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceaeaeae, Rosaceaeaeae, Rosaceaeaeae Tetragoniacea, Theaceae, Umbelliferae.
  • Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants, such as, for example, the family of the Gramineae such as rice, corn, wheat or other types of cereals such as barley, millet, rye, triticale or oats as well as sugar cane and all types of grass.
  • the family of the Gramineae such as rice, corn, wheat or other types of cereals such as barley, millet, rye, triticale or oats as well as sugar cane and all types of grass.
  • the invention is very particularly preferably applied from dicotyledonous plant organisms.
  • Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example
  • Asteraceae such as sunflower, tagetes or calendula and others
  • Cucurbitaceae such as melon, pumpkin or zucchini and others
  • Rubiaceae preferably of the subclass Lamiidae such as Coffea arabica or Coffea liberica (coffee bush) and others,
  • Solanaceae especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tüberosum (potato) and melongena (eggplant) as well as tobacco or peppers and others,
  • Sterculiaceae preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) and others,
  • Theaceae preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others, - Umbelliferae, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (Seiarie)) and others; and the genus Capsicum, especially the species annu (pepper) and others,
  • ornamental plants useful or ornamental trees, flowers, cut flowers, shrubs or lawn.
  • examples include, but are not limited to, angiosperms, bryophytes such as hepaticae (liverwort) and musci (moss), pteridophytes such as ferns, horsetail and lycopods; Gymnosperms such as conifers, cycads, ginkgo and gnetals, the families of rosaceae such as rose, ericaceae such as rhododendrons and azaleas, euphorbiaceae such as poinsettias and croton, caryophyllaceae such as cloves, solanaceae such as petunias, Gesneriaceae such as the Usamalsamineaeaid as the Usambaramineae , Iridaceae like gladiolus, iris, freesia and crocus, Compositae like mari
  • Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as algae, cyanobacteria and mosses.
  • Preferred algae are green algae, such as, for example, algae of the genus Hae atococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocy ⁇ tis is particularly preferred.
  • plants which are suitable for oil production such as, for example, arabidopsis, rapeseed, sunflower, sesame, safflower, olive tree, soybean, corn, wheat or various types of nuts, such as, for example, walnut almond.
  • the dicot plants, in particular rapeseed, soybeans and sunflower are particularly preferred.
  • Oil includes neutral and / or polar lipids and mixtures thereof. Examples listed but not restrictive are those listed in Table 1. Tab. Plant lipid classes
  • Neutral lipids preferably means triacylglycerides. Both the neutral and the polar lipids can contain a wide range of different fatty acids. The fatty acids listed in Table 2 should be mentioned as examples, but not by way of limitation.
  • Oils preferably means seed oils.
  • Oil content means the sum of all oils as defined above, preferably the sum of the triacylglycerides. "Increasing” the oil content means increasing the content of oils in a plant or a part, tissue or organ thereof, preferably in the seed organs of the plant. The oil content is at least 5%, preferably at least 10%, particularly preferably at least 15%, very particularly preferably at least 20%, most preferably at least 25, in comparison to a starting plant which is not subjected to the process according to the invention but is otherwise unchanged, under otherwise identical general conditions % elevated. Framework conditions means all conditions relevant to the germination, cultivation or growth of the plant such as soil, climate or light conditions, fertilization, irrigation, plant protection measures etc.
  • Storage protein generally means a protein which has at least one of the following essential properties:
  • Storage proteins are essentially only expressed in the embryo during seed development. "Essentially” means that at least 50%, preferably at least 70%, very particularly preferably at least in the said stage
  • Storage proteins are broken down again during seed germination.
  • the degradation during germination is at least 20%, preferably at least 50%, very particularly preferably at least 80%.
  • Storage proteins make up a significant proportion of the total protein content of the non-germinating seed.
  • the storage protein in the non-germinating seed of the wild-type plant preferably makes up more than 5% by weight of the total protein, particularly preferably at least 10% by weight), very particularly preferably at least 20% by weight, most preferably at least 30% by weight .-%.
  • Storage proteins preferably have 2 or all of the above-mentioned essential properties a), b) or c).
  • Storage proteins can be divided into subgroups according to other characteristic properties, such as their sedimentation coefficient or their solubility in different solutions (water, saline, alcohol).
  • the determination of the sedimentation coefficient can be carried out in the manner familiar to the person skilled in the art by means of ultracentrifugation (for example described in Correia JJ (2000) Methods in Enzymology 321: 81-100).
  • the storage protein is preferably selected from the classes of 2S-albumin (similar to napin), 7S-globulin (similar to phaseolin), HS / 12S-globulin (similar to legumin / cruciferin) or zein-prolamine.
  • Particularly preferred 2S albumins include
  • 2S albumins from Arabidopsis very particularly preferably the 2S albumins with SEQ ID NO: 2, 4, 6 or 8, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 1, 3, 5 or 7 .
  • 2S albumins from species of the genus Bras ⁇ ica, such as, for example, Brassica napus, Brassica nigra, Brassica juncea, Brassica oleracea or Sinapis alba, very particularly preferably the 2S albumins with SEQ ID NO: 32, 34, 36, 38, 40 , 46 or 48, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 31, 33, 35, 37, 39, 45 or 47,
  • 2S albumins from soybeans, very particularly preferably the 2S albumins with SEQ ID NO: 42 or 44, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 41 or 43,
  • 2S albumins from sunflower (Helianthus annus), very particularly preferably the 2S albumins with SEQ ID NO: 50 or 52, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 49 or 51,
  • rapeseed sunflower, flax, sesame, safflower, olive tree, soya or various types of nut.
  • functional equivalents are preferably distinguished by characteristic properties such as a 2S sedimentation coefficient and / or by solubility in water.
  • functional equivalents of the 2S-albumins have a homology of at least 60%, preferably at least 80%, very particularly preferably at least 90%, most preferably at least 95% to one of the protein sequences with SEQ ID NO: 2, 4 , 6, 8, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 where the homology preferably extends over a length of at least 30 amino acids, preferably at least 50 amino acids, particularly preferably over 100 amino acids most preferably extends over the entire length of the respective proteins, and have the same essential properties of a storage protein and - preferably - the characteristic properties of a 2S storage protein.
  • nucleic acid sequences coding for them at least one, preferably two, particularly preferably 3, most preferably all of the sequence motifs selected from in each case one of the following groups I, II, III or IV:
  • the functional equivalents in their nucleic acid sequences particularly preferably have at least one of the following sequence motifs selected from a specific one of the following groups V, VI, VII or VIII:
  • Particularly preferred 7S globulins include those from Arabidopsis or soybeans, very particularly preferably the proteins with SEQ ID NO: 155 or 157, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 154 or 156.
  • functional equivalents are preferably characterized by characteristic ones Properties such as a 7S sedimentation coefficient and / or by solubility in saline. As a further characteristic property, 7S globulins cannot contain any cysteine residues.
  • functional equivalents of the 7S globulins have a homology of at least 60%, preferably at least 80%, very particularly preferably at least 90%, most preferably at least 95% to one of the protein sequences with SEQ ID NO: 155 or 157 wherein the homology preferably extends over a length of at least 30 amino acids, preferably at least 50 amino acids, particularly preferably over 100 amino acids, most preferably over the entire length of the respective proteins, and have the same essential properties of a storage protein and - preferably - the characteristic properties of a 7S storage protein.
  • HS / 12S globulins preferably comprise 11S globulins from rapeseed, soybeans and Arabidopsis in particular
  • HS globulins from soya with SEQ ID NO: 20, 22, 24, 26 or 28, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 19, 21, 23, 25 or 27,
  • HS globulins from Arabidopsis thaliana with SEQ ID NO: 112, 114, 116, 118, 120 or 122 most preferably those encoded by the nucleic acids according to SEQ ID NO: 111, 113, 115, 117, 119 or 121 proteins,
  • rapeseed sunflower, flax, sesame, safflower, olive tree, soybean or various types of nuts
  • sunflower IIS storage protein SEQ ID NO: 30
  • functional equivalents are preferably distinguished by characteristic properties such as an IIS or 12S sedimentation coefficient and / or by solubility in saline solution (PBS; phosphate-buffered saline solution) and / or poor solubility in water.
  • PBS phosphate-buffered saline solution
  • functional equivalents of the IIS or 12S albumins have a homology of at least 60%, preferably at least 80%, very particularly preferably at least 90%, most preferably at least 95% to one of the protein sequences with SEQ ID NO: 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 112, 114, 116, 118, 120 or 122 where the homology is particularly preferred over a length of at least 30 amino acids, preferably at least 50 amino acids preferably over 100 amino acids, most preferably over the entire length of the respective proteins, and have the same essential properties of a storage protein and - preferably - the characteristic properties of an 11S or 12S storage protein.
  • Arabidopsis thaliana 12S cruciferin storage protein (ATCRU3) according to SEQ ID NO: 112 and its homologues from other plant species, such as, for example, rapeseed, soybean or sunflower, are particularly preferred, these preferably having a homology of at least 65%, preferably at least 80%, entirely particularly preferably have at least 90%, most preferably at least 95% of one of the protein sequences with SEQ ID NO: 112.
  • rapeseed 11S / 12S storage proteins contain at least one, preferably two, particularly preferably 3 of the sequence motifs selected from group IX or selected from group X in the nucleic acid sequences coding for them:
  • the functional equivalents of the rapeseed HS storage proteins particularly preferably have a sequence motif with the SEQ ID NO: 93 in their nucleic acid sequences:
  • soybean 11S / 12S storage proteins contain at least one, preferably two, particularly preferably 3, most preferably 4, 5 or 6 of the sequence motifs selected from the group XI or selected from the group in the nucleic acid sequences coding for them XII:
  • Arabidopsis thaliana HS / 12S storage proteins contain in the nucleic acid sequences coding for them at least one, preferably two, particularly preferably 3, most preferably 4, 5 or 6 of the sequence motifs selected from group XIII:
  • the functional equivalents of the Arabidopsis HS / 12S storage proteins particularly preferably have a sequence motif with the SEQ ID NO: 129 in their nucleic acid sequences:
  • Particularly preferred zein prolamines preferably include those from monocotyledonous plants, in particular maize, raisins, oats, barley or wheat. Corn is particularly preferred Zein prolamines described by SEQ ID NO: 159, 161 ,.
  • functional equivalents of the zein prolamines have a homology of at least 60%, preferably at least 80%, very particularly preferably ' at least 90%, most preferably at least 95% to one of the protein sequences with SEQ ID NO: 159, 161, 163, 165, 167, 169, 171, 172 or 174 where the homology preferably extends over a length of at least 30 amino acids, preferably at least 50 amino acids, particularly preferably over 100 amino acids, most preferably over the entire length of the respective proteins, and have the same essential properties of a storage protein and - preferably - the characteristic properties of a zein prolamine.
  • Functional equivalents mean, in particular, natural or artificial mutations of the above-mentioned storage proteins as well as homologous polypeptides from other plants which have the same essential and — preferably — characteristic properties. Homologous polypeptides from preferred plants described above are preferred.
  • storage proteins disclosed homologous sequences from other plants - for example those whose genomic sequence is known in whole or in part, such as, for example, from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum or Solanum tuberosum - can be found from databases by homology comparisons, for example by searching the database or screening genes -Banks - can easily be found using the exemplary storage protein sequences as a search sequence or probe.
  • Mutations include substitutions, additions, deletions, inversions, or insertions of one or more amino acid residues.
  • Homology between two nucleic acid sequences is understood to mean the identity of the nucleic acid sequence over the respective entire sequence length, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25: 3389ff) using the following parameters:
  • Gap Weight 50 Length Weight: 3
  • a sequence which has a homology of at least 80% based on nucleic acid with the sequence SEQ ID NO: 1 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set, has a homology of has at least 80%.
  • GAP Garnier ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • Gap Weight 8 Length Weight: 2
  • a sequence which has a homology of at least 80% on a protein basis with the sequence SEQ ID NO: 2 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set 'has a homology of at least 80%.
  • Functional equivalents also include those proteins which are encoded by nucleic acid sequences which, under standard conditions, have one of the sequences represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 111, 113, 115, 117, 119, 121, 132, 154, 156, 158, 160, 162, 164, 166, 168, 170 or 173 described for a nucleic acid sequence coding for storage proteins, which hybridize to this complementary nucleic acid sequence or parts of the aforementioned and have the essential properties of a storage protein and - preferably - further characteristic properties.
  • Standard hybridization conditions is to be understood broadly and means stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al. , in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley S-Sons, N.Y. (1989), 6.3.1-6.3.6. described.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2X SSC at 50 ° C.) and those with high stringency (with approximately 0.2X SSC at 50 ° C., preferably at 65 ° C. C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0).
  • the temperature during the washing step can be raised from low stringent conditions at room temperature, about -22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can also be kept constant and only the other can be varied. Denaturing agents such as formamide or SDS can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C.
  • Hybridization conditions can be selected from the following conditions, for example:
  • Washing steps can be selected, for example, from the following conditions:
  • the reduction in the expression of a storage protein can be implemented in a variety of ways.
  • Amount of protein means the amount of a storage protein polypeptide in an organism, a tissue, a cell or a cell compartment.
  • the amount of protein preferably means the amount of a certain storage protein in the seed of a plant.
  • “Decreasing" the amount of protein means reducing the amount of a storage protein in an organism, a tissue, a cell or a cell compartment - for example by one of the methods described below - compared to the wild type of the same genus and species to which this method has not been applied , otherwise under the same general conditions (such as culture conditions, age of plants, etc.)
  • Reduction preferably means the reduction in the amount of protein in the seed of a plant.
  • the reduction is at least 10%, preferably at least 10% or at least 20%, particularly preferably by at least 40% or 60%, very particularly preferably by at least 70% or 80%, most preferably by at least 90% or 95%.
  • Methods for determining the amount of protein are known to the person skilled in the art.
  • the micro-biuret method (Goa. J (1953) Scand J Clin Lab Invest 5: 218-222), the Folin-Ciocalteu method (Lowry OH et al. (1951) J Biol Chem 193: 265 -275) or the measurement of the adsorption of CBB G-250 (Bradford MM (1976) Analyt Biochem 72: 248-254).
  • the amount of protein is reduced by more than one storage protein.
  • the reduced storage proteins can belong to the same or different classes, such as 2S albumins, 7S globulins, 11S / 12S globulins or zein prolamines. Storage proteins from more than one of these classes are preferably reduced in their protein quantity at the same time.
  • the storage proteins to be reduced can be highly homologous or less homologous to one another. At least two of the storage proteins reduced in their protein quantity preferably have a homology of less than 90%, preferably less than 70%, particularly preferably less than 60%, very particularly preferably less than 50%.
  • Reduction or “decrease” is to be interpreted broadly in connection with a storage protein (or the amount of a storage protein or the amount of RNA coding for it) and includes the partial or essentially complete prevention or blocking of the expression of a based on different semasiological mechanisms Storage protein in a plant or a part, tissue, organ, cells or seeds derived from it.
  • a reduction in the sense of the invention comprises the quantitative reduction of a storage protein up to an essentially complete absence of the storage protein (i.e. a lack of immunological detectability of the storage protein).
  • the expression of a specific storage protein in a cell or an organism is preferably reduced by more than 50%, particularly preferably by more than 80%, very particularly preferably by more than 90%.
  • RNA nucleic acid sequence as a result of "SP-dsRNA"
  • the double-stranded RNA sequence includes subsequent elements i) at least one “sense” ribonucleotide sequence which is essentially identical to at least part of the “sense” RNA transcript of a storage protein nucleic acid sequence and
  • RNA nucleic acid sequences introduction of a storage protein antisense RNA nucleic acid sequences or an expression cassette ensuring their expression, the storage protein antisense RNA nucleic acid sequence being essentially complementary to at least part of the “sense” RNA transcript of a storage protein nucleic acid sequence.
  • a storage protein gene that is to say genomic DNA sequences
  • a storage protein gene transcript that is to say RNA sequences.
  • ⁇ -Anomeric nucleic acid sequences are also included.
  • the storage protein sense RNA nucleic acid sequence being essentially identical to at least part of the "sense" RNA transcript a storage protein nucleic acid sequence
  • SP-dsRNA RNA nucleic acid sequence
  • double-stranded RNA interference double-stranded RNA interference
  • dsKNAi double-stranded RNA interference
  • dsRNAi methods are based on the phenomenon that the simultaneous introduction of complementary strand and counter strand of a gene transcript causes a highly efficient suppression of the expression of the corresponding gene. The phenotype caused is very similar to that of a corresponding knock-out mutant (Waterhouse PM et al. (1998) Proc Natl Acad Sei USA 95: 13959-64).
  • the d ⁇ RNAi method has proven to be particularly efficient and advantageous in reducing the storage protein expression. As, inter alia, in WO . 99/32619, dsRNAi approaches are clearly superior to classic antisense approaches.
  • Another object of the invention therefore relates to double-stranded RNA molecules (dsRNA molecules) which, when introduced into a plant (or a cell, tissue, organ or seed derived therefrom) bring about the reduction of a storage protein.
  • dsRNA molecules double-stranded RNA molecules
  • dsRNA sequence can also have insertions, deletions and individual point mutations compared to the target protein sequence and nevertheless bring about an efficient reduction in expression.
  • the homology according to the above definition is preferably at least 65%, preferably at least 75%, very particularly preferably at least 90%, most preferably 95% between the "sense" ribonucleotide sequence of a dsRNA and at least part of the "sense" RNA transcript Storage protein nucleic acid sequence.
  • RNA transcript of a storage protein nucleic acid sequence means fragments of an RNA or mRNA transcribed from a nucleic acid sequence coding for a storage protein, preferably from a storage protein gene.
  • the fragments preferably have a sequence length of at least 20 bases, preferably at least 50 bases, particularly preferably at least 100 bases, very particularly preferably at least 200 bases, most preferably at least 500 bases.
  • the complete transcribed RNA or mRNA is also included.
  • “Essentially complementary” means that the “antisense” ribonucleotide sequence can also have insertions, deletions and individual point mutations in comparison to the complement of the “sense” ribonucleotide sequence.
  • the homology is preferably at least 80%, preferably at least 90%, very particularly preferably at least 95%, most preferably 100% between the "antisense” ribonucleotide sequence and the complement of the
  • an "essentially identical" dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with part of a storage protein gene transcript (eg in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50 ° C or 70 ° C for 12 to 16 h).
  • a storage protein gene transcript eg in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50 ° C or 70 ° C for 12 to 16 h.
  • dsRNA preferably comprises sequence regions of storage protein gene transcripts, which correspond to conserved regions of the individual storage protein families. Said conserved areas can be derived from sequence comparisons (cf. FIGS. La-7d).
  • the “sense” ribonucleotide sequence of the dsRNA contains at least one of the sequence motifs selected from one of the groups of sequence motifs I, II, III, IV, V, VI, VII, VIII, IX, X, XI or XII defined above or the sequence motifs with SEQ ID NO: .93 or 129.
  • Double-stranded RNA molecules are most preferably described by the ribonucleic acid sequence according to SEQ ID NO: 106, 108 or 110.
  • the dsRNA contains a plurality of sequence sections which bring about simultaneous suppression of a plurality of storage proteins, preferably storage proteins from different classes, such as, for example, a 2S albumin, 7S globulin, HS / 12S globulin or the zein prolamine.
  • the dsRNA preferably comprises
  • RNA transcript of which the "sense" ribonucleotide sequences are essentially identical have a homology of less than 90%, preferably less than 80%, very particularly preferably less than 60%, most preferably less than 50% over the entire length of their coding nucleotide sequence, and
  • the "sense" ribonucleotide sequences and “antisense” ribonucleotide sequences can be present as separate molecules or - preferably - as a single, self-complementary RNA molecule, in the latter case the two strands preferably via a connecting sequence ("linker "), which very particularly preferably represents an intron, are connected to one another.
  • linker which very particularly preferably represents an intron
  • Double-stranded RNA molecules are most preferably described by the ribonucleic acid sequence according to SEQ ID NO: 145 or 147.
  • dsRNA molecules each comprising one of the ribonucleotide sequence sections defined above, can also be introduced into the cell or the organism.
  • the dsRNA can consist of one or more strands of polymerized ribonucleotides.
  • modifications of both the sugar-phosphate structure and the nucleosides For example, the phosphodiester bonds of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom.
  • Bases can be modified such that the activity is restricted by adenosine deaminase, for example. Such and other modifications are described below in the methods for stabilizing antisense RNA.
  • the dsRNA can be produced enzymatically or in whole or in part chemically and synthetically.
  • the double-stranded dsRNA structure can be formed from two complementary, separate RNA strands or - preferably - from a single, self-complementary RNA strand.
  • RNA strands of the dsRNA are to be brought together in a cell or plant, this can be done in different ways:
  • the dsRNA structure is formed by a single, self-complementary strand of RNA.
  • "Sense" and “antisense” ribonucleotide sequences can be linked here by a connecting sequence ("linker") and, for example, form a hairpin structure.
  • the connecting sequence is preferably an intron.
  • the nucleic acid sequence coding for a dsRNA can contain further elements, such as, for example, transcription termination signals or polyadenylation signals.
  • the formation of the RNA duplex can be initiated either outside the cell or inside the cell.
  • the dsRNA can also comprise a hairpin structure by connecting the “sense” and “antisense” strand by means of a “linker” (for example an intron).
  • linker for example an intron.
  • the self-complementary dsRNA structures are preferred because they only require the expression of a construct and always comprise the complementary strands in an equi-olar ratio.
  • the expression cassettes coding for the “antisense” or “sense” strand of a dsRNA or for the self-complementary strand of the dsRNA are preferably inserted into a vector and are stable using the methods described below (for example using selection markers) inserted into the genome of a plant to ensure permanent expression of the dsRNA.
  • the dsRNA can be introduced using an amount that allows at least one copy per cell. Higher quantities (e.g. at least 5, 10, 100, 500 or 1000 copies per cell) can possibly result in an efficient reduction.
  • the dsRNA can be synthesized either in vivo or in vitro.
  • a DNA sequence coding for a dsRNA can be placed in an expression cassette under the control of at least one genetic control element (such as promoter, enhancer, silencer, splice donor or acceptor, polyadenylation signal).
  • at least one genetic control element such as promoter, enhancer, silencer, splice donor or acceptor, polyadenylation signal.
  • a dsRNA can be synthesized chemically or enzymatically.
  • Cellular RNA polymerases or bacteriophage RNA polymerases (such as T3, T7 or SP6 RNA polymerase) can be used for this.
  • Corresponding methods for in vitro expression of RNA are described (WO 97/32016; US 5,593,874; US 5,698,425, US 5,712,135, US 5,789,214, US 5,804,693).
  • a dsRNA synthesized chemically or enzymatically in vitro can be extracted from the reaction mixture, for example by extraction, precipitation, electrophoresis, before being introduced into a cell, tissue or organism. Chromatography or combinations of these processes are wholly or partially purified.
  • the dsRNA can be introduced directly into the cell or can also be applied extracellularly (for example in the inter ⁇ titial space).
  • the plant is preferably transformed stably with an expression construct that realizes the expression of the d ⁇ RNA. Corresponding methods are described below.
  • Hybridization can occur in a conventional manner via the formation of a stable duplex or - in the case of genomic DNA - by binding of the antisense nucleic acid molecule with the duplex of the genomic DNA through specific interaction in the major groove of the DNA helix.
  • An antisense nucleic acid sequence suitable for reducing a storage protein contains an "antisense" RNA strand comprising at least one ribonucleotide sequence which is essentially complementary to at least part of the "sense" RNA transcript of a storage protein nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 3 ' 5, 37, 39, 41, 43, 45, 47, 49, 51, 111, 113, 115, 117, 119, 121, 132, 154, 156, 158, 160, 162, 164, 166, 168, 170 or 173.
  • Essentially complementary means that the antisense RNA sequence can also have insertions, deletions and individual point mutations in comparison to the complement of the storage protein target sequence.
  • the homology according to the above definition is preferably 75%, preferably at least 85%, very particularly preferably at least 95%, most preferably 98% between the "antisense" RNA molecule and the complement at least part of the "sense" RNA Transkripte ⁇ a storage protein nucleic acid sequence.
  • the complement can be derived in accordance with the base pair rules of Watson and Crick in the manner familiar to the person skilled in the art from the corresponding sequences.
  • RNA transcript of a storage protein nucleic acid sequence means fragments of an RNA or mRNA transcribed from a nucleic acid coding for a storage protein, preferably from a storage protein gene.
  • the fragments preferably have a sequence length of at least 20 bases, preferably at least 50 bases, particularly preferably at least 100 bases, very particularly preferably at least 200 bases, most preferably at least 500 bases.
  • the complete transcribed RNA or mRNA is also included.
  • the antisense nucleic acid sequence can be complementary to the entire transcribed mRNA of said protein, be limited to the coding region or consist only of an oligonucleotide which is complementary to a part of the coding or non-coding sequence of the mRNA.
  • the oligonucleotide can be complementary to the region that comprises the start of translation for said protein.
  • Antisense nucleic acid sequences can have a length of, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides, but can also be longer and at least 100, 200, 500, 1000, 2000 or 5000 nucleotides include.
  • Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically using methods known to the person skilled in the art. Natural or modified nucleotides can be used in chemical synthesis.
  • a storage protein in a further preferred embodiment, can be inhibited by nucleotide sequences which are complementary to the regulatory region of a storage protein gene . (eg a storage protein promoter and / or enhancer) and form triple-helical structures with the DNA double helix there, so that the transcription of the storage protein gene is reduced.
  • nucleotide sequences which are complementary to the regulatory region of a storage protein gene .
  • a storage protein promoter and / or enhancer eg. storage protein promoter and / or enhancer
  • the antisense nucleic acid molecule can be an ⁇ -anomeric nucleic acid.
  • Such ⁇ -anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA in which - in contrast to the conventional ⁇ -nucleic acids - the two strands run parallel to one another (Gautier C et al. (1987) Nucleic Acids Res 15: 6625-6641) ,
  • the antisense nucleic acid olecule may also include 2'-O-methylribonucleotides (Inoue et al. (1987) Nucleic Acids Re ⁇ 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBS Lett 215 : 327-330).
  • the antisense strategy described above can advantageously be coupled with a ribozyme method.
  • Catalytic RNA molecules or ribozymes can be adapted to any target RNA and cleave the phosphodiester framework at specific positions, whereby the target RNA is functionally deactivated (Tanner NK (1999) FEMS Microbiol Rev 23 (3): 257-275 ).
  • the ribozyme is not itself modified thereby, but is able to analogously cleave further target RNA molecules, as a result of which the properties of an enzyme are obtained.
  • ribozyme sequences into “antisense” RNAs gives these "antisense” RNAs this enzyme-like, RNA-cleaving property and thus increases their efficiency in inactivating the target RNA.
  • the production and use of corresponding ribozyme "antiseise” RNA molecules is described, for example, by Haseloff et al. (1988) Nature 334: 585-591.
  • ribozymes eg "Hammerhead”ribozymes; Ha ⁇ elhoff and Gerlach (1988) Nature 334: 585-591
  • Ribozyme technology can increase the efficiency of an antiseise strategy.
  • Methods for expressing ribozymes to reduce certain proteins are described in (EP 0 291 533, EP 0 321 201, EP 0 360 257). Ribozyme expression is also described in plant cells (Steinecke P et al. (1992) EMBO J 11 (4): 1525-1530; de Feyter R et al.
  • Suitable target sequences and ribozymes can, for example, as described in "Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. Eds, Acade ic Press, Inc. (1995), pp. 449-460", by secondary structure calculations of ribozyme and target RNA and their interaction (Bayley CC et al. (1992) Plant Mol Biol 18 (2): 353-361; Lloyd AM and Davis RW et al. (1994) Mol Gen Genet 242 (6): 653-657).
  • Tetrahymena L-19 IVS RNA can be constructed which have regions complementary to the mRNA of the storage protein to be suppressed (see also US Pat. No. 4,987,071 and US Pat. No. 5,116,742).
  • ribozymes can also be identified via a selection process from a library of diverse ribozymes (Bartel D and Szostak JW (1993) Science 261: 1411-1418).
  • ⁇ en ⁇ e RNA with homology to an endogenous gene can reduce or switch off the expression of the same, similar to what has been described for antiseenic approaches (Jorgen ⁇ en et al. (1996) Plant Mol Biol 31 (5): 957-973; Goring et al. (1991) Proc Natl Acad Sei USA 88: 1770-1774; Smith et al. (1990) Mol Gen Genet 224: 447-481; Napoli et al. (1990) Plant Cell 2: 279-289; Van der Krol et al.
  • a “sense" nucleic acid sequence suitable for reducing a storage protein contains a "sense" RNA strand comprising at least one ribonucleotide sequence which is essentially identical to at least a part of the "sen ⁇ e" RNA transcript of a storage protein nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 , 49, 51, 111, 113, 115, 117, 119, 121, 132, 154, 156, 158, 160, 162, 164, 166, 168, 170 or 173.
  • RNA sequence can also have insertions, deletions and individual point mutations in comparison to the complement of the storage protein target sequence.
  • the homology according to the above definition is preferably at least 65%, preferably at least 75%, very particularly preferably at least 90%, most preferably 95% between the “sense” RNA molecule and the “sense” RNA transcript of at least part of a storage protein nucleic acid sequence.
  • RNA transcript of a storage protein nucleic acid sequence means fragments of an RNA or mRNA transcribed from a nucleic acid sequence coding for a storage protein, preferably from a storage protein gene.
  • the fragments preferably have a sequence length of at least 20 bases, preferably at least 50 bases, particularly preferably at least 100 bases, very particularly preferably at least 200 bases, most preferably at least 500 bases.
  • the complete transcribed RNA or mRNA is also included.
  • a reduction of a storage protein gene expression is also possible with specific DNA-binding factors, for example, by factors of the zinc finger transcription factors. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory areas, and bring about repression of the endogenous gene.
  • the use of such a method enables the expression of an endogenous storage protein gene to be reduced without its sequence having to be genetically manipulated. Appropriate processes for the production of such factors are described (Dreier B et al. (2001) J Biol Chem 276 (31): 29466-78; Dreier B et al. (2000) J Mol Biol 303 (4): 489-502; Beerli RR et al.
  • This section is preferably located within the Storage proteins conserved sequence regions or in the region of the promoter region. For gene suppression, however, it can also be in the area of the coding exons or introns.
  • the DNA-binding factors can, for example, be directed against sequences which are conserved in various storage protein genes.
  • the DNA-binding factor is preferably contained against a sequence motif selected from one of the groups of sequence motifs I, II, III, IV, V, VI, VII, VIII, IX, X, XI or XII or the sequence motifs with the SEQ ID defined above NO: 93 or 129 directed.
  • sequences in the promoter area can also be used which occur with many storage proteins. To give examples of, but not by way of limitation, the following sequences:
  • the gene expression can also be suppressed by tailor-made, low molecular weight synthetic compounds, for example of the polyamide type (Dervan PB and Bürli RW (1999) Current Opinion in Chemical Biology 3: 688-693; Gottesfeld JM et al. (2000) Gene Expr 9 (1-2).-77-91).
  • These oligomers consist of the building blocks 3- (dimethylamino) propylamine, N-methyl-3-hydroxypyrrole, N-methylimidazole and N-methylpyrrole and can be adapted to any piece of double-stranded DNA in such a way that they bind sequence-specifically into the major groove and block the expression of the gene sequences there. Corresponding methods are described (see, inter alia, Bremer RE et al.
  • the storage protein expression can also be effectively achieved by induction of the specific storage protein RNA degradation by the plant with the aid of a viral expression system (amplicon) (Angell SM et al. (1999) Plant J 20 (3): 357-362).
  • amplicon Angell SM et al. (1999) Plant J 20 (3): 357-362
  • VIPGS viral induced gene silencing
  • nucleic acid construct which contains at least part of an endogenous storage protein gene, which is changed by deletion, addition or substitution of at least one nucleotide so that the functionality is reduced or completely eliminated becomes.
  • the change can also affect the regulatory elements (e.g. the promoter) of the gene, so that the coding sequence remains unchanged, but expression (transcription and / or translation) is omitted and reduced.
  • the changed region at its 5 'and 3' ends is flanked by further nucleic acid sequences, which must have a sufficient length to enable the recombination.
  • the length is usually in a range from several hundred bases to several kilobases (Thoma ⁇ KR and Capecchi MR (1987) Cell 51: 503; Strepp et al. (1998) Proc Natl Acad Sei USA 95 (8): 4368-4373) ,
  • the host organism - for example a plant - is transformed with the recombination construct using the methods described below, and successfully recombined clones are selected using, for example, an antibiotic or herbicide resistance.
  • Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate.
  • One possibility of removing the randomly integrated sequences and thus enriching cell clones with a correct homologous recombination is to use a sequence-specific recombination system as described in US Pat. No. 6,110,736, by means of which unspecific integrated sequences can be deleted again, which makes the selection successful via homologous recombination of integrated events facilitated.
  • a large number of sequence-specific recombination systems can be used, examples being the Cre / lox system of bacteriophage Pl, the FLP / FRT system of yeast, the gin recombinase of Mu phage, the pin recombinase from E. coli and the R / RS system of the pSRI plasmid called.
  • Preferred are bacteriophages Pl Cre / lox and the yeast FLP / FRT system.
  • the FLP / FRT and cre / lox recombinase system has already been used in plant systems (Odell et al. (1990) Mol Gen Genet 223: 369-378)
  • RNA / DNA oligonucleotides into the plant
  • knockout mutants with the aid of, for example, T-DNA mutagenesis (Koncz et al. (1992) Plant Mol Biol 20 (5): 963-976), ENU- (N-ethyl-N-nitrosourea) - Mutagenesis or homo-recombination (Hohn B and Puchta (1999) H Proc Natl Acad Sei USA 96: 8321-8323.).
  • Point mutations can also be generated using DNA-RNA hybrids, also known as "chimeraplasty” (Cole-Strauss et al. (1999) Nucl Acid ⁇ Re ⁇ 27 (5): 1323-1330; K iec (1999) Gene therapy American Science 87 (3): 240-247).
  • PTGS post-transcriptional gene silencing
  • La to 7d can be expected using a specific storage protein nucleic acid sequences, the expression of homologous storage proteins in 'the same or other species effectively suppress without das ⁇ the I ⁇ olitation, structure elucidation and Kon ⁇ trutechnisch Corresponding suppression constructs for storage protein homologs occurring there would be absolutely necessary. This greatly simplifies the workload.
  • anti-SP anti-SP
  • anti-SP compounds which directly or indirectly reduce the amount of protein, RNA or gene activity of at least one storage protein, and thus directly or indirectly reduce the amount of protein in at least one storage protein, are consequently combined under the name "anti-SP” compounds .
  • the term “anti-SP” compound explicitly includes the nucleic acid sequences, peptides, proteins or other factors used in the methods described above.
  • introduction includes all methods which are suitable for introducing or generating an "anti-SP” compound, directly or indirectly, into a plant or a cell, compartment, tissue, organ or seed thereof. Direct and indirect processes are included.
  • the introduction can lead to a temporary (transient) presence of an “anti-SP” connection (for example a dsRNA) or else to a permanent (stable) one.
  • the "anti-SP” compound can perform its function directly (for example by insertion into an endogenous storage protein gene).
  • the function can also be done indirectly after transcription into a RNA (for example in the case of antisense approaches) or after transcription and translation into a protein (for example in the case of binding factors).
  • Both direct and indirect acting "anti-SP" compounds are included according to the invention.
  • Introducing includes, for example, methods such as transfection, transduction or transformation.
  • Anti-SP compounds thus also include, for example, recombinant expression constructs that express (ie transcribe and possibly translate), for example, a storage protein dsRNA or a storage protein "antisense” RNA - preferably in a plant or part, tissue, organ or Seeds of the same - condition.
  • nucleic acid molecule whose expression (transcription and possibly translation) generates an "anti-SP" connection, preferably in a functional link with at least one genetic control element (for example a promoter) which expresses in an organism, preferably in Plants, guaranteed.
  • a genetic control element for example a promoter
  • plant-specific genetic control elements for example promoters
  • the "anti-SP” compound can also be generated in other organisms or in vitro and then introduced into the plant. In this, all prokaryotic or eukaryotic genetic control elements (for example promoters) are preferred which allow expression in the organism chosen for the production.
  • a functional link is understood to mean, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example an "anti-SP" compound) and possibly other regulatory elements such as a terminator such that each of the regulatory elements is its own Can perform function in the transgenic expression of the nucleic acid sequence, depending on the arrangement of the nucleic acid sequences, or anti-sense RNA. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed is positioned behind the sequence functioning as a promoter, so that both Sequences are covalently linked.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed is less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.
  • sequences for the expression of The expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed, can preferably be integrated in a vector and inserted into a plant genome by, for example, transformation.
  • An expression cassette is, however, also to be understood as such constructions in which a promoter is placed behind an endogenous storage protein gene, for example by homologous recombination, and the expression of an antisense storage protein RNA causes the reduction of a storage protein according to the invention.
  • an "anti-SP" compound for example a nucleic acid sequence coding for a storage protein d ⁇ RNA or a storage protein antisense RNA
  • an anti-SP for example a nucleic acid sequence coding for a storage protein d ⁇ RNA or a storage protein antisense RNA
  • Both approaches lead to expression cassettes in the sense of the invention.
  • Plant-specific promoters basically means any promoter which can control the expression of genes, in particular foreign genes, in plants or plant parts, cells, tissues or crops.
  • the expression can, for example, be constitutive, inducible or development-dependent.
  • “Constitutive” promoters mean those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development
  • a plant promoter or a promoter derived from a plant virus is preferably used in particular.
  • the promoter of the 35S transcript of the CaMV cauliflower mosaic virus is particularly preferred (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) or the 19S CaMV promoter (US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8: 2195- 2202).
  • SSU Rostorf S et al. (1995) Plant Mol Biol “ 29: 637-649), the Ubiquitin 1 promoter (Christtensen et al. (1992) Plant Mol Biol 18 : 675-689; Bruce et al.
  • promoters with specificities for seeds such as, for example, the promoter of phaseoline (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53), of 2S albumen gene (Jo ⁇ eff ⁇ on LG et al. ( 1987) J Biol Chem 262: 12196-12201), de ⁇ Legumin ⁇ (Shir ⁇ at A et al. (1989) Mol Gen Genet 215 (2): 326-331), de ⁇ USP (unknown seed protein; Bäumiein H et al. (1991 ) Mol Gen Genet 22 ' 5 (3): 459-67), the Napin gene (US 5,608,152; Stalberg K et al.
  • phaseoline US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53
  • 2S albumen gene Jo ⁇ eff ⁇ on LG et al. ( 1987) J Biol Chem 262: 12196-12201
  • seed-specific promoters are those of Genes coding for "high molecular weight glutenin” (HMWG), gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch synthase. Also preferred are promoters that allow seed-specific expression in monocots such as corn, barley, wheat, rye, rice, etc.
  • HMWG high molecular weight glutenin
  • AGPase ADP glucose pyrophosphatase
  • starch synthase starch synthase.
  • promoters that allow seed-specific expression in monocots such as corn, barley, wheat, rye, rice, etc.
  • the promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzine gene, etc.) can advantageously be used Prola in gene, the gliadin gene, the glutelin gene, the zein gene, the kasirin gene or the secalin gene).
  • the expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al.
  • promoters e.g. the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), promoter inducible by salicylic acid (WO 95/19443), a promoter inducible by benzenesulfonamide (EP 0 388 186), one by tetracycline - inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), a promoter inducible by abscisic acid
  • Constitutive and seed-specific promoters are particularly preferred.
  • promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable expression in other plant tissues or in other organisms, such as E. coli bacteria.
  • all promoters described above can be used as plant promoters.
  • the nucleic acid sequences contained in the expression cassettes or vectors according to the invention can be functionally linked to further genetic control sequences in addition to a promoter.
  • the term “genetic control sequences” is to be understood broadly and means all those sequences which have an influence on the occurrence or the function of the expression cassette according to the invention. Genetic control sequences modify, for example, the transcription and translation in prokaryotic or eukaryotic organisms.
  • the expression cassettes according to the invention preferably comprise a plant-specific promoter 5 'upstream of the respective nucleic acid sequence to be expressed transgenically and a terminator sequence 3' downstream as an additional genetic control sequence, and optionally further customary regulatory elements, each functionally linked to the transgene nucleic acid sequence to be expressed.
  • Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. Genetic control sequences, for example, allow tissue-specific expression to additionally depend on certain stress factors. Corresponding elements are, for example, for water stress, absinic acid (Lam. E and Chua NH, J Biol Chem 1991; 266 (26): 17131-17135) and heat stress (Schoffl F et al. (1989) Mol Gen Genetics 217 (2 -3): 246-53).
  • control sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
  • Genetic control sequences also include the 5'-untranslated regions, introns or non-coding 3 'regions of genes, such as the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been shown that they can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can enhance the transient expression of heterologous genes.
  • An example of translation enhancers is the 5 'leader sequence from the tobacco mosaic virus (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. They can also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).
  • the expression cassette can advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable an increased transgenic expression of the nucleic acid sequence.
  • additional adhesive sequences are inserted, such as further regulatory elements or terminators.
  • One or more copies of the nucleic acid sequences to be expressed can be contained in the gene construct.
  • Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular Gen 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J 3: 835 ff) or functional equivalents thereof.
  • Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.
  • Control sequences are further to be understood as those which enable homologous recombination or insertion into the genome of a host organism or which allow removal from the genome.
  • the coding sequence of a specific endogenous gene can be specifically exchanged for the coding sequence for a dsRNA.
  • Methods such as cre / lox technology allow tissue-specific, possibly inducible removal of the expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14 (4): 381-92).
  • certain flanking sequences are added to the target gene (lox sequences), which later enable removal by means of the cre recombinase.
  • an expression cassette and the vectors derived from it can contain further functional elements.
  • the term functional element is to be understood broadly and means all such elements which have an influence on the production, multiplication or function of the expression cassettes, vectors or transgenic organisms according to the invention. Examples include, but are not limited to:
  • Metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc.
  • herbicides such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc.
  • Particularly preferred selection markers are those which confer resistance to herbicides.
  • Examples include ⁇ eien: DNA sequences which encode phosphinothricin (PAT) and Glutaminsyntha ⁇ einhibitoren inactivate (bar and pat genes), 5-Enolpyruvyl ⁇ hikimat-3-pho ⁇ phat ⁇ yntha ⁇ egene (EPSP Syntha ⁇ egene), which confer resistance to glyphosate ® (N- (phosphonomethyl) glycine) confer that for da ⁇ Glyphosat ® degrading enzymes encoding the gox gene (glyphosate oxidoreductase), the deh gene (encoding a dehalogenase that inactivates dalapon), sulfonylurea and imidazolinone inactivating acetolactate synthases and bxn genes that code for bromoxynil-degrading nitrilase enzymes, as a gene against the antibiotic apectinomycin, the streptomycin phosphotransferase
  • reporter genes which code for easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an evaluation of the transformation efficiency or of the expression location or time.
  • Reporter proteins Schoenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (l): 29-44) such as "green fluorescence protein” (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784), the chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234: 856-859), the aequorin gene (Prasher et al.
  • GFP green fluorescence protein
  • origins of replication which ensure an increase in the expression cassettes or vectors according to the invention in, for example, E. coli.
  • examples include OR (origin of DNA replication), pBR322 ori or P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2 d ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 ).
  • a selectable marker which gives the successfully recombined cells resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglucose-6- gives phosphate (WO 98/45456) or an antibiotic.
  • the selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5: 81-84).
  • transgenic expression cassette or expression vectors can contain nucleic acid sequences which do not lead to a reduction in at least one storage protein and whose transgene expression leads to an additional increase in fatty acid biosynthesis (as a result of proOIL).
  • This additionally transgenically expressed proOIL nucleic acid sequence can be selected by way of example but not by way of limitation from nucleic acids coding for acetyl-CoA carboxylase (ACCase), glycerol-3-phosphate acyltransferase (GPAT), lyophosphatidate-acyltransferase (LPAT), diac ) and phospholipid: diacylglycerol acyltransferase (PDAT).
  • ACCase acetyl-CoA carboxylase
  • GPAT glycerol-3-phosphate acyltransferase
  • LPAT lyophosphatidate-acyltransferase
  • PDAT diacylglycerol acy
  • an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably into plants or plant cells, tissues, organs, parts or seeds) can advantageously be implemented using vectors in which the Expression cassettes are included.
  • Vectors can be, for example, plasmids, cosmids, phages, viruses or even agrobacteria.
  • the expression cassette can be introduced into the vector (preferably a plasmid vector) via a suitable restriction interface.
  • the resulting vector is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombining vector obtained using methods familiar to the person skilled in the art. Restriction analysis and sequencing can be used to check the cloning step.
  • Preferred vectors are those which enable stable integration of the expression cassette into the host genome.
  • RNA or protein be introduced into the corresponding host cell.
  • transformation or transduction or transfection
  • the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
  • the cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can pass through Diffusion can get into the cell.
  • the DNA can also be carried out by protoplast fusion with other DNA-containing units such as micelles, cells, lysoomes or liposomes.
  • Electroporation is another suitable method for introducing DNA, in which the cells are reversibly permeabilized by an electrical impulse.
  • Corresponding methods are described (for example in Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol Gen Genet 228: 104-112; Guerche et al. (1987) Plant Science 52: 111- 116; Neuhause et al. (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature 327: 70-73; Howell et al.
  • Suitable methods include protoplast transformation by polyethylene glycol-induced DNA uptake, the biological method with the gene gun, the so-called “particle bo bardment” method, electroporation, the incubation of dry embryos in DNA-containing solution and microinjection.
  • a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • the Agrobacteri ⁇ m-mediated transformation is best suited for dicotyledonous plant cells. The methods are described, for example, by Horsch RB et al. (1985) Science 225: 1229f).
  • the expression cassette has to be integrated into special plasmids, either into an intermediate vector (English: shuttle or intermediate vector) or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right boundary, but mostly the right and left boundary of the Ti or Ri plasmid T-DNA as a flanking region, is connected to the expression cassette to be introduced.
  • Binary vectors are preferably used.
  • Binary vectors can replicate in both E. coli and Agrobacterium. They usually contain a selection marker gene and one Left or polylinker flanked by the right and left T-DNA delimitation sequence. They can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163: 181-187).
  • the selection marker gene allows selection of transformed agrobacteria and is, for example, the nptll gene which confers resistance to kanamycin.
  • the Agrobacterium which acts as the host organism in this case should already contain a plasmid with the vir region. This is necessary for the transfer of T-DNA to the plant cell. An Agrobacterium transformed in this way. can be used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and described (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanter ⁇ BV, Alblasserdam, Chapter V; An et al. (1985) EMBO J 4: 277-287).
  • Various binary vectors are known and some are commercially available, for example pBHOl.2 or pBIN19 (Clontech Laboratorie ⁇ , Inc. USA).
  • Direct transformation techniques are suitable for every organism and cell type.
  • plasmid used in the case of injection or electroporation of DNA or RNA into plant cells.
  • Simple plasmids such as the pUC series can be used. If complete plants are to be regenerated from the transformed cells, then it is necessary that there is an additional selectable marker gene on the plasmid.
  • Stably transformed cells ie those which contain the inserted DNA integrated into the DNA of the host cell, can be selected from untransformed cells if a selectable marker is part of the inserted DNA.
  • a selectable marker is part of the inserted DNA.
  • any gene can act as a marker that can confer resistance to antibiotics or herbicides (such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc.) (see above).
  • Transformed cells that express such a marker gene are able to survive in the presence of concentrations of an appropriate antibiotic or herbicide that kill an untransformed wild type. Examples are mentioned above and preferably comprise the bar gene which confers resistance to the herbicide phosphinotricin (Rathore KS et al.
  • the selection marker allows the selection of transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Report 5: 81-84). The plants obtained can be grown and crossed in a conventional manner. Two or more generations should be cultivated to ensure that the genomic integration is stable and inheritable.
  • the above-mentioned methods are described, for example, in Jene ⁇ B et al. (1993) Technique ⁇ for Gene Transfer, in: T.ran ⁇ genic Plant ⁇ , Vol. 1, Engineering and Utilization, edited by SD Kung and R Wu, Academic Pre ⁇ s, p.128 -143 and in Potrykus (1991) Annu.Rev Plant Physiol Plant Molec Biol 42: 205-225).
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al. (1984) Nucl Acids Re ⁇ 12: 8711f).
  • a whole plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown.
  • transgene means all such constructions, either in which the genetic engineering methods are used, in which
  • Natural genetic environment means a natural chromosomal locus in the organism of origin or the presence in a genomic library.
  • the natural, genetic environment of the nucleic acid sequence is preferably at least partially preserved.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, very particularly preferably at least 5000 bp.
  • non-natural, synthetic (“artificial") methods such as, for example, mutagenization.
  • Corresponding methods are described (US 5,565,350; WO 00/15815; see also above).
  • Preferred host or starting organisms as transgenic organisms are, above all, plants as defined above. All genera and species are Einge ⁇ chlos ⁇ en in the context of the invention, higher and lower plants of the plant kingdom, in particular plants that are used for the extraction of oils such as rapeseed, sunflower, sesame, safflower, olive tree, 'soya, maize, wheat and nut species. Also included are the mature plants, seeds, sprouts and seedlings, as well as parts, propagation material and cultures derived from them, for example cell cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
  • the production of the transgenic organisms can be carried out using the processes described above for transforming or transfecting organisms.
  • Another object of the invention relates to the use of the transgenic organisms according to the invention and the cells, cell cultures, parts derived therefrom - such as roots, leaves etc. for transgenic plant organisms - and transgenic propagation material such as seeds or fruits for the production of Food or feed, pharmaceuticals or fine chemicals, in particular of oils, fats, fatty acids or derivatives of the aforementioned.
  • SEQ ID NO: 15 partial nucleic acid sequence coding for B.napu ⁇ cruciferin cru4 ububit (GenBank Acc.-No .: X57848)
  • SEQ ID NO: 16 partial protein sequence coding for B.napus cruciferin cru4 subunit
  • Protein sequence coding for Bras ⁇ ica oleracea 2S storage protein 37 Protein sequence coding for Bras ⁇ ica oleracea 2S storage protein 37. SEQ ID NO: 37
  • SEQ ID NO: 39 partial nucleic acid sequence coding for Sinapis alba sinl storage protein (GenBank Acc. -No.: X91799)
  • SEQ ID NO: 40 partial protein sequence coding for Sinapis alba sinl • storage protein
  • SEQ ID NO: 51 partial nucleic acid sequence coding for sunflower (Helianthus annuus) 2S albumin (GenBank Acc.-No.: X76101)
  • SEQ ID NO: 52 partial protein sequence coding for sunflower (Helianthus annuus) 2S albumin
  • Nucleic acid sequence coding for dsRNA for the suppression of Arabidopsis thaliana 12S storage protein AtCru3 (insert of vector pCR2.1-AtCRU3-RNAi)
  • Nucleic acid sequence coding for Arabidopsis thaliana 12S cruciferin storage protein (ATCRU3; GenBank Acc. -No.: U66916)
  • ATCRU3 Protein sequence coding for Arabidopsis thaliana 12S cruciferin storage protein
  • CRA1 A.thaliana 12S storage protein
  • Nucleic acid sequence coding for Arabidopsis 12S storage protein (CRB; GenBank Acc.-No .: X14313; M37248)
  • Nucleic acid sequence coding for Arabidopsis thaliana putative 12S storage protein (from GenBank. Acc. -No.: AC003027)
  • Nucleic acid sequence coding for Arabidopsi ⁇ thaliana cruciferin 12S Spwicherprotein (Atlg03890) (GenBank Acc.-No .: AY065432) 122.
  • SEQ ID NO: 122 Nucleic acid sequence coding for Arabidopsi ⁇ thaliana cruciferin 12S Spwicherprotein (Atlg03890) (GenBank Acc.-No .: AY065432) 122. SEQ ID NO: 122
  • SEQ ID NO: 134 oligonucleotide primer OPN1
  • SEQ ID NO: 135 oligonucleotide primer 0PN2
  • SEQ ID NO: 138 oligonucleotide primer OPN5
  • SEQ ID NO: 139 oligonucleotide primer OPN6
  • SEQ ID NO: 140 oligonucleotide primer OPN7
  • SEQ ID NO: 142 oligonucleotide primer OPN9
  • SEQ ID NO: 143 oligonucleotide primer OPN10
  • Ribonucleic acid sequence coding for sRNAi4-dsRNA for the suppression of several storage proteins 146 SEQ ID NO: 146
  • SEQ ID NO: 148 oligonucleotide primer OPN11
  • SEQ ID NO: 150 oligonucleotide primer OPN13
  • SEQ ID NO: 151 oligonucleotide primer OPN15
  • SEQ ID NO: 152 oligonucleotide primer OPN16
  • SEQ ID NO: 153 oligonucleotide primer OPN17
  • Nucleic acid sequence coding for Zea may 19kD Zein (GenBank Acc.-No .: E01144)
  • Protein sequence coding for zea may 19kD zein
  • Nucleic acid sequence coding for Zea mays 19kD alpha Zein Bl (GenBank Acc.-No .: AF371269) 161.
  • SEQ ID NO: 161 Nucleic acid sequence coding for Zea mays 19kD alpha Zein Bl (GenBank Acc.-No .: AF371269) 161.
  • SEQ ID NO: 164 SEQ ID NO: 164
  • Protein sequence part 1 coding for Hordeum vulgar C-hordein
  • Protein sequence part 2 coding for Hordeum vulgar C-hordein
  • Nucleic acid sequence coding for Triticum aetivum LMW Glutenin-IDL (GenBank Acc. -No.: X13306) 174. SEQ ID NO: 174
  • Protein sequence encoding a fusion protein from the Arabidosis thaliana ACCase (GenBank Acc.-No .: D34630) and the plastid signal sequence of the transketolase from tobacco
  • Nucleotide sequence coding for Arabido ⁇ i ⁇ thaliana ACCase (GenBank Acc.-No .: D34630) as a fusion protein with the platinum signal sequence of the tobacco transketolase under the control of the napin promoter
  • nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
  • nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
  • Fig. 3a-b Alignment of Brassica nigra, Sinapis alba and
  • nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
  • Fig. 4a-b Alignment of Helianthus annuus 2S albumins.
  • nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
  • Fig. 5 Alignment of Arabidopsis thaliana 2S albumins.
  • the nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. : gi 1 . 166609: 951-1445 (M22032) At2Sl (SEQ ID.NO: 1) gijl66611: 212-706 (M22035) At2S3 (SEQ ID NO: 3) gi
  • Fig. 6a-c DNA alignment of Bras ⁇ ica napus IIS memory
  • nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
  • GLC1 M36686; D00566 GMGLYBSU_2 (SEQ ID NO: 19)
  • GLC2 X15122 GMGY2_8 (SEQ ID NO: 21)
  • GLC3 X02626 GMGLYRl_3 (SEQ ID NO: 23)
  • GLC4 Ml0962 GMGLYAB_4 (SEQ ID NO: 25)
  • GLC5 X15123; S44896 GMGY3_7 (SEQ ID NO: 27)
  • oligonucleotides can be carried out, for example, in a known manner, using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the cloning steps carried out in the context of the present invention such as, for example, restriction cleavages, 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, multiplication of phages and sequence analysis of recombinant DNA are as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Pre ⁇ ; ISBN 0-87969-309-6 described performed.
  • the sequencing of recombinant DNA molecules is carried out using a laser fluorescence DNA sequencer from ABI using the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sei USA 74: 5463-5467).
  • the Arabidopsis thaliana plant represents a member of the higher plants (seed plants). This plant is closely related to other plant species from the cruciferous family such as Bras ⁇ ica napus, but also with other dicotyledonous plant families. Because of the high degree of homology of their DNA sequences or polypeptide sequences, Arabidopsis thaliana can be used as a model plant for other plant species.
  • the plants are either grown on Murashige-Skoog medium with 5% sucrose (Ogas et al. (1997) Science 277: 91-94) or on earth (Focks & Benning (1998) Plant Physiol 118: 91-101).
  • the seeds are stratified at 4 ° C for two days after plating or sprinkling on soil.
  • the pods are marked. According to the markings, pods are harvested 6 to 20 days after flowering.
  • RNA or polyA + RNA is isolated for the production of suppression constructs.
  • RNA was isolated from pods of Arabidopsis plants according to the following procedure: pod material from 6 to 20 days after flowering was harvested and snap-frozen in liquid nitrogen. The material was stored at -80 ° C before further use. 75 mg of the material was ground in a cooled mortar to a fine powder and mixed with 200 ⁇ L of the lysis buffer from the Ambion RNAqueo ⁇ kit. The isolation of the total RNA was then carried out according to the manufacturer's instructions. The RNA was eluted with 50 ⁇ L elution buffer (Ambion) and the concentration was determined by absorbing a solution diluted 1 to 100 on a photometer (Eppendorf) at 260 nm.
  • elution buffer Ambion
  • RNA 40 ⁇ g / ml RNA corresponds to an absorption of 1.
  • the RNA solutions were with RNA ⁇ e free water adjusted to a concentration of 1 ⁇ g / ⁇ L. The concentrations were checked by agarose gel electrophoresis.
  • oligo (dT) cellulose from Amer ⁇ ham Pharmacia according to the manufacturer's instructions was used. RNA or polyA + RNA was stored at -70 ° C.
  • the nucleotides were removed by phenol / chloroform extraction and Sephadex G50 centrifugation columns.
  • EcoRI / XhoI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends using T4-DNA-Liga ⁇ e (Röche, 12 ° C, overnight), cut with Xhol and by incubation with polynucleotide kinase (Röche, 37 ° C , 30 min) phosphorylated. This mixture was subjected to separation on a low-melting agarose gel.
  • DNA molecules over 300 base pairs were eluted from the gel, phenol-extracted, concentrated on Elutip-D columns (Schleicher and Schüll, Dassel, Germany) and ligated to vector arms and in la bda-ZAPII phage or lambda-ZAP -Expre ⁇ s phages packaged using the Gigapack Gold Kit (Stratagene, Amsterdam, the Netherlands), using the manufacturer's material and following its instructions.
  • the mixture is extracted with 1 ml of chlorofor / octanol (24: 1, shaken with IM Tris / HCl, pH 8.0) by slow inverting and centrifuged for 5 min at 8,500 rpm (7,500 xg) and room temperature , The aqueous phase is then extracted again with 1 ml of chloroform / octanol, centrifuged and carefully mixed by inverting with 1/10 volume of CTAB II buffer preheated to 65 ° C.
  • chlorofor / octanol 24: 1, shaken with IM Tris / HCl, pH 8.0
  • the mixture is mixed carefully with 1 ml of chloroform / octanol mixture (see above) and centrifuged for 5 min at 8,500 rpm (7,500 xg) and room temperature to separate the phases again.
  • the aqueous lower phase is transferred to a fresh Eppendorf tube and the upper organic phase is centrifuged again in a fresh Eppendorf tube for 15 min at 8,500 rpm (7,500 xg) and room temperature.
  • the resulting aqueous phase is combined with the aqueous phase of the previous centrifugation step and the entire batch is mixed with exactly the same volume of preheated CTAB III buffer. This is followed by incubation at 65 ° C until the DNA precipitates in flakes.
  • the sediment resulting from the subsequent centrifugation step (5 min, 2000 rpm (500 ⁇ g), 4 ° C.) is mixed with 250 ⁇ l of CTAB IV buffer preheated to 65 ° C. and added for at least 30 minutes or until the sediment is completely dissolved Incubated at 65 ° C.
  • the solution for precipitating the DNA is then mixed with 2.5 volumes of ice-cold ethanol and incubated for 1 h at -20 ° C.
  • the batch can be mixed with 0.6 volumes of isopropanol and centrifuged immediately for 15 min at 8,500 rpm (7,500 xg) and 4 ° C without further incubation.
  • the sedimented DNA is washed twice with 1 ml of 80% ice-cold ethanol by inverting the Eppendorf tube, centrifuged again after each washing step (5 min, 8,500 rpm (7,500 xg), 4 ° C) and then air-dried for approx. 15 min , Finally, the DNA is resuspended in 100 ⁇ l TE with 100 ⁇ g / ml RNase and incubated for 30 min at room temperature. After a further incubation phase at 4 ° C overnight, the DNA solution is homogeneous and can be used for further experiments.
  • Solution IV (high-salt TE) (for 200 ml): 10 mM Tris / HC1 pH 8.0 (0.242 g) 0.1 mM EDTA (40 ⁇ l 0.5 M stock solution) 1 M NaCl (11.69 g)
  • genomic Arabidopsis thaliana DNA with a subsequent pair of oligonucleotides was used to convert an exon region with the complete subsequent intron, including the splice acceptor sequence following the intron (base pair 1947 to 2603 of the sequence with the gene B Acc.-No: U66916) amplified:
  • the PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-1 vector and the sequence checked.
  • the PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-2 vector and the sequence checked.
  • vector pCR2.1-l 0.5 ⁇ g of vector pCR2.1-l were incubated with the restriction enzyme BamHI (New England Biolabs) for 2 hours according to the manufacturer's instructions and then dephosphorylated for 15 min with alkaline phosphatase (New England Biolabs). The vector prepared in this way (1 ⁇ L) was then ligated with the fragment obtained from vector pCR2.1-2.
  • BamHI New England Biolab ⁇
  • DNA fragments were separated by gel electrophoresis.
  • the 489 bp piece created next to the vector (3.9 kb) was cut out of the gel and cut with the "Gel purification" kit (Qiagen) purified according to the manufacturer's instructions and eluted with 50 ⁇ L elution buffer. 10 ⁇ L of the eluate were ligated with vector pCR2.1-l (see above) overnight at 16 ° C. (T4 ligase, New England Biolabs). The ligation products were then transformed into TOP10 cells (Stratagene) according to the manufacturer's instructions and selected accordingly. Positive clones were verified with the primer pair ONP1 and 0NP2 by PCR.
  • the vector pCR2.l-AtCRU3-RNAi • obtained was then incubated for 2 hours with Notl (New England Biolabs), "blunted” with Klenow fragment and the DNA fragments analyzed by gel-electrophoresis.
  • the 1155 bp fragment was then ligated into the StuI-cut, dephosphorylated binary vector pSUN2-USPl, 2, 3 (SEQ ID NO: 178; see Example 5).
  • the vector pSUN2-USPl, 2, 3 is a derivative of the vector pPZPlll ((Hajdukiewicz, P et al.
  • an exon region (base pair 601 to 1874 of the sequence with GenBank Acc.-No: M37248) from Arabidop ⁇ i ⁇ thaliana cDNA is amplified with the following oligonucleotide primer pair:
  • ONP5 (SEQ ID NO: 138):
  • ONP6 (SEQ ID NO: 139):
  • the PCR product is cloned into the pCR2.1-T0P0 vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-3 vector and the sequence checked.
  • Arabidopsis thaliana genomic DNA becomes an intron with the corresponding splice acceptor and donor sequences of the flanking exons (Base pair 1874 to 2117 of the sequence with GenBank Acc.-No: M37248) amplified with the following primer pair:
  • the PCR product is cloned into the pCR2.1-T0P0 vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-4 vector and the sequence checked.
  • AtCRB The construct for AtCRB is created in a strategy similar to that described for AtCRU3.
  • Vector pCR2.1-3 is incubated with Xhol (New England Biolabs) for 2 hours and dephosphorylated (alkaline phosphate, New England Biolabs).
  • Vector pCR2.1-4 is also incubated with Xhol in the same way and the gel fragments separated by gel electrophoresis. The corresponding fragments are purified and ligated in the manner described under AtCRU3, resulting after bacterial transformation in the vector pCR2.1-AtCRB exon / intron.
  • This vector is incubated with Xbal (NEB) for 2 hours, then with Klenow fragment (NEB) for 15 minutes, then with SalI for 2 hours and finally treated with alkaline phosphatase (NEB) for 15 minutes.
  • the vector pCR2.1-3 is incubated with BamHI (NEB), then for 15 min with Klenow fragment and then for 2 hours with Xhol (NEB).
  • BamHI NEB
  • Xhol N-Xhol
  • the exon fragment of AtCRB is isolated after gel electrophoresis, purified and used for ligation. Both fragments are then ligated and the vector pCR2.1-AtCRB-RNAi results.
  • the vector pCR2.1-AtCRB-RNAi obtained is then digested with HindiII and Pvul for 2 hours and incubated for 15 min with Klenow fragment (blunted).
  • the cut-out fragment is isolated by gel electrophoresis and then used for the ligation.
  • the corresponding fragment is then ligated into the dephosphorylated vector pSUN2-USP-RNAi-a cut with EcoRV (see above).
  • Vectors with the desired orientation of the insert are determined by means of restriction digestion and sequencing.
  • the resulting construct is called pSUN2-USP-RNAi-b.
  • the nucleic acid sequence coding for the dsRNA is described by SEQ ID NO: 107.
  • Suppress ⁇ ion ⁇ strukt for Arabidopsi ⁇ thaliana 2S storage protein At2S3 (SEQ ID NO: 3 or 4; GenBank Acc.-No: M22035):
  • an exon region (base pair 212 to 706 of the sequence with the GenBank Acc.-No: M22035) is amplified from Arabidop ⁇ i ⁇ thaliana cDNA with the subsequent pair of oligonucleotides and primers:
  • ONP10 SEQ ID NO: 1433:
  • the PCR product is cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-5 vector and the sequence checked.
  • At2S3 The construct for At2S3 is created in a strategy similar to that explained for AtCRU3.
  • Vector pCR2.1-5 is incubated with Xhol (New England Biolabs) for 2 hours and dephosphorylated (alkaline phosphate, New England Biolabs).
  • Vector pCR2.1-3 is also incubated with Xhol in the same way and the gel fragments separated by gel electrophoresis. The corresponding fragments are purified and ligated in the manner described under At-CRU3, resulting after bacterial transformation in the vector pCR2.1-At2S3 exon / intron.
  • This vector is incubated for 2 hours with SalI (NEB), then for 15 min with Klenow fragment (NEB) and lastly treated for 15 min with alkaline phosphate (NEB).
  • the vector pCR2.1-5 is incubated with BamHI (NEB) and then for 15 min with Klenow fragment.
  • BamHI NEB
  • the exon fragment of At2S3 is isolated after gel electrophoresis, purified and used for ligation. Both fragments are then ligated and the vector pCR2. l-At2S3-RNAi resulted.
  • the vector pCR2 obtained. I-At2S3-RNAi is then digested for 2 hours with HindIII and Xbal (New England Biolabs) and incubated for 15 min with Klenow fragment (blunted). The cut-out fragment is isolated by gel electrophoresis and then used for the ligation. The corresponding fragment is then ligated into the vector pSUN2-USP-RNAi-b digested and dephosphed with Smal (see above). vectorial Ren with the desired orientation of the insert are determined by means of restriction digestion and sequencing. The resulting construct is called pSUN2-USP-RNAil. The nucleic acid sequence coding for the dsRNA is described by SEQ ID NO: 109.
  • Binary vectors such as pBinAR can be used for plant transformation (Höfgen and Willmitzer (1990) Plant Science 66: 221-230).
  • the binary vectors can be constructed by ligating the cDNA in sense or anti-sense orientation in T-DNA. 5 'of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is located 3 'from the cDNA.
  • the tissue-specific expression can be achieved using a tissue-specific promoter.
  • seed-specific expression can be achieved by cloning in the napin or LeB4 or USP promoter 5 'of the cDNA. Any other seed-specific promoter element can also be used.
  • the CaMV-35S promoter can be used for constitutive expression in the whole plant.
  • Another example of a binary vector is the vector pSUN2-USPL, 2, 3, in which the fragments from Example 2 were cloned, and pSUN2-USP.
  • the vector pSUN2-USP contains the USP promoter and the OCS terminator.
  • pSUN2-USPl, 2, 3, contains three times the USP promoter.
  • the fragments from Example 2 were cloned into the multiple cloning site of the vector pSUN2-USPL, 2, 3 in order to enable the seed-specific expression of the suppression constructs.
  • the Agrobacterium -mediated plant transformation can be carried out, for example, using the Agrobacterium tumefacien ⁇ strains GV3101 (pMP90) (Koncz and Schell (1986) Mol Gen Genet 204: -383- 396) or LBA4404 (Clontech).
  • the transformation can be carried out by standard transformation techniques (Deblaere et al. (1984) Nucl Acids Res 13: 4777-4788).
  • Example 5 Plant transformation
  • Agrobacterium-mediated plant transformation can be carried out using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995 , in Sect., Ringbuc Central signature: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thomp ⁇ on, John E., Method ⁇ in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).
  • rapeseed can be transformed using cotyledon or hypocotyl transformation (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701).
  • the use of antibiotics for Agrobacterium and plant selection depends on the binary vector and Agrobacterium strain used for the transformation. Rapeseed selection is usually carried out using kanamycin as a selectable plant marker.
  • the Agrobacterium -mediated gene transfer in linseed can be carried out using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 perform the technique described.
  • soya can be carried out using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University Toledo).
  • Example 6 Examination of the expression of a recombinant gene product in a transformed organism
  • the activity of a recombinant gene product in the transformed host organism was measured at the transcription and / or translation level.
  • a suitable method for determining the amount of transcription of the gene is to carry out a Northern blot as explained below (for reference see Au ⁇ ubel et al.
  • RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, the binding and the extent of the binding of the probe, the presence and also the amount of mRNA for this Gene indicates.
  • This information indicates the degree of transcription of the transformed gene.
  • Total cellular RNA can be obtained from cells, tissues or organs using several methods, all of which are known in the art, such as, for example, by Bormann, ER, et al. (1992) Mol. Microbiol. 6: 317-326.
  • RNA hybridization 20 ⁇ g of total RNA or 1 ⁇ g of poly (A) + RNA were used by means of gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in A asino (1986, Anal. Biochem. 152, 304), transferred by capillary attraction using 10 x SSC to positively charged nylon membranes (Hybond N +, A ersham, Braunschweig), immobilized using UV light and 3 hours at 68 ° C using hybridization buffer (10% dextran sulfate wt . / Vol., 1 M NaCl, 1% SDS, 100 mg herring ⁇ perma DNA) prehybridized.
  • hybridization buffer 10% dextran sulfate wt . / Vol., 1 M NaCl, 1% SDS, 100 mg herring ⁇ perma DNA
  • the DNA probe was labeled with the Highprime DNA labeling kit (Röche, Mannheim, Germany) during the pre-hybridization using alpha- 32 P-dCTP (Amersham Pharmacia, Braunschweig, Germany).
  • the hybridization was carried out after adding the labeled DNA probe in the same buffer at 68 ° C. overnight.
  • the washing steps were carried out twice for 15 min using 2 ⁇ SSC and twice for 30 min using 1 ⁇ SSC, 1% SDS, at 68 ° C.
  • the closed filters were exposed at -70 ° C for a period of 1 to 14 days.
  • Standard techniques such as a Western blot can be used to examine the presence or the relative amount of protein translated from this mRNA (see, for example, Auubel et al. (1988) Current Protocol in Molecular Biology, Wiley: New York).
  • a probe such as an antibody
  • This probe is usually provided with a chemiluminescent or colorimetric label that is easy to detect. The presence and amount of the label observed indicates the presence and amount of the desired mutant protein present in the cell.
  • Example 7 Analysis of the effect of the recombinant proteins on the production of the desired product
  • the effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound can be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and that Medium and / or the cellular components for the increased production of the desired product (ie lipids or a fatty acid) is examined.
  • suitable conditions such as those described above
  • These analysis techniques are known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Eneyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and p.
  • plant lipids are made from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Be. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145, described extracted.
  • the qualitative and quantitative lipid or fatty acid analysis is described by Christie, William W., Advances in Lipid Methodology, Ayr / Scotland: Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 S. (Oily Pres ⁇ Lipid Library; 1), - "Progre ⁇ in Lipid Research, Oxford: Pergamon Pres ⁇ , 1 (1952) - 16 (1977) udT: Progress in the Chemistry of Fat ⁇ and Other Lipid ⁇ CODEN.
  • the analysis methods include measurements of the amounts of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways and measurements of gases generated during fermentation.
  • nutrients in the medium e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions
  • FAME fatty acid methyl ester
  • GC-MS gas liquid chromatography / mass spectrometry
  • TAG triacylglycerol
  • TLC thin layer chromatography
  • the unambiguous detection of the presence of fatty acid products can be obtained by analysis of recombinant organisms according to standard analysis methods: GC, GC-MS or TLC, as described variously by Christie and the literature therein (1997, in: Advances on Lipid Methodology, Fourth Edition. : Christie, Oily Pres ⁇ , Dundee, 119-169; 1998, gas chromatography-mass spectrometry method, lipids 33: 343-353).
  • the material to be analyzed can be broken up by ultrasound treatment, grinding in a glass mill, liquid nitrogen and grinding, or by other applicable methods.
  • the material must be centrifuged after breaking up.
  • the sediment is detached in aqua. resuspended, heated at 100 ° C. for 10 min, cooled on egg and centrifuged again, followed by extraction in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90 ° C., to give hydrolyzed oil and lipid - leads compounds that give trans-methylated lipids.
  • fatty acid methyl esters are extracted in petroleum ether and finally a GC analysis using a capillary column (chrome pack, WCOT fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ° C and 240 ° C for 20 min and subjected to 5 min at 240 ° C.
  • the identity of the fatty acid methyl esters obtained has to be defined using standards which are available from commercial sources (ie Sigma).
  • the extraction of lipids from seeds is carried out according to the method of Bligh & Dyer (1959) Can J Biochem Physiol 37: 911.
  • 5 mg Arabidopsi ⁇ seeds in 1.2 ml Qiagen microtubes (Qiagen, Hilden) are weighed out on a Sartorius (Göttingen) microbalance.
  • the seed material is mixed with 500 uL of chloroform / methanol (2: 1; includes mono-C17-glycerol from Sigma as internal standard) was homogenized in the Rusch ⁇ chmühle MM300 from Retsch ( ⁇ aan) and incubated for 20 min at RT "After addition of 500 uL.
  • the phase is separated by 50 mM potassium phosphate buffer pH 7.5, 50 ⁇ L are removed from the organic phase, diluted with 1500 ⁇ L chloroform and 5 ⁇ L applied to the capillaries Chromarod ⁇ SIII from Iatroscan (SKS, Bechenheim) this for 15 min in a thin layer chamber, which is saturated with 6: 2: 2 chloroform: methanol: toluene, separated in a first step, after which the capillaries were dried for 4 min at room temperature and then for 22 min in a thin film chamber saturated with 7: 3 n-hexane: ethyl ether After a further drying step for 4 min at room temperature, the samples are mixed in an Iatro ⁇ can MK-5 (SKS, Bechenheim) in accordance with F ra ⁇ er & Taggart, 1988 J.
  • Chromatogr. 439: 404 analyzed. The following parameters were set for the measurements: Slice width 50 msec, Tre ⁇ hold 20 mV, noisy ⁇ e 30, Skim ratio 0. The data were quantified using the internal standard Mono-C17-glycerin (Sigma) and a calibration curve with Tri-C17- glycerin (Sigma) using the ChromStar program (SKS, Beichenheim).
  • the vectors pCR2. l-AtCRU3-RNAi and pCR2.1-4 are incubated with the restriction enzymes Xhol and Sall for 2 hours at 37 ° C., the DNA fragments separated by agarose gel electrophoresis and both the vector and the PCR Inert cut out of pCR2.1-4 and purified with the "Gelpurification" kit from Qiagen according to the manufacturer's instructions and eluted with 50 ⁇ L elution buffer. 1 ⁇ L of the vector and 8 ⁇ L of the eluate from the PCR insert of pCR2.1-4 are used for the ligation, resulting in the construct pCR2.1-sRNAil.
  • This vector is incubated for 2 hours with the restriction enzyme Xhol and then for 15 min with the Klenow fragment.
  • the vector pCR2.1-AtCRB-RNAi (see example 2) is incubated with the enzyme EcoRI for 2 hours and also treated with Klenow fragment for 15 minutes. Both incubation approaches are separated by gel electrophoresis and the vector (pCR2.1- ⁇ RNAil) or the insert (from pCR2.1-AtCRB-RNAi) is cut out of the agarose gel and the DNA fragments are purified as described above.
  • 1 ⁇ L of the eluate from the vector and 8 ⁇ L of the eluate from the insert are used and incubated at 4 ° C. overnight.
  • the resultant construct is made with pCR2.
  • l-sRNAi2 The resulting vector is incubated with the enzyme Xbal and then with Klenow fragment.
  • the vector pCR2.1-4 is incubated with the enzymes EcoRV and Xbal and then with the Klenow fragment. After gel electrophoresis and purification, the fragment from pCR2.1-4 with the vector pCR2. l- ⁇ RNAi2 ligated, resulting in the construct pCR2. l-sRNAi3.
  • the resulting vector is then incubated with the Apal enzyme for 2 hours and then with the Klenow fragment for 15 minutes.
  • the vector pCR2 is used as an insert. Incubate l-At2S3-RNAi with the EcoRI enzyme for 2 hours and then with the Klenow fragment for 15 minutes.
  • l-sRNAi4 This vector then becomes the sRNAi4 fragment (SEQ ID NO: 144), coding for the super-suppressing dsRNA, by incubation with HindIII and Pvul cut out and ligated into the binary vector pSUN-USP (SEQ ID NO: 179).
  • the construct serves the simultaneous suppression of Arabidopsis thaliana storage proteins CRB (SEQ ID NO: 4), CRU3 (SEQ ID NO: 112) and At2S3 (SEQ ID NO: 118).
  • a fragment from the storage protein AtCRU3 (SEQ ID NO: 111, 112) is amplified with the following pair of oligonucleotides-primers under the PCR conditions given in Example 2:
  • OPN 11 5 -AAAAGGCCTGTGTTCCATTTGGCCGGAAACAAC-3 '(SEQ ID NO: 148)
  • OPN 12 5 '-AAAGATATCACCCTGGAGAACGCCACGAGTG-3' (SEQ ID NO: 149).
  • the fragment obtained is cloned into the vector pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-6 and the sequences checked.
  • OPN 13 5 '-AAAAGGCCTATGGCTAACAAGCTCTTCCTCGTC-3' (SEQ ID NO: 150)
  • OPN 14 5 -AAAGATATCCTAGTAGTAAGGAGGGAAGAAAG-3 '(SEQ ID NO: 151).
  • the fragment obtained is cloned into the vector pCR2.1-T0P0 vector (Invitrogen) according to the manufacturer's instructions, resulting in the pCR2.1-7 and the sequences checked.
  • the constructs from pCR2.1-3, pCR2.1-4 (see Example 2) and pCR2.1-6 and pCR2.1-7 are then ligated together as follows:
  • the vector pCR2.1-3 is used for 2 hours with EcoRV incubated and then depho ⁇ phorylated for 15 min with alkaline phosphate.
  • the vector pCR2.1-6 is incubated with the enzymes StuI and EcoRV for 2 hours and the PCR insert is isolated by gel electrophoresis and purification.
  • Vector pCR2.1-3 and insert from pCR2.1-6 are then ligated overnight at 4 ° C, resulting in the construct pCR 2.
  • This vector is then incubated with EcoRV and dephosphorylated and ligated with the StuI / EcoRV incubated and gel-purified fragment from pCR2.1-7, resulting in the construct pCR2. l- ⁇ RNAi6.
  • This vector is then incubated with Xhol and dephosphorylated.
  • the vector pCR2.1-4 is incubated with SalI and Xhol and the inert to pCR2.1-4 with the prepared vector pCR2. l- ⁇ RNAi6 ligated, resulting in the construct pCR2.l-sRNAi7.
  • a PCR is carried out with the following primer pair under the conditions given in Example 2:
  • OPN 15 5 CCGCTCGAGCTCAGGGTCTTTTCTTGCCCACT (SEQ ID NO: 152)
  • OPN 16 5 ⁇ -CCGGTCGACCTAGTAGTAAGGAGGGAAGAAAG (SEQ ID NO: 153).
  • the resulting PCR product is incubated with the enzymes Xhol and Sall.
  • the fragment is then ligated into the vector pCR2.l-sRNAi7 (incubated with Xhol), resulting in the construct pCR2.1-sRNAi ⁇ .
  • the sRNAi8 fragment (SEQ ID NO: 146), coding for the super-suppressing dsRNA, is then cut out of this vector by incubation with HindIII and Xbal and ligated into the binary vector pSUN-USP (SEQ ID NO: 179).
  • the construct serves for the simultaneous suppression of Arabidopsi thaliana storage proteins CRB (SEQ ID NO: 4), CRU3 (SEQ ID NO: 112) and At2S3 (SEQ ID NO: 118).

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Abstract

L'invention concerne des procédés permettant d'augmenter la teneur en huile de plantes, par diminution d'une ou de plusieurs protéines de stockage. L'invention concerne en outre l'utilisation de plantes à teneur en protéine de stockage réduite pour produire des produits alimentaires, des denrées fourragères, des semences, des produits pharmaceutiques ou des produits chimiques fins, notamment pour produire des huiles.
PCT/EP2003/002733 2002-03-20 2003-03-17 Procedes pour augmenter la teneur en huile de plantes WO2003077643A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1713908A2 (fr) * 2004-02-10 2006-10-25 Monsanto Technology, LLC Adn de recombinaison de suppression genique
EP1799833A1 (fr) * 2004-08-11 2007-06-27 Monsanto Technology, LLC Reduction accrue de zeine dans des graines de mais transgenique
WO2008027592A3 (fr) * 2006-08-31 2008-09-04 Monsanto Technology Llc Petits arn en phase
US8461418B2 (en) 2004-08-11 2013-06-11 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
JP2013226094A (ja) * 2012-04-26 2013-11-07 Toyota Motor Corp 植物の種子総量の増産方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0787801A2 (fr) * 1996-02-01 1997-08-06 Mitsubishi Corporation Procédé d'augmentation de la teneur en lipide de graines de plantes
WO1998026064A2 (fr) * 1996-12-09 1998-06-18 Dekalb Genetics Corporation Methodes d'accroissement du contenu nutritionnel de plantes
WO1999053050A1 (fr) * 1998-04-08 1999-10-21 Commonwealth Scientific And Industrial Research Organisation Procedes et moyens d'obtention de phenotypes modifies
WO2002000904A2 (fr) * 2000-06-23 2002-01-03 E. I. Du Pont De Nemours And Company Constructions recombinees et leur utilisation pour reduire l'expression de genes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620281A3 (fr) * 1993-03-31 1995-05-03 Mitsubishi Corp Plantes à graines oléagineuses produisant des sémences de valeur avec des compositions d'acides aminé et d'acides gras modifiées.
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0787801A2 (fr) * 1996-02-01 1997-08-06 Mitsubishi Corporation Procédé d'augmentation de la teneur en lipide de graines de plantes
WO1998026064A2 (fr) * 1996-12-09 1998-06-18 Dekalb Genetics Corporation Methodes d'accroissement du contenu nutritionnel de plantes
WO1999053050A1 (fr) * 1998-04-08 1999-10-21 Commonwealth Scientific And Industrial Research Organisation Procedes et moyens d'obtention de phenotypes modifies
WO2002000904A2 (fr) * 2000-06-23 2002-01-03 E. I. Du Pont De Nemours And Company Constructions recombinees et leur utilisation pour reduire l'expression de genes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAMMOND S M ET AL: "POST-TRANSCRIPTIONAL GENE SILENCING BY DOUBLE-STRANDED RNA" NATURE REVIEWS GENETICS, MACMILLAN MAGAZINES, GB, Bd. 2, Nr. 2, Februar 2001 (2001-02), Seiten 110-119, XP001146153 *
MATZKE M A ET AL: "Transgene silencing by the host genome defense: Implications for the evolution of epigenetic control mechanisms in plants and vertebrates" PLANT MOLECULAR BIOLOGY, NIJHOFF PUBLISHERS, DORDRECHT, NL, Bd. 43, Nr. 2-3, Juni 2000 (2000-06), Seiten 401-415, XP002189062 ISSN: 0167-4412 *
WESLEY S VARSHA ET AL: "Construct design for efficient, effective and high-throughput gene silencing in plants" PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, Bd. 27, Nr. 6, September 2001 (2001-09), Seiten 581-590, XP002187670 ISSN: 0960-7412 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9913487B2 (en) 2004-02-10 2018-03-13 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
EP1713908A4 (fr) * 2004-02-10 2008-08-20 Monsanto Technology Llc Adn de recombinaison de suppression genique
EP1713908A2 (fr) * 2004-02-10 2006-10-25 Monsanto Technology, LLC Adn de recombinaison de suppression genique
US7855323B2 (en) 2004-02-10 2010-12-21 Monsanto Technology Llc Recombinant DNA for gene suppression
EP2365072A1 (fr) * 2004-02-10 2011-09-14 Monsanto Technology LLC ADN recombinant pour la suppression de gènes
US10772276B2 (en) 2004-02-10 2020-09-15 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
US9006414B2 (en) 2004-02-10 2015-04-14 Monsanto Technology Llc Recombinant DNA for gene suppression
US9976139B2 (en) 2004-02-10 2018-05-22 Monsanto Technology Llc Recombinant DNA for gene suppression
EP3290516A1 (fr) * 2004-02-10 2018-03-07 Monsanto Technology LLC Adn recombinant pour la suppression de gènes
EP1799833A1 (fr) * 2004-08-11 2007-06-27 Monsanto Technology, LLC Reduction accrue de zeine dans des graines de mais transgenique
EP1799833A4 (fr) * 2004-08-11 2008-07-02 Monsanto Technology Llc Reduction accrue de zeine dans des graines de mais transgenique
US8461418B2 (en) 2004-08-11 2013-06-11 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
WO2008027592A3 (fr) * 2006-08-31 2008-09-04 Monsanto Technology Llc Petits arn en phase
US9309512B2 (en) 2006-08-31 2016-04-12 Monsanto Technology Llc Phased small RNAs
US10301623B2 (en) 2006-08-31 2019-05-28 Monsanto Technology Llc Phased small RNAs
JP2013226094A (ja) * 2012-04-26 2013-11-07 Toyota Motor Corp 植物の種子総量の増産方法

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AU2003209737A1 (en) 2003-09-29
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AU2003209737A8 (en) 2003-09-29
DE10212893A9 (de) 2004-09-09

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