WO2003077643A2 - Methods for increasing the oil content in plants by reducing the content of one or more storage proteins - Google Patents

Methods for increasing the oil content in plants by reducing the content of one or more storage proteins 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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French (fr)
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WO2003077643A3 (en
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Jörg BAUER
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Basf Plant Science Gmbh
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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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

The invention relates to methods for increasing the oil content in plants by reducing the content of storage proteins by one or more. The invention also relates to the use of plants having a reduced content of storage proteins for producing foodstuffs, feedstuffs, seeds, pharmaceutics or fine chemicals, particularly for producing oils.

Description

Verfahren zum Erhöhen des Ölgehaltes in PflanzenProcess for increasing the oil content in plants
Beschreibungdescription
Die Erfindung betrifft Verfahren zum Erhöhen des Ölgehaltes in Pflanzen durch Verminderung eines oder mehrerer Speicherproteine. Die Erfindung betrifft ferner die Verwendung von Pflanzen mit einer verminderten Speicherproteingehalt zur Herstellung von Nahrungs-, Futtermitteln, Saatgut, Pharmazeuti a oder Feinchemikalien, insbesondere zur Herstellung von Ölen.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.
Die Erhöhung des Ölgehalts in Pflanzen und insbesondere in Pflanzensamen ist für die klassische wie für die modernen Pflanzenzüchtung und insbesondere die pflanzliche Biotechnologie von großem Interessen. Bedingt durch den steigenden Verbrauch von Pflanzenölen für Ernährung bzw. industrielle Anwendungen sind Möglichkeiten zur Steigerung bzw. Modifikation von Pflanzenölen zunehmend Gegenstand aktueller Forschung (z.B. Töpfer et al . (1995) Science 268:681-686). Ziel ist dabei insbesondere die Erhöhung des Gehaltes an Fettsäuren in Samenölen.Increasing the oil content in plants and especially in plant seeds is of great interest for classic as well as modern plant breeding and especially plant biotechnology. Due to the increasing consumption of vegetable oils for food and industrial applications, options for increasing or modifying vegetable oils are increasingly the subject of current research (e.g. Töpfer et al. (1995) Science 268: 681-686). The main goal is to increase the fatty acid content in seed oils.
Auch die aus den pflanzlichen Ölen erhältlichen Fettsäuren sind von besonderem Interesse. Sie kommen beispielsweise als Grundstoffe für Weichmacher, Schmierstoffe, Tenside, Kosmetika usw. zum Einsatz oder werden in der Lebens- und Futtermittelindustrie als wertvoll Grundstoffe eingesetzt. So ist beispielsweise die Bereitstellung von Rapsölen mit Fettsäuren mittlerer Kettenlänge von besonderem Interesse, da diese besonderes in der Tensid- herstellung begehrt sind.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. For example, 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.
Durch die gezielte Modulation pflanzlicher Stoffwechselwege mittels gentechnische Verfahren kann der pflanzlichen Metabolismus in einer Weise vorteilhaft verändert werden, die durch klassische Züchtungsmethoden nur über langwierige Schritte bzw. überhaupt nicht zu erreichen wären. So werden ungewöhnliche Fettsäuren, beispielsweise bestimmte polyungesättigte Fettsäuren, nur in bestimmten Pflanzen bzw. ■ überhaupt nicht in Pflanzen synthetisiert und können deshalb nur nur durch Expression des entsprechenden Enzyms in transgenen Pflanzen hergestellt werden (z.B. Millar et al . (2000) Trends Plant Sei 5:95-101).Through the targeted modulation of plant metabolic pathways by means of genetic engineering processes, plant metabolism can be advantageously changed in a way that would be difficult or impossible to achieve with traditional breeding methods. For example, 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).
Lipide werden aus Fettsäuren synthetisiert und ihre Synthese kann in zwei Teilmechanismen unterteilt werden, einen quasi "prokaryotischen" und einen quasi "eukaryotisehen" ' (Browse et al . (1986) Biochemical J 235:25-31; Ohlrogge & Browse (1995) Plant Cell 7:957-970) . Der prokaryotische Mechanismus ist in den Piastiden lokalisiert und umfasst die Biosynthese der freien Fettsäuren, die in das Cytosol exportiert werden, wo sie als i'ettsäureacyl-CoA-Ester in den eukaryotischen Mechanismus eingehen und mit Glycerol-3-phosphat zu Phosphatidsäure (PA) ver- -stert werden. PA ist der Ausgangspunkt für die Synthese von leutralen und polaren Lipiden. Die neutralen Lipide werden labei über den Kennedy-Weg synthetisiert (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) .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 'ettsäureacyl CoA esters in the eukaryotic mechanism and -stert having glycerol-3-phosphate to phosphatidic acid (PA) comparable. 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 sind insbesondere darauf angewiesen Energie und Grund- oausteine zu speichern, um z.B. eine spätere Keimung zu gewährleisten. Die Speicherung erfolgt in Form von Speicherlipiden, Speicherproteinen oder Stärke (Speicherkohlenhydrat) . Je nach Pflanze variieren dabei die Verhältnisse der drei Speichermoleküle zueinander. So enthalten Rapssorten durchschnittlich etwa 48 % Speicherlipide, 19 % Stärke und 21 % Speicherproteine, während Sojabohne 22 % Lipide, 12 % Stärke und 37 % Proteine (jeweils bezogen auf die Trockenmasse) enthält (Biochemistry & Molecular Biology of the Plant ed. Buchanan, Gruissem, Jones 2000, American Society of Plant Physiologists). Die Speichermoleküle werden während der Embryoentwicklung des Samen akkumuliert.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). Depending on the plant, 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.
Speicherproteine (infolge auch SP) im Embryo dienen zur Speicherung von Kohlenstoff, Stickstoff und Schwefel, die für das schnelle heterotrophe Wachstum bei Keimung von Samen oder Pollen benötigt werden. Sie haben meist keine enzymatische Aktivität. Diese Proteine werden dabei nur im Embryo während der Samenentwicklung synthetisiert. SP akkumulieren dabei zum einen in Proteinspeichervakuolen (PSV) von unterschiedlich differenzierten Zellen im Embryo bzw. Endosperm. Als weitere Form können sie auch als Proteinkörper assoziiert mit den endo- plasmatischen Retikulum (ER) vorliegen (Her an & Larkins (1999) Plant Gell 11:601-613). Dabei werden alle Speicherproteine ursprünglich am rauen ER synthetisiert (Bollini & Chrispeels (1979) Planta 146:487-501). Sie können im ER verbleiben oder können über den Golgiapparat zu anderen Kompartimenten in der Zelle transportiert werden. Im ER erfolgt die Prozessierung und korrekte Faltung der Speicherproteine, sowie deren Oligo- erisierung (Vitale & Denecke (1999) Plant Gell 11:615-628). Speicherproteine in Samen wurden historisch gesehen auf Grund ihrer verschiedenen Löslichkeiten eingeordnet. Proteine wurden dabei sequentiell mit verschiedenen Lösungsmitteln aus den Samen extrahiert. Wasserlösliche Proteine wurden dabei als Albumine bezeichnet. Proteine, die in einer Salzlösung löslich sind, wurden als Globuline, und Proteine, die in einem Ethanol-Wasser- Gemisch löslich sind, wurden als Prolamine bezeichnet. Gluteline, die letzte Gruppe, können nur durch Behandlung mit verdünnten Säuren extrahiert werden (Biochemistry &. Molecular Biology of the Plant ed. Buchanan, Gruissem, Jones 2000, American Society of Plant Physiologists) . Prolamine kommen dabei spezifisch nur im Endosperm von Gräsern (Poaceae) vor, wo sie die Hauptspeicherproteine darstellen (Ausnahmen sind Reis und Hafer, wo Glutelin- ähnliche und Globuline überwiegen) . In Dikotyledonen dominieren dagegen Globuline.Storage proteins (as a result also 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). They can remain in the ER or can be transported to other compartments in the cell via the Golgi apparatus. In the ER, the storage proteins are processed and folded correctly, as well as their oligomerization (Vitale & Denecke (1999) Plant Gell 11: 615-628). Storage proteins in seeds have historically been classified based on their different solubilities. Proteins were extracted sequentially from the seeds using various solvents. Water-soluble proteins were referred to as albumins. Proteins that are soluble in a saline solution were called globulins, and proteins that were in an ethanol-water Mixtures that are soluble were called prolamines. Gluteline, the last group, can only be extracted by treatment with dilute acids (Biochemistry &. Molecular Biology of the Plant ed. Buchanan, Gruissem, Jones 2000, American Society of Plant Physiologists). 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.
Insgesamt können vier große Genfamilien für Speicherproteine aufgrund ihrer Sequenzen zugeordnet werden: 2S-Albumine (Napinähnlich) , 7S-Globuline (Phaseolin-ähnlich) , HS/12S-Globuline (Legumin-/Cruciferin-ähnlich) und die Zein-Prolamine .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 Albumine sind weit verbreitet in Samen von Dikotyledonen, einschließlich wichtiger kommerzieller Pflanzenfamilien wie Fabaceae (z.B. Sojabohne), Brassicaceae (z.B. Raps), Euphorbiaceae (z.B. Rizinus) oder Asteraceae (z.B. Sonnenblume). 2S Albumine sind kompakte globuläre Proteine mit konservierten Cysteinresten, die oft Heterodimere bilden.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-Globuline liegen in trimerer Form vor und enthalten keine Cysteinreste. Nach ihrer Synthese werden sie wie die 2S-Albumine in kleinere Fragmente gespalten und glykosyliert . Trotz Unterschiede in der Polypeptidgröße sind die verschiedenen 7S-Globu- line hoch konserviert und gehen vermutlich wie die 2S-Albumine auf einen gemeinsames Vorläuferprotein zurück. Die 7S-Globuline sind nur in geringen Mengen in Monokotyledonen vorhanden. In Dikotyledonen ist ihr Anteil immer kleiner verglichen mit den HS/12S-Globulinen.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-Globuline stellen neben den 2S-Albuminen die Hauptfraktion der Speicherproteine in Dikotyledonen. Die hohe Ähnlichkeit der verschiedenen HS-Globuline aus verschiedenen Pflanzengattungen lassen wiederum auf einen gemeinsames Vorläuferprotein in der Evolution schließen.In addition to the 2S albumins, 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.
Alle Speichermoleküle in Samen werden direkt oder indirekt aus Kohlenhydrat-Vorstufen gebildet. Dabei ist Saccharose die Primärquelle von Kohlenstoff und Energie welche von den Blättern in die sich entwickelnden Samen transportiert wird. So wird z.B. Saccharose zu Glucose-6-phosphat und Pyruvat umgesetzt, die in die Plastiden transportiert und dort zur Synthese von Acetyl-CoA dienen, welches das Ausgangsprodukt für die Synthese der Fettsäuren ist. Wie in den einzelnen Pflanzen das Gleichgewicht zwischen den einzelnen Speichermolekülen reguliert wird, ist nicht im einzelnen bekannt. So führt die Reduktion der Lipidsynthese zur Erhöhung der Speicherproteine (O'Hara et al . (2000) Trans Biochem Soc 613- 615) .All storage molecules in seeds are formed directly or indirectly from carbohydrate precursors. Sucrose is the primary source of carbon and energy which is transported from the leaves to the developing seeds. For example, 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. It is not known in detail how the balance between the individual storage molecules is regulated in the individual plants. The reduction of lipid synthesis leads to an increase in the storage proteins (O'Hara et al. (2000) Trans Biochem Soc 613-615).
EP-A 0 591 530 beschreibt die Verminderung der Expression eines Speicherproteins in Samen, insbesondere des Speicherproteins Glutelin in Reis, mit dem Ziel die Verwertbarkeit von Reis in Fermentationsprozessen zur Herstellung alkoholischer Getränke zu optimieren. In diesen Prozessen sind Proteine als solche hinderlich. Eine Auswirkung der Verminderung auf den Gehalt anderen pflanzlicher Inhaltstoffe ist nicht beschrieben.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 beschreibt die Verminderung von einem oder mehreren Speicherproteinen im Samen von Sojabohnen durch Antisense- Technologie. Weiterhin beschrieben ist ein Verfahren, wobei neben dem Speicherprotein noch die Expression eines Gen der FettsäurebioSynthese (mikrosomale Δ-12 Desaturase [Fad 2-1] Gene) vermindert wird. In Beispiel 2 (S.25/ Z.4-9) geschieht dies durch Cosuppression. Es resultieren transgene So abohnenpflanzen mit verminderten Gehalt eines Speicherproteins und einem Gehalt an Ölsäure an der Gesamtmenge von Fettsäuren, das im Verhältnis zu anderen Fettsäuren relativ höher ist als in nicht transgenen Sojabohnenpflanzen. Die Veränderung im Fettsäureprofil ist auf die Suppression des Fad2-1 Gens und nicht des Speicherproteins zurückzuführen. Auch wird keine Veränderung im Gesamtgehalt der Fettsäuren beschrieben.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 Δ-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 beschreibt Speicherproteine und Verfahren zur Erhöhung des Gehaltes bestimmter Aminosäuren in Pflanzen durch Expression besagter Speicherproteine. Eine Beschreibung der Auswirkung auf andere pflanzliche Metaboliten ist nicht offenbart. WO 97/41239 beschreibt ähnliche Verfahren bezogen auf schwefelreiche Speicherproteine.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 beschreibt Verfahren zur Verminderung eines oder mehrerer Speicherproteine, bevorzugt in Mais. Beschrieben sind ferner Pflanzen mit einem erhöhten oder veränderten Gehalt an Aminosäuren oder Stärke. Eine Auswirkung auf den Ölgehalt ist nicht beschrieben.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 beschreibt die Modifikation des Gehaltes von Metaboliten in den SpeieherOrganen von Pflanzen durch Expression von schwefelreichen Proteinen mit mehr als 10 % schwefelhaltigen Aminosäuren, insbesondere dem Samenalbumin der Sonnenblume (SSA; sunflower seed albumin) . Dabei bewirkt die Expression in Lupine eine Erhöhung des Ölgehaltes (Beispiel 1; S.28/Z.4-5, 17). Umgekehrt bewirkt die Expression des gleichen Gen in Erbse (Pisu sativum) eine Erniedrigung (Beispiels 2; S.32/Z.21).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). The expression in lupine an increase in the oil content (Example 1; p.28 / Z.4-5, 17). Conversely, the expression of the same gene in pea (Pisu sativum) causes a decrease (Example 2; p.32 / Z.21).
EP-A 0 620 281 beschreibt eine Veränderung in der Lipidzusammen- setzung (Fettsäuremuster) in Raps durch Verminderung der Expression eines Speicherproteins (Napin) mittels Antisense- Technologie. Beispiel 6 (S.ll/Z.12-15) beschreibt, dass sich lediglich das Verhältnis der einzelnen Fettsäuren dahingehend veränderte, dass der Gehalt an Ölsäure sank und der Gehalt an Linol- und Linolensäure stieg. Es wird explizit daraufhin gewiesen, dass der Gesamtgehalt an Fettsäure unverändert blieb. Entsprechende Daten sind auch in der korrespondierenden Veröffentlichung der Erfinder offenbart (Kohno-Murase J et al . (1994) Plant Mol Biol 26(4): 1115-1124) .WO 01/81604 beschreibt transgene Pflanzen die eine zytosolische Acetyl-CoA-carboxylase (ACCase) exprimieren.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).
Es stellte sich daher die Aufgabe alternative Verfahren zur Erhöhung des Ölgehaltes in Pflanzen bereitzustellen.It was therefore the task of providing alternative methods for increasing the oil content in plants.
Diese Aufgabe wird durch die vorliegende Erfindung gelöst. Ein erster Gegenstand der Erfindung umfasst ein Verfahren zum Erhöhen des Gesamtölgehalt in pflanzlichen Organismen, dadurch gekenn- zeichnet, dass nachfolgende Arbeitsschritte umfasst sindThis object is achieved by the present invention. 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
a) Verminderung der Proteinmenge eines oder mehrerer Speicherproteine in einem pflanzlichen Organismus oder einem Gewebe, Organ, Teil oder Zelle des besagten pflanzlichen Organismus unda) reducing the amount of protein of one or more storage proteins in a plant organism or a tissue, organ, part or cell of said plant organism and
b) Auswahl von pflanzlichen Organismen, bei denen - im Unterschied oder Vergleich zur AusgangsOrganismus - der Gesamtölgehalt in dem besagten pflanzlichen Organismus oder einem Gewebe, Organ, Teil oder Zelle des besagten pflanzlichen Organismus erhöht ist.b) Selection of plant organisms in which - in contrast to or compared to the parent organism - the total oil content in said plant organism or in a tissue, organ, part or cell of said plant organism is increased.
Das erfindungsgemäße Verfahren kann im Prinzip auf alle Pflanzenarten angewendet werden. Bevorzugt auf solche in denen natür- licherweise ein Speicherprotein exprimiert wird. Die zu den im Rahmen dieser Erfindung offenbarten Speicherproteinsequenzen homologen Sequenzen aus anderen Pflanzen können z.B. durch Datenbanksuche oder Durchmustern von Gen-Banken leicht aufgefunden werden. "Pflanzlicher Organismus oder von diesem abgeleitete Zellen" meint allgemein jede Zelle, Gewebe, Teile oder Vermehrungsgut (wie Samen oder Früchte) eines Organismus, der zur Photosynthese befähigt ist. Eingeschlossen sind im Rahmen der Erfindung alle Gattungen und Arten höherer und niederer Pflanzen des Pflanzenreiches. Einjährige, mehrjährige, monocotyledone und dicotyledone Pflanzen sind bevorzugt. Eingeschlossen sind reife Pflanze, Saatgut, Sprosse und Keimlinge, sowie davon abgeleitete Teile, Vermehrungsgut (zum Beispiel Knollen, Samen oder Früchte) und Kulturen, zum Beispiel Zeil- oder Kalluskulturen. Reife Pflanzen meint Pflanzen zu jedem beliebigen EntwicklungsStadium jenseits des Keimlings. Keimling meint eine junge, unreife Pflanze in einem frühen EntwicklungsStadium.The method according to the invention can in principle be applied to all types of plants. Preferred for those in which a storage protein is naturally expressed. The sequences from other plants which are homologous to the storage protein sequences disclosed in the context of this invention can easily be found, for example, by searching the database or by screening gene banks. "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. Included are mature plants, seeds, shoots and seedlings, as well as parts derived from them, propagation material (for example tubers, seeds or fruits) and cultures, for example row or callus 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.
"Pflanze" im Rahmen der Erfindung meint alle Gattungen und Arten höherer und niederer Pflanzen des Pflanzenreiches. Eingeschlossen unter dem Begriff sind die reifen Pflanzen, Saatgut, Sprossen und Keimlinge, sowie davon abgeleitete Teile, Vermehrungsgut, Pflanzenorgane, Gewebe, Protoplasten, Kallus und andere Kulturen, zum Beispiel Zellkulturen, sowie alle anderen Arten von Gruppierungen von Pflanzenzellen zu funktionellen oder strukturellen Einheiten. Reife Pflanzen meint Pflanzen zu jedem beliebigen Entwicklungsstadium jenseits des Keimlings. Keimling meint eine junge, unreife Pflanze in einem frühen Entwicklungsstadium. "Pflanze" umfasst alle einjährigen und mehrjährige, monokotyledonen und dikotyledonen Pflanzen und schließt beispielhaft jedoch nicht einschränkend solche der Gattungen 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, Heterocallis, Nemesis, Pelar- gonium, Panieum, Pennisetum, Ranunculus , Senecio, Salpiglossis, Cucu is, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Seeale, Triticum, Sorghum, Picea und Populus ein."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, phaseolus, lolium, oryza, zea, avena, hordeum, seeale, triticum, sorghum, picea and populus.
Bevorzugt sind Pflanzen nachfolgender Pflanzenfamilien: Amarant- haceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubia- ceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae. Bevorzugte monokotyle Pflanzen sind insbesondere ausgewählt aus den monokotylen Kulturpflanzen, wie zum Beispiel der Familie der Gramineae wie Reis, Mais, Weizen oder andere Getreidearten wie Gerste, Hirse, Roggen, Triticale oder Hafer sowie dem Zuckerrohr sowie alle Arten von Gräsern.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, 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.
Die Erfindung wird ganz besonders bevorzugt aus dikotyledone pflanzliche Organismen angewendet. Bevorzugte dikotyle Pflanzen sind insbesondere ausgewählt aus den dikotylen Kulturpflanzen, wie zum BeispielThe 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 wie Sonnenblume, Tagetes oder Calendula und andere mehr,- Asteraceae such as sunflower, tagetes or calendula and others,
- Compositae, besonders die Gattung Lactuca, ganz besonders die Art sativa (Salat) und andere mehr,- Compositae, especially the genus Lactuca, especially the species sativa (salad) and others,
- Cruciferae, besonders die Gattung Brassica, ganz besonders die Arten napus (Raps) , campestris (Rübe) , oleracea cv Tastie (Kohl) , oleracea cv Snowball Y (Blumenkohl) und oleracea cv- Cruciferae, especially the genus Brassica, especially the species napus (rape), campestris (turnip), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv
Emperor (Broccoli) und weitere Kohlarten; und der Gattung Arabidopsis, ganz besonders die Art thaliana sowie Kresse oder Canola und andere mehr,Emperor (broccoli) and other types of cabbage; and the genus Arabidopsis, especially the species thaliana as well as cress or canola and others,
- Cucurbitaceae wie Melone, Kürbis oder Zucchini und andere mehr,- Cucurbitaceae such as melon, pumpkin or zucchini and others,
- Leguminosae besonders die Gattung Glycine, ganz besonders die Art ax (Sojabohne) Soja sowie Alfalfa, Erbse, Bohnengewächsen oder Erdnuss und andere mehr- Leguminosae, especially the genus Glycine, especially the type ax (soybean), soybean as well as alfalfa, peas, beans or peanuts and others
- Rubiaceae, bevorzugt der Unterklasse Lamiidae wie beispielsweise Coffea arabica oder Coffea liberica (Kaffeestrauch) und andere mehr,Rubiaceae, preferably of the subclass Lamiidae such as Coffea arabica or Coffea liberica (coffee bush) and others,
- Solanaceae besonders die Gattung Lycopersicon, ganz besonders die Art esculentum (Tomate) und die Gattung Solanum, ganz besonders die Art tüberosum (Kartoffel) und melongena (Aubergine) sowie Tabak oder Paprika und andere mehr,- 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, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Theobroma cacao (Kakaostrauch) und andere mehr,Sterculiaceae, preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) and others,
- Theaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Camellia sinensis oder Thea sinensis (Teestrauch) und andere mehr, - Umbelliferae, besonders die Gattung Daucus (ganz besonders die Art carota (Karrotte) ) und Apium (ganz besonders die Art graveolens dulce (Seiarie) ) und andere mehr; und die Gattung Capsicum, ganz besonders die Art annu (Pfeffer) und andere mehr,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,
sowie Lein, Soja, Baumwolle, Hanf, Flachs, Gurke, Spinat, Möhre, Zuckerrübe und den verschiedenen Baum-, Nuss- und Weinarten, insbesondere Banane und Kiwi .as well as flax, soybeans, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various types of trees, nuts and wines, in particular banana and kiwi.
Umfasst sind ferner Schmuckpflanzen, Nutz- oder Zierbäume, Blumen, Schnittblumen, Sträucher oder Rasen. Beispielhaft aber nicht einschränkend seien zu nennen Angiospermen, Bryophyten wie zum Beispiel Hepaticae (Leberblümchen) und Musci (Moose) ,- Pteridophyten wie Farne, Schachtelhalm und Lycopoden; Gymnospermen wie Koniferen, Cycaden, Ginkgo und Gnetalen, die Familien der Rosaceae wie Rose, Ericaceae wie Rhododendrons und Azaleen, Euphorbiaceae wie Weihnachtssterne und Kroton, Caryophyllaceae wie Nelken, Solanaceae wie Petunien, Gesneriaceae wie das Usambaraveilchen, Balsaminaceae wie das Springkraut, Orchidaceae wie Orchideen, Iridaceae wie Gladiolen, Iris, Freesie und Krokus, Compositae wie Ringelblume, Geraniaceae wie Geranien, Liliaceae wie der Drachenbaum, Moraceae wie Ficus, Araceae wie Philodendron und andere mehr.Also included are 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 marigold, Geraniaceae like geranium, Liliaceae like the dragon tree, Moraceae like Ficus, Araceae like Philodendron and others.
Pflanzliche Organismen im Sinne der Erfindung sind weiterhin weitere photosynthetisch aktive befähigte Organismen, wie zum Beispiel Algen, Cyanobakterien sowie Moose. Bevorzugte Algen sind Grünalgen, wie beispielsweise Algen der Gattung Hae atococcus , Phaedactylum tricornatum, Volvox oder Dunaliella. Insbesondere bevorzugt ist Synechocyεtis.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. Synechocyεtis is particularly preferred.
Am meisten bevorzugt sind Pflanzen, die zur Olproduktion geeignet sind, wie beispielsweise Arabidopsis, Raps, Sonnenblume, Sesam, Färberdistel, Ölbaum, Soja, Mais, Weizen oder verschiedene Nuss- arten wie beispielsweise Walnuss Mandel. Unter diesen wieder besonders bevorzugt sind die dikotyledonen Pflanzen, insbesondere Raps, Soja und Sonnenblume.Most preferred are 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. Among these, the dicot plants, in particular rapeseed, soybeans and sunflower, are particularly preferred.
"Öle" umfasst neutrale und/oder polare Lipiden und Mischungen derselben..Beispielhaft jedoch nicht einschränkend seien die in Tabelle 1 aufgeführten zu nennen. Tab. Pflanzliche Lipidklassen"Oils" includes neutral and / or polar lipids and mixtures thereof. Examples listed but not restrictive are those listed in Table 1. Tab. Plant lipid classes
Figure imgf000010_0001
Figure imgf000010_0001
Neutrale Lipide meint bevorzugt Triacylglyceride . Sowohl dre neutralen als auch die polaren Lipide können ein breites Spektrum an verschiedenen Fettsäuren enthalten. Beispielhaft jedoch nicht einschränkend seien die in Tabelle 2 aufgeführten Fettsäuren zu nennen.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.
Tab. 2: Übersicht über verschiedene Fettsäuren (Auswahl) 1 Kettenlänge:Anzahl der Doppelbindungen * nicht natürlicherweise in Pflanzen vorkommendTab. 2: Overview of different fatty acids (selection) 1 chain length: number of double bonds * not naturally occurring in plants
Figure imgf000010_0002
Figure imgf000010_0002
Öle meint bevorzugt Samenöle.Oils preferably means seed oils.
"Ölgehalt" meint die Summe aller Öle nach obiger Definition, bevorzugt die Summe die Triacylglyceride. "Erhöhung" des Ölgehaltes meint die Steigerung des Gehaltes an Ölen in einer Pflanze oder einem Teil, Gewebe oder Organ derselben, bevorzugt in den Samenorganen der Pflanze. Dabei ist der Ölgehalt im Vergleich zu einer nicht dem erfindungsgemäßen Verfahren unterworfenen, aber ansonsten unveränderten Ausgangspflanze unter ansonsten gleichen Rahmenbedingungen um mindestens 5 %, bevorzugt mindestens 10 %, besonders bevorzugt mindestens 15 %, ganz besonders bevorzugt mindestens 20 %, am meisten bevorzugt mindestens 25 % erhöht. Rahmenbedingungen meint dabei alle für die Keimung, Kultivierung oder Wachstum der Pflanze relevanten Bedingungen wir Boden-, Klima- oder Lichtverhältnisse, Düngung, Bewässerung, Pflanzenschutzmaßnahmen usw."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.
"Speicherprotein" meint allgemein ein Protein, das mindestens eine der nachfolgenden wesentlichen Eigenschaften aufweist:"Storage protein" generally means a protein which has at least one of the following essential properties:
a) Speicherproteine werden im wesentlichen nur im Embryo während der Samenentwicklung exprimiert. "Im wesentlichen" bedeutet dabei, dass in dem besagten Stadium mindestens 50 %, bevorzugt mindestens 70 %, ganz besonders bevorzugt mindestensa) 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
90 %, am meisten bevorzugt mindestens 95 % der Gesamt- expression über die Lebensdauer einer Pflanze hinweg stattfindet.90%, most preferably at least 95% of the total expression takes place over the life of a plant.
b) Speicherproteine werden während der Keimung des Samen wieder abgebaut . Dabei beträgt der Abbau während der Keimung mindestens 20 %, bevorzugt mindestens 50 %, ganz besonders bevorzugt mindestens 80 %.b) 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%.
c) Speicherproteine machen einen wesentlichen Anteil am Gesamtproteingehalt des nicht-keimenden Samens aus . Bevorzugt macht das Speicherprotein in dem nicht-keimenden Samen der Wildtyp- Pflanze mehr als 5 Gew.-% des Gesamtproteins aus, besonders bevorzugt mindestens 10 Gew.-%) ganz besonders bevorzugt mindestens 20 Gew.-%, am meisten bevorzugt mindestens 30 Gew.-%.c) 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 .-%.
Bevorzugt weisen Speicherproteine 2 oder alle der oben genannten wesentlichen Eigenschaften a) , b) oder c) auf.Storage proteins preferably have 2 or all of the above-mentioned essential properties a), b) or c).
Speicherproteine können in Untergruppen entsprechend weiterer charakteristischer Eigenschaften, wie beispielsweise ihrem Sedimentationskoeffizienten oder der Löslichkeit in verschiedenen Lösungen (Wasser, Salzlösung, Alkohol) aufgeteilt werden. Die Bestimmung des Sedimentationskoeffizienten kann in der dem Fachmann vertrauten Weise mittels Ultrazentrifugation durchgeführt werden (z.B. beschrieben bei Correia JJ (2000) Methods in Enzymology 321:81-100). Bevorzugt ist das Speicherprotein ausgewählt aus den Klassen der 2S-Albumine (Napin-ähnlich) , 7S-Globuline (Phaseolin-ähnlich) , HS/12S-Globuline (Legumin-/Cruciferin-ähnlich) oder Zein-Prol- amine.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.
Besonders bevorzugte 2S-Albumine umfassenParticularly preferred 2S albumins include
a) 2S-Albumine aus Arabidopsis, ganz besonders bevorzugt die 2S-Albumine mit der SEQ ID NO: 2, 4, 6 oder 8, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 1, 3, 5 oder 7 kodierten Proteine,a) 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 .
b) 2S-Albumine aus Arten der Gattung Brasεica, wie beispielsweise Brassica napus, Brassica nigra, Brassica juncea, Brassica oleracea oder Sinapis alba, ganz besonders bevorzugt die 2S-Albumine mit der SEQ ID NO: 32, 34, 36, 38, 40, 46 oder 48, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 31, 33, 35, 37, 39, 45 oder 47 kodierten Proteine,b) 2S albumins from species of the genus Brasεica, 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,
c) 2S-Albumine aus Soja, ganz besonders bevorzugt die 2S-Albumine mit der SEQ ID NO: 42 oder 44, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 41 oder 43 kodierten Proteine,c) 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,
d) 2S-Albumine aus Sonnenblume (Helianthus annus) , ganz besonders bevorzugt die 2S-Albumine mit der SEQ ID NO: 50 oder 52, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 49 oder 51 kodierten Proteine,d) 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,
sowie die entsprechenden Homologen und funktionellen Äquivalente zu a) oder b) oder c) oder d) aus identischen oder anderen Pflanzenarten, insbesondere Raps) Sonnenblume, Lein, Sesam, Färberdistel, Ölbaum, Soja oder verschiedene Nussarten. Funktionelle Äquivalente zeichnen sich bevorzugt neben den oben genannten wesentlichen Eigenschaften durch charakteristische Eigenschaften wie einen 2S-Sedimentationskoeffizienten und/oder durch eine Löslichkeit in Wasser aus.as well as the corresponding homologues and functional equivalents to a) or b) or c) or d) from identical or other plant species, in particular rapeseed) sunflower, flax, sesame, safflower, olive tree, soya or various types of nut. In addition to the essential properties mentioned above, functional equivalents are preferably distinguished by characteristic properties such as a 2S sedimentation coefficient and / or by solubility in water.
Funktionelle Äquivalente der 2S-Albumine haben in einer weiteren bevorzugten Ausführungsform eine Homologie von mindestens 60 %, bevorzugt mindestens 80 %, ganz besonders bevorzugt mindestens 90 %, am meisten bevorzugt mindestens 95 % zu einer der Proteinsequenzen mit der SEQ ID NO: 2, 4, 6, 8, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 oder 52 wobei die Homologie sich bevorzugt über eine Länge von mindestens 30 Aminosäuren, bevorzugt mindestens 50 Aminosäuren besonders bevorzugt über 100 Aminosäuren, am meisten bevorzugt über die gesamte Länge der jeweiligen Proteine erstreckt, und weisen die gleichen wesentlichen Eigenschaften eines Speicherproteins und - bevorzugt- die charakteristischen Eigenschaften eines 2S-Speicherproteins auf.In a further preferred embodiment, 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.
Weiterhin bevorzugte funktionelle Äquivalente enthalten in den für sie kodierenden Nukleinsäuresequenzen mindestens eins, bevorzugt zwei , besonders bevorzugt 3 , am meisten bevorzugt alle der Sequenzmotive ausgewählt aus jeweils einer bestimmten der nachfolgenden Gruppen I , II, III oder IV:Further preferred functional equivalents contain in the 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:
Gruppe I :Group I:
1. 5 ' -TTCCTCGTCTGCGCA-3 ' (SEQ ID NO 53)1.5 '-TTCCTCGTCTGCGCA-3' (SEQ ID NO 53)
2. 5 ' -TCCTCACCAACGCTTCC-3 ' (SEQ ID NO 54)2.5 '-TCCTCACCAACGCTTCC-3' (SEQ ID NO 54)
3. 5 ' -CACCTAAGAGCTTGCCAG-3 ' (SEQ ID NO 55)3.5 '-CACCTAAGAGCTTGCCAG-3' (SEQ ID NO 55)
4. 5 ' -GTTTGCCCCACCTTGA-3 ' (SEQ ID NO 56)4.5 '-GTTTGCCCCACCTTGA-3' (SEQ ID NO 56)
Gruppe IIGroup II
5 ' -ATGGCGAACAAGCTCTTCCTCG-3 ' (SEQ ID NO 57) 5 ' -GCCTTCTTCTTCCTTCTCACCAA-3 ' (SEQ ID NO 58) 5 ' -CCAAAATGTAGGAAGGAGTTTCA-3 ' (SEQ ID NO 59) 5 ' -CTTTGCGTTTGCCCAACCTTGAAAG-3 ' (SEQ ID NO 60) 5 ' -GCAAGTTAGCGTTTGTCCCTTCCAG-3 ' (SEQ ID NO 61) 5 ' -GTCCCTTCCAGAAGACCATGCCTGG-3 ' (SEQ ID NO 62)5 '-ATGGCGAACAAGCTCTTCCTCG-3' (SEQ ID NO 57) 5 '-GCCTTCTTCTTCCTTCTCACCAA-3' (SEQ ID NO 58) 5 '-CCAAAATGTAGGAAGGAGTTTCA-3' (SEQ ID NO 59) 5 '-CTTTGCACTTGAQ 60) 5 '-GCAAGTTAGCGTTTGTCCCTTCCAG-3' (SEQ ID NO 61) 5 '-GTCCCTTCCAGAAGACCATGCCTGG-3' (SEQ ID NO 62)
Gruppe III:Group III:
1. 5 ' -CACATCATGGAGAAGATCCAAG-3 ' (SEQ ID NO 63)1.5 '-CACATCATGGAGAAGATCCAAG-3' (SEQ ID NO 63)
2. 5 ' -AGAAGAAGGACACATGCAGAAGTGCTGCA-3 ' (SEQ ID NO 64)2.5 '-AGAAGAAGGACACATGCAGAAGTGCTGCA-3' (SEQ ID NO 64)
3. 5 ' -ATGCCAGTGCAAAGCGCT-3 ' (SEQ ID NO 65)3.5'-ATGCCAGTGCAAAGCGCT-3 '(SEQ ID NO 65)
4. 5 ' -AGATCATCACTGGTGAATGGTGATCGTGTA-3 ' (SEQ ID NO 66)4.5 'AGATCATCACTGGTGAATGGTGATCGTGTA-3' (SEQ ID NO 66)
5. 5 ' -GGCAATGGAAGCACTTAGAGTGTGCTTTGTG-3 (SEQ ID NO 67)5.5 '-GGCAATGGAAGCACTTAGAGTGTGCTTTGTG-3 (SEQ ID NO 67)
Gruppe IV:Group IV:
1. 5 ' -GACGAGAACCCGATCTCCG-3 ' (SEQ ID NO 68)1.5 '-GACGAGAACCCGATCTCCG-3' (SEQ ID NO 68)
2. 5 ' -TGAACCAGTGTCGCATGTTCCTCCAGCA-3 ' (SEQ ID NO 69)2.5 '-TGAACCAGTGTCGCATGTTCCTCCAGCA-3' (SEQ ID NO 69)
3. 5 ' -CTCCAGCAGTGTTGTCAAGAGCT-3 ' (SEQ ID NO 70)3.5 '-CTCCAGCAGTGTTGTCAAGAGCT-3' (SEQ ID NO 70)
4. 5 ' -CAGTGCCAATGTGAGGCGGTGAAGCAGGTG-3 ■ (SEQ ID NO 71)4. 5 '-CAGTGCCAATGTGAGGCGGTGAAGCAGGTG-3 ■ (SEQ ID NO 71)
5. 5 ' -GGCTCAGATTCTCCCTAACGTA-3 ' (SEQ ID NO 72)5.5 '-GGCTCAGATTCTCCCTAACGTA-3' (SEQ ID NO 72)
6. 5 ' -TGCAACCTTCAATCAAGACGATG-3 ' (SEQ ID NO 73) Besonders bevorzugt weisen die funktionelle Äquivalente in ihren Nukleinsäuresequenzen mindestens eines der nachfolgenden Sequenzmotive ausgewählt aus einer bestimmten der nachfolgenden Gruppen V, VI, VII oder VIII auf:6.5 '-TGCAACCTTCAATCAAGACGATG-3' (SEQ ID NO 73) 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:
Gruppe V:Group V:
1. 5-ATGGCDAACAAGYTSTTCCTCGTCTGCGCRACTYTCGCYCTSTGYTTCMTCCTCACCAA CGCTTCCTRTCTAYCGCACYGTYGTYGARTTCGAMGAAGATGACGCCASYAACCCCRTRGG YCCAADA-3' (SEQ ID NO: 74)1.5-ATGGCDAACAAGYTSTTCCTCGTCTGCGCRACTYTCGCYCTSTGYTTCMTCCTCACCAA CGCTTCCTRTCTAYCGCACYGTYGTYGARTTCGAMGAAGATGACGCCASYAACCCCRTRGG YCCAADA-3 '(SEQ ID NO: 74)
2. 5 ' -AAGGAGTTTCAGMAAKMMCARCACCTAAGAGCTTGCCAG-3 ' (SEQ ID NO: 75)2.5 'AAGGAGTTTCAGMAAKMMCARCACCTAAGAGCTTGCCAG-3' (SEQ ID NO: 75)
3. 5 ' -TCCAGMAGTGCTGCARCGAGCTTCGCCARGAAGAGCCAGWTTGYGTTTGCCCCACCTT3. 5 '-TCCAGMAGTGCTGCARCGAGCTTCGCCARGAAGAGCCAGWTTGYGTTTGCCCCACCTT
GARACAAGCTGCCARGGCMGTTAGRYTCCAGGGACARCA-3' (SEQ ID NO: 76)GARACAAGCTGCCARGGCMGTTAGRYTCCAGGGACARCA-3 '(SEQ ID NO: 76)
4. 5 ' -CAGGAAAATTTACMAGDCAGCYAAGYACTTGCCYAACRTTTGCRAVATCCMGCAAGTT4. 5 '-CAGGAAAATTTACMAGDCAGCYAAGYACTTGCCYAACRTTTGCRAVATCCMGCAAGTT
G-3' (SEQ ID NO: 77)G-3 '(SEQ ID NO: 77)
Gruppe VI :Group VI:
1. 5 ' -AGCCGTATCTACCAGACYGCTACGCACTTACCTARAGTTTGCAACATYMSGCAAGTTA GCGTTTGTCCCTTCCAGAAGACCATGCCTGGRCCCTCC-3' (SEQ ID NO: 78)1.5'-AGCCGTATCTACCAGACYGCTACGCACTTACCTARAGTTTGCAACATYMSGCAAGTTA GCGTTTGTCCCTTCCAGAAGACCATGCCTGGRCCCTCC-3 '(SEQ ID NO: 78)
2. 5 ' -ACCAGGAAGAGCCMCTTTGCGTTTGCCCAACCTTGAAAGGAGCATCCAAAGCSGTTAA2. 5 'ACCAGGAAGAGCCMCTTTGCGTTTGCCCAACCTTGAAAGGAGCATCCAAAGCSGTTAA
ACA-3' (SEQ ID NO: 79)ACA-3 '(SEQ ID NO: 79)
3. 5 ' -CAGCCGGCCCATTTRGGATYCCAAAATGTAGGAAGGAGTTTCARCAAGCACAACACCT3.5 '-CAGCCGGCCCATTTRGGATYCCAAAATGTAGGAAGGAGTTTCARCAAGCACAACACCT
-3' (SEQ ID NO: 80)-3 '(SEQ ID NO: 80)
Gruppe VII :Group VII:
1. 5 ' -ATCGCCCACACYTGCWGCGCCTCCAAATGGCA-3 ' (SEQ ID NO: 81) 2. 5 ' -GAAATGAGCGAGCTGARAAGCCCCAWATGCCAGTGCAAAGCGCT-3 '1.5 'ATCGCCCACACYTGCWGCGCCTCCAAATGGCA-3' (SEQ ID NO: 81) 2.5 'GAAATGAGCGAGCTGARAAGCCCCAWATGCCAGTGCAAAGCGCT-3'
(SEQ ID NO: 82)(SEQ ID NO: 82)
Gruppe VIII:Group VIII:
1. 5 ' -ATCACCACCACCATCGAMGACGAGAACCCGATCTCCG-3 ' (SEQ ID NO: 83)1.5 'ATCACCACCACCATCGAMGACGAGAACCCGATCTCCG-3' (SEQ ID NO: 83)
2. 5 '-TACAGGGACAAAGGYTGAACCAGTGTCGCATGTTCCTCCAGCA-3 '2.5 '-TACAGGGACAAAGGYTGAACCAGTGTCGCATGTTCCTCCAGCA-3'
(SEQ ID NO: 84)(SEQ ID NO: 84)
3. 5 ' -CAAAACATCGAAGGRCAGTGCCAATGTGAGGCGGTGAAGCAGGTG-3 '3.5 '-CAAAACATCGAAGGRCAGTGCCAATGTGAGGCGGTGAAGCAGGTG-3'
(SEQ ID NO: 85)(SEQ ID NO: 85)
4. 5 ' -CAACAGTTGAAGCAGARGGCTCAGATTCTCCCTAACGTATGCAACCTTCAAT-3 '4.5 '-CAACAGTTGAAGCAGARGGCTCAGATTCTCCCTAACGTATGCAACCTTCAAT-3'
(SEQ ID NO: 86)(SEQ ID NO: 86)
Besonders bevorzugte 7S-Globuline umfassen solche aus Arabidopsis oder Soja, ganz besonders bevorzugt die Proteine mit der SEQ ID NO: 155 oder 157, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 154 oder 156 kodierten Proteine. Funktionelle Äquivalente zeichnen sich bevorzugt neben den oben genannten wesentlichen Eigenschaften durch charakteristische Eigenschaften wie einen 7S-Sedimentationskoeffizienten und/oder durch eine Löslichkeit in Salzlösung aus. Als weitere charakteristische Eigenschaft können 7S-Globuline keine Cystein- reste enthalten.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. In addition to the essential properties mentioned above, 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.
Funktionelle Äquivalente der 7S-Globuline haben in einer weiteren bevorzugten Ausführungsform eine Homologie von mindestens 60 %, bevorzugt mindestens 80 %, ganz besonders bevorzugt mindestens 90 %, am meisten bevorzugt mindestens 95 % zu einer der Proteinsequenzen mit der SEQ ID NO: 155 oder 157 wobei die Homologie sich bevorzugt über eine Länge von mindestens 30 Aminosäuren, bevorzugt mindestens 50 Aminosäuren besonders bevorzugt über 100 Aminosäuren, am meisten bevorzugt über die gesamte Länge der jeweiligen Proteine erstreckt, und weisen die gleichen wesentlichen Eigenschaften eines Speicherproteins und - bevorzugt- die charakteristischen Eigenschaften eines 7S-Speicherproteins auf.In a further preferred embodiment, 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.
Besonders bevorzugte HS/12S-Globuline umfassen bevorzugt 11S- Globuline aus Raps, Soja und Arabidopsis insbesondereParticularly preferred HS / 12S globulins preferably comprise 11S globulins from rapeseed, soybeans and Arabidopsis in particular
a) HS-Globuline aus Raps mit der SEQ ID NO: 10, 12, 14, 16 oder 18, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 9, 11, 13, 15 oder 17 kodierten Proteine,a) HS globulins from rapeseed with SEQ ID NO: 10, 12, 14, 16 or 18, most preferably the proteins encoded by the nucleic acids according to SEQ ID NO: 9, 11, 13, 15 or 17,
b) die HS-Globuline aus Soja mit der SEQ ID NO: 20, 22, 24, 26 oder 28, am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 19, 21, 23, 25 oder 27 kodierten Proteine,b) the 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,
c) die HS-Globuline aus Arabidopsis thaliana mit der SEQ ID NO: 112, 114, 116, 118, 120 oder 122 am meisten bevorzugt die durch die Nukleinsäuren gemäß SEQ ID NO: 111, 113, 115, 117, 119 oder 121 kodierten Proteine,c) the 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,
sowie die entsprechenden Homologen und funktionellen -Äquivalente aus anderen Pflanzenarten, insbesondere Raps, Sonnenblume, Lein, Sesam, Färberdistel, Ölbaum, Soja oder verschiedene Nuss- arten, wie beispielsweise das Sonnenblume IIS Speicherprotein (SEQ ID NO: 30) , insbesondere das durch die Nukleinsäuresequenz gemäß SEQ ID NO: 29 kodierte Protein. Funktionelle Äquivalente zeichnen sich bevorzugt neben den oben genannten wesentlichen Eigenschaften durch charakteristische Eigenschaften wie einen IIS- oder 12S-Sedimentationskoeffizienten und/oder durch eine Löslichkeit in Salzlösung (PBS; phosphatgepufferte Salzlösung) und/oder eine schlechte Löslichkeit in Wasser aus. ISas well as the corresponding homologues and functional equivalents from other plant species, in particular rapeseed, sunflower, flax, sesame, safflower, olive tree, soybean or various types of nuts, such as the sunflower IIS storage protein (SEQ ID NO: 30), in particular the one by Nucleic acid sequence according to SEQ ID NO: 29 encoded protein. In addition to the above-mentioned essential properties, 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. IS
Funktionelle Äquivalente der IIS- oder 12S Albumine haben in einer weiteren bevorzugten Ausführungsform eine Homologie von mindestens 60 %, bevorzugt mindestens 80 %, ganz besonders bevorzugt mindestens 90 %, am meisten bevorzugt mindestens 95 % zu einer der Proteinsequenzen mit der SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 112, 114, 116, 118, 120 oder 122 wobei die Homologie sich bevorzugt über eine Länge von mindestens 30 Aminosäuren, bevorzugt mindestens 50 Aminosäuren besonders bevorzugt über 100 Aminosäuren, am meisten bevorzugt über die gesamte Länge der jeweiligen Proteine erstreckt, und weisen die gleichen wesentlichen Eigenschaften eines Speicherproteins und - bevorzugt - die charakteristischen Eigenschaften eines 11S- oder 12S-Speicherproteins auf.In a further preferred embodiment, 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.
Insbesondere bevorzugt ist das Arabidopsis thaliana 12S Crucife- rin Speicherprotein (ATCRU3) gemäß SEQ ID NO: 112 sowie seine Homologen aus anderen Pflanzenarten wie beispielsweise Raps, Soja oder Sonnenblume, wobei diese bevorzugt eine Homologie von mindestens 65 %, bevorzugt mindestens 80 %, ganz besonders bevorzugt mindestens 90 %, am meisten bevorzugt mindestens 95 % zu einer der Proteinsequenzen mit der SEQ ID NO: 112 aufweisen.The 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.
Weiterhin bevorzugte funktionelle Äquivalente der Raps 11S/12S- Speicherproteine enthalten in den für sie kodierenden Nuklein- säuresequenzen mindestens eins, bevorzugt zwei, besonders bevorzugt 3 der Sequenzmotive ausgewählt aus der Gruppe IX oder ausgewählt aus der Gruppe X:Further preferred functional equivalents of the 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:
Gruppe IX:Group IX:
1. 5 ' -GAGGCTGGTCGCATCGA-3 ' ( SEQ ID NO : 87 )1.5 '-GAGGCTGGTCGCATCGA-3' (SEQ ID NO: 87)
2. 5 ' -TTCCGTGATATGCACCAGAA-3 ' ( SEQ ID NO : 88 )2.5 '-TTCCGTGATATGCACCAGAA-3' (SEQ ID NO: 88)
3. 5 ' -TGGTTAACGACAACGGTGA-3 ' ( SEQ ID NO : 89 )3.5 '-TGGTTAACGACAACGGTGA-3' (SEQ ID NO: 89)
Gruppe X:Group X:
1. 5 ' -GAGGCTGGTCGGGTCGA-3 ' (SEQ ID NO : 90 )1.5 '-GAGGCTGGTCGGGTCGA-3' (SEQ ID NO: 90)
2. 5 ' -TTCCGTGACATGCACCAGAA-3 ' ( SEQ ID NO : 91 )2.5 '-TTCCGTGACATGCACCAGAA-3' (SEQ ID NO: 91)
3. 5 ' -TGGTGAACGACAACGGacA-3 ' ( SEQ ID NO : 92 )3.5 '-TGGTGAACGACAACGGacA-3' (SEQ ID NO: 92)
Besonders bevorzugt weisen die funktionelle Äquivalente der Raps HS-Speicherproteine in ihren Nukleinsäuresequenzen ein Sequenzmotive mit der SEQ ID NO: 93 auf: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:
5 ' -CARGGBTTCCGTGAYATGCACCAGAARGTVGARCA-3 ' (SEQ ID NO: 93) Weiterhin bevorzugte funktionelle Äquivalente der Soja 11S/12S- Speicherproteine enthalten in den für sie kodierenden Nuklein- säuresequenzen mindestens eins, bevorzugt zwei, besonders bevorzugt 3, am meisten bevorzugt 4, 5 oder 6 der Sequenzmotive ausgewählt aus der Gruppe XI oder ausgewählt aus der Gruppe XII:5 '-CARGGBTTCCGTGAYATGCACCAGAARGTVGARCA-3' (SEQ ID NO: 93) Further preferred functional equivalents of the 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:
Gruppe XI:Group XI:
1. 5 '-ATTGAGACATGGAACCCTAA-3 ' (SEQ ID NO: 94)1.5 '-ATTGAGACATGGAACCCTAA-3' (SEQ ID NO: 94)
2. 5 '-TGGATGTACAACAATGAAGA-3 ' (SEQ ID NO: 95)2.5 '-TGGATGTACAACAATGAAGA-3' (SEQ ID NO: 95)
3. 5 ' -CACTCCTGTTGTTGCCGTTTCT-3 ' (SEQ ID NO: 96)3.5 '-CACTCCTGTTGTTGCCGTTTCT-3' (SEQ ID NO: 96)
4. 5 ' -ATTCTATCTTGCTGGGAACCAAGA-3 ' (SEQ ID NO: 97)4.5'-ATTCTATCTTGCTGGGAACCAAGA-3 '(SEQ ID NO: 97)
5. 5 ' -GCCATTGTGACAGTGAAAGGAGGTCT-3 ' (SEQ ID NO: 98)5.5 '-GCCATTGTGACAGTGAAAGGAGGTCT-3' (SEQ ID NO: 98)
6. 5 ' -AGGGAATGCAGTGTTCGACGGTGAGC-3 ' (SEQ ID NO: 99)6.5 '-AGGGAATGCAGTGTTCGACGGTGAGC-3' (SEQ ID NO: 99)
Gruppe XII :Group XII:
1. 5 ' -AAACATGGAACTCTCAACAC-3 ' (SEQ ID NO 100)1.5 '-AAACATGGAACTCTCAACAC-3' (SEQ ID NO 100)
2. 5 ' -TGGACCTATAACACTGGCGA-3 ' (SEQ ID NO 101)2.5 '-TGGACCTATAACACTGGCGA-3' (SEQ ID NO 101)
3. 5 ' -TGAACCAGTTGTTGCCATCAGT-3 ' (SEQ ID NO 102)3.5 'TGAACCAGTTGTTGCCATCAGT-3' (SEQ ID NO 102)
4. 5 ' -ATTTTACCTTGCTGGGAACCCAGA-3 ' (SEQ ID NO 103)4.5 '-ATTTTACCTTGCTGGGAACCCAGA-3' (SEQ ID NO 103)
5. 5 ' -GGTGAGAGAGTGTTTGATGGAGAGCT-3 ' (SEQ ID NO 104)5.5 '-GGTGAGAGAGTGTTTGATGGAGAGCT-3' (SEQ ID NO 104)
Weiterhin bevorzugte funktionelle Äquivalente der Arabidopsis thaliana HS/12S-Speicherproteine enthalten in den für sie kodierenden Nukleinsäuresequenzen mindestens eins, bevorzugt zwei, besonders bevorzugt 3, am meisten bevorzugt 4, 5 oder 6 der Sequenzmotive ausgewählt aus der Gruppe XIII:Further preferred functional equivalents of the 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:
Gruppe XIII:Group XIII:
1. 5 ' -AACGAGTGCCAGCTCGA-3 ' (SEQ ID NO 123)1.5 '-AACGAGTGCCAGCTCGA-3' (SEQ ID NO 123)
2. 5 ' -TTCCGTGACATGCACCAG-3 ' (SEQ ID NO 124)2.5 '-TTCCGTGACATGCACCAG-3' (SEQ ID NO 124)
3. 5 ' -CAGAACCAGCTTGACCGCA-3 ' (SEQ ID NO 125)3.5 '-CAGAACCAGCTTGACCGCA-3' (SEQ ID NO 125)
4. 5 ' -TAGCCGGAAACAACCCACAAGG-3 ' (SEQ ID NO 126)4.5 'TAGCCGGAAACAACCCACAAGG-3' (SEQ ID NO 126)
5. 5 '-AACCTCGATGACCCGTC-3 ' (SEQ ID NO 127)5.5 'AACCTCGATGACCCGTC-3' (SEQ ID NO 127)
6. 5 ' -TGCTGACGTGTACAAGCCA-3 ' (SEQ ID NO 128)6.5 '-TGCTGACGTGTACAAGCCA-3' (SEQ ID NO 128)
Besonders bevorzugt weisen die funktionelle Äquivalente der Arabidopsis HS/12S-Speicherproteine in ihren Nukleinsäuresequenzen ein Sequenzmotive mit der SEQ ID NO: 129 auf: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:
5 ' -GACATGCACCAGAARKTRGA-3 ' (SEQ ID NO: 129)5 '-GACATGCACCAGAARKTRGA-3' (SEQ ID NO: 129)
Besonders bevorzugte Zein-Prolamine umfassen bevorzugt solche aus monokotyledonen Pflanzen, insbesondere Mais, Rais, Hafer, Gerste oder Weizen. Ganz besonders bevorzugt sind die Mais Zein-Prolamine beschrieben durch SEQ ID NO: 159, 161,. 163 oder 165 - insbesondere die durch SEQ ID NO 158, 160, 162 oder 164 kodierten Protein -, das Reis Prolamin gemäß SEQ ID NO: 167 - insbesondere das durch SEQ ID NO 166 kodierte Protein -, das Hafer Prolamin gemäß SEQ ID NO: 169 - insbesondere das durch SEQ ID NO 168 kodierte Proteine-, das Gerste Prolamin gemäß SEQ ID NO: 171 und/oder 172 - insbesondere das durch SEQ ID NO: 170 kodierte Protein - und das das Weizen Prolamin gemäß SEQ ID NO: 174 - insbesondere das durch SEQ ID NO 173 kodierte Protein. Funktionelle Äquivalente zeichnen sich bevorzugt durch eine Löslichkeit in 70%iger ethanolischer Lösung und eine schlechte Löslichkeit in Wasser oder Salzlösung aus.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 ,. 163 or 165 - in particular the protein encoded by SEQ ID NO 158, 160, 162 or 164 -, the rice prolamin according to SEQ ID NO: 167 - in particular the protein encoded by SEQ ID NO 166 -, the oat prolamine according to SEQ ID NO: 169 - in particular the protein encoded by SEQ ID NO 168, the barley prolamine according to SEQ ID NO: 171 and / or 172 - in particular the protein encoded by SEQ ID NO: 170 - and the wheat prolamine according to SEQ ID NO: 174 - in particular the protein encoded by SEQ ID NO 173. Functional equivalents are preferably characterized by solubility in 70% ethanolic solution and poor solubility in water or saline.
Funktionelle Äquivalente der Zein-Prolamine haben in einer weiteren bevorzugten Ausführungsform eine Homologie von mindestens 60 %, bevorzugt mindestens 80 %, ganz besonders bevorzugt 'mindestens 90 %, am meisten bevorzugt mindestens 95 % zu einer der Proteinsequenzen mit der SEQ ID NO: 159, 161, 163, 165, 167, 169, 171, 172 oder 174 wobei die Homologie sich bevorzugt über eine Länge von mindestens 30 Aminosäuren, bevorzugt mindestens 50 Aminosäuren besonders bevorzugt über 100 Aminosäuren, am meisten bevorzugt über die gesamte Länge der jeweiligen Proteine erstreckt, und weisen die gleichen wesentlichen Eigenschaften eines Speicherproteins und - bevorzugt- die charakteristischen Eigenschaften eines Zein-Prolamine auf.In a further preferred embodiment, 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.
Dabei haben die einzelnen Buchstaben in den genannten Sequenzen nachfolgende dem Fachmann vertraute Bedeutung:The individual letters in the sequences mentioned have the following meaning familiar to the person skilled in the art:
Figure imgf000018_0001
Figure imgf000018_0001
Funktionelle Äquivalente meint insbesondere natürliche oder künstliche Mutationen der obengenannten Speicherproteine sowie homologe Polypeptide aus anderen Pflanzen, die die gleichen wesentlichen und - bevorzugt - charakteristischen Eigenschaften aufweisen. Bevorzugt sind homologe Polypeptide aus oben beschriebenen bevorzugten Pflanzen. Die zu den im Rahmen dieser Erfindung offenbarten Speicherproteinen homologen Sequenzen aus anderen Pflanzen - beispielsweise solchen deren genomische Sequenz ganz oder teilweise bekannt ist, wie beispielsweise aus Arabidopsis thaliana, Brassica napus, Nicotiana tabacum oder Solanum tuberosum - durch Homologievergleiche aus Datenbanken auffinden, können z.B. durch Datenbanksuche oder Durchmustern von Gen-Banken - unter Verwendung der beispielhaft aufgeführten Speicherprotein-Sequenzen als Suchsequenz bzw. Sonde - leicht aufgefunden werden.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. The one in the frame In this invention, 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.
Mutationen umfassen Substitutionen, Additionen, Deletionen, Inversion oder Insertionen eines oder mehrerer Aminosäurereste.Mutations include substitutions, additions, deletions, inversions, or insertions of one or more amino acid residues.
Unter Homologie zwischen zwei Nukleinsäuresequenzen wird die Identität der Nukleinsäuresequenz über die jeweils gesamte Sequenzlänge verstanden, die durch Vergleich mit Hilfe des Programmalgorithmus GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG) , Madison, USA; Altschul et al . (1997) Nucleic Acids Res. 25:3389ff) unter Einstellung folgender Parameter berechnet wird: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: 3Gap Weight: 50 Length Weight: 3
Average Match: 10 Average Mismatch:0Average Match: 10 Average Mismatch: 0
Beispielhaft wird unter einer Sequenz, die eine Homologie von mindestens 80 % auf Nukleinsäurebasis mit der Sequenz SEQ ID NO: 1 aufweist, eine Sequenz verstanden, die bei einem Vergleich mit der Sequenz SEQ ID NO: 1 nach obigem Programmalgorithmus mit obigem Parametersatz eine Homologie von mindestens 80 % aufweist.By way of example, 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%.
Unter Homologie zwischen zwei Polypeptiden wird die Identität der Aminosäuresequenz über die jeweils gesamte Sequenzlänge verstanden, die durch Vergleich mit Hilfe des Programmalgorithmus GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG) , Madison, USA) unter Einstellung folgender Parameter berechnet wird:Homology between two polypeptides is understood to mean the identity of the amino acid sequence over the entire entire length of the sequence, 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) with the following parameters is calculated:
Gap Weight: 8 Length Weight: 2Gap Weight: 8 Length Weight: 2
Average Match: 2,912 Average Mismatch:-2 , 003Average Match: 2,912 Average Mismatch: -2, 003
Beispielhaft wird unter einer Sequenz, die eine Homologie von mindestens 80 % auf Proteinbasis mit der Sequenz SEQ ID NO: 2 aufweist, eine Sequenz verstanden, die bei einem Vergleich mit der Sequenz SEQ ID NO: 2 nach obigem Programmalgorithmus mit obigem Parametersatz eine Homologie von mindestens 80' % aufweist.By way of example, 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%.
Funktionelle Äquivalente umfasst auch solche Proteine, die durch Nukleinsäuresequenzen kodiert werden, die unter Standardbedingungen mit einer der durch 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 oder 173 beschriebenen, für ein Speicherproteine kodierenden Nukleinsäuresequenz, der zu dieser komplementären Nukleinsäuresequenz oder Teilen der vorgenannten hybridisieren und die wesentlichen Eigenschaften eines Speicherproteins und - bevorzugt - weitere charakteristische Eigenschaften aufweisen.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.
"Standardhybridisierungsbedingungen" ist breit zu verstehen und meint stringente als auch weniger stringente Hybridisierungs- bedingungen. Solche Hybridisierungsbedingungen sind unter anderem bei Sambrook J, Fritsch EF, Maniatis T et al . , in Molecular Cloning (A Laboratory Manual), 2. Auflage, Cold Spring Harbor Laboratory Press, 1989, Seiten 9.31-9.57) oder in Current Protocols in Molecular Biology, John Wiley S-Sons, N.Y. (1989) , 6.3.1-6.3.6. beschrieben."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.
Beispielhaft können die Bedingungen während des Waschschrittes ausgewählt sein aus dem Bereich von Bedingungen begrenzt von solchen mit geringer Stringenz (mit ungefähr 2X SSC bei 50°C) und solchen mit hoher Stringenz (mit ungefähr 0,2X SSC bei 50°C bevorzugt bei 65°C) (20X SSC: 0,3 M Natriumeitrat, 3 M NaCl, pH 7,0). Darüberhinaus kann die Temperatur während des Waschschrittes von niedrig stringenten Bedingungen bei Raumtemperatur, ungefähr -22°C, bis zu stärker stringenten Bedingungen bei ungefähr 65°C angehoben werden. Beide Parameter, Salzkonzentration und Temperatur, können gleichzeitig variiert werden, auch kann einer der beiden Parameter konstant gehalten und nur der andere variiert werden. Während der Hybridisierung können auch denaturierende Agenzien wie zum Beispiel Formamid oder SDS eingesetzt werden. In Gegenwart von 50 % Formamid wird die Hybridisierung bevorzugt bei 42°C ausgeführt. Einige beispielhafte Bedingungen für Hybridisierung und Waschschritt sind infolge gegeben:By way of example, 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). Furthermore, 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. Some exemplary conditions for hybridization and washing step are given as a result:
(1) Hybridisierungbedingungen zum Beispiel aus nachfolgenden Bedingungen ausgewählt sein:(1) Hybridization conditions can be selected from the following conditions, for example:
a) 4X SSC bei 65°C, b) 6X SSC bei 45°C, c) 6X SSC, 100 μg/ml denaturierter, fragmentierte Fischsperma-DNA bei 68°C, f) 50 % Formamid, 4X SSC bei 42°C, h) 2X oder 4X SSC bei 50°C (schwach stringente Bedingung) , i) 30 bis 40 % Formamid, 2X oder 4X SSC bei 42°C (schwach stringente Bedingung) .a) 4X SSC at 65 ° C, b) 6X SSC at 45 ° C, c) 6X SSC, 100 μg / ml denatured, fragmented fish sperm DNA at 68 ° C, f) 50% formamide, 4X SSC at 42 ° C, h) 2X or 4X SSC at 50 ° C (weakly stringent condition), i ) 30 to 40% formamide, 2X or 4X SSC at 42 ° C (weakly stringent condition).
(2) Waschschritte können zum Beispiel aus nachfolgenden Bedingungen ausgewählt sein:(2) Washing steps can be selected, for example, from the following conditions:
a) 0,015 M NaCl/0,0015 M Natriumcitrat/0, 1 % SDS bei 50°C. b) 0,1X SSC bei 65°C. c) 0,1X SSC, 0,5 % SDS bei 68°C. d) 0,1X SSC, 0,5 % SDS, 50 % Formamid bei 42°C. e) 0,2X SSC, 0,1 % SDS bei 42°C. f) 2X SSC bei 65°C (schwach stringente Bedingung) .a) 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% SDS at 50 ° C. b) 0.1X SSC at 65 ° C. c) 0.1X SSC, 0.5% SDS at 68 ° C. d) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C. e) 0.2X SSC, 0.1% SDS at 42 ° C. f) 2X SSC at 65 ° C (weakly stringent condition).
Die Verminderung der Expression eines Speicherproteins kann auf vielfältige Art und Weise realisiert werden.The reduction in the expression of a storage protein can be implemented in a variety of ways.
"Proteinmenge" meinte die Menge eines Speicherprotein-Poly- peptides in einem Organismus, einem Gewebe, einer Zelle oder einem Zellkompartiment . Bevorzugt meint Proteinmenge die Menge eines bestimmten Speicherproteins im Samen einer Pflanze.“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.
"Verminderung" der Proteinmenge meint die mengenmäßige Verminderung der Menge eines Speicherproteins in einem Organismus, einem Gewebe, einer Zelle oder einem Zellkompartiment - beispielsweise durch eines der unten beschriebenen Verfahren - im Vergleich zu dem Wildtyp derselben Gattung und Art auf den dieses Verfahren nicht angewendet wurde, unter ansonst gleichen Rahmenbedingungen (wie beispielsweise Kulturbedingungen, Alter der Pflanzen etc . ) . Bevorzugt meint Verminderung die Verminderung der Proteinmenge im Samen einer Pflanze ."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.
Der Verminderung beträgt dabei mindestens 10 %, bevorzugt mindestens 10 % oder mindestens 20 %, besonders bevorzugt um mindestens 40 % oder 60 %, ganz besonders bevorzugt um mindestens 70 % oder 80 %, am meisten bevorzugt um mindestens 90 % oder 95 %. Verfahren zur Bestimmung der Proteinmenge sind dem Fachmann bekannt. Beispielhaft seien zu nennen: Das Mikro-Biuret Verfahren (Goa. J (1953) Scand J Clin Lab Invest 5:218-222), die Folin-Ciocalteu-Methode (Lowry OH et al . (1951) J Biol Chem 193:265-275) oder die Messung der Adsorption von CBB G-250 (Bradford MM (1976) Analyt Biochem 72:248-254). Bevorzugt erfolgt die Verminderung des oder der Speicherproteine und/oder die Erhöhung der Olproduktion im Samen einer Pflanze .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). Preferably done reducing the storage protein (s) and / or increasing oil production in the seed of a plant.
In einer weiteren bevorzugten Ausführungsform wird die Proteinmenge von mehr als einem Speicherprotein vermindert. Dabei können die verminderten Speicherproteine der gleichen oder unterschiedlichen Klassen, wie 2S-Albuminen, 7S-Globulinen, 11S/12S- Globulinen oder Zein-Prolaminen, angehören. Bevorzugt werden Speicherproteine aus mehr als einer dieser Klassen gleichzeitig in ihrer Proteinmenge vermindert. Die zu vermindernden Speicherproteine können stark homolog oder weniger stark homolog zu einander sein. Bevorzugt haben mindesten zwei der in ihrer Proteinmenge verminderten Speicherproteine eine Homologie geringer als 90 %, bevorzugt geringer als 70 %, besonders bevorzugt geringer als 60 %, ganz besonders bevorzugt geringer als 50 %.In a further preferred embodiment, 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%.
"Verminderung" oder "vermindern" ist im Zusammenhang mit einem Speicherprotein (bzw. der Menge eines Speicherprotein oder der für dieses kodierende RNA Menge) weit auszulegen und umfasst die teilweise oder im wesentlichen vollständige, auf unterschiedliche semasiologische Mechanismen beruhende Unterbindung oder Blockierung der Expression eines Speicherproteins in einer Pflanze oder einem davon abgeleiteten Teil, Gewebe, Organ, Zellen oder Samen."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.
Eine Verminderung im Sinne der Erfindung umfasst die mengenmäßige Verringerung eines Speicherproteins bis hin zu einem im wesentlichen vollständigen Fehlen des Speicherproteins (d.h. fehlende immunologische Nachweisbarkeit des Speicherproteins) . Dabei wird die Expression eines bestimmten Speicherproteins in einer Zelle oder einem Organismus bevorzugt um mehr als 50 %, besonders bevorzugt um mehr als 80 %, ganz besonders bevorzugt um mehr als 90 % vermindert.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%.
Erfindungsgemäß sind verschiedene Strategien zur Verminderung der Expression eines Speicherproteins umfasst. Der Fachmann erkennt, dass eine Reihe verschiedener Methoden zur Verfügung stehen, um die Expression eines Speicherproteins in gewünschter Weise zu beeinflussen. Eine Verminderung der Speicherproteinmenge kann beispielsweise jedoch nicht einschränkend unter Verwendung nachfolgender Verfahren realisiert werden:According to the invention, various strategies for reducing the expression of a storage protein are included. Those skilled in the art will recognize that a number of different methods are available to influence the expression of a storage protein in the desired manner. However, a reduction in the amount of storage protein can, for example, not be implemented restrictively using the following methods:
a) Einbringung einer doppelsträngigen RNA-Nukleinsäuresequenz (infolge "SP-dsRNA"), wobei die doppelsträngige RNA-Sequenz; nachfolgende Elemente umfasst i) mindestens eine "sense"-Ribonukleotidsequenz, die im wesentlichen identisch ist zu mindestens einem Teil des "sense" RNA-Transkriptes einer Speicherprotein-Nuklein- säuresequenz unda) introduction of a double-stranded 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
ii) "antisense"-Ribonukleotidsequenzen, die zu besagtenii) "antisense" ribonucleotide sequences to be said
"sense"-Ribonukleotidsequenzen unter i) im wesentlichen komplementären sind"sense" ribonucleotide sequences under i) are essentially complementary
oder einer die SP-dsRNA Expression gewährleistenden Expressionskassette oder Expressionskassetten;or an expression cassette or cassettes which ensure SP-dsRNA expression;
b) Einbringung einer Speicherprotein antisense-RNA-Nukleinsäure- sequenzen oder einer deren Expression gewährleistenden Expressionskassette, wobei die Speicherprotein antisense- RNA-Nukleinsäuresequenz im wesentlichen komplementär ist zu mindestens einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nukleinsäuresequenz . Umfasst sind solche Verfahren bei denen die antisense-RNA-Nukleinsäuresequenz gegen ein Speicherprotein-Gen (also genomische DNA-Sequenzen) oder ein Speicherprotein-Gentranskript (also RNA-Sequenzen) gerichtet ist. Umfasst sind auch α-anomere Nukleinsäuresequenzen.b) 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. These include methods in which the antisense RNA nucleic acid sequence is directed against a storage protein gene (that is to say genomic DNA sequences) or a storage protein gene transcript (that is to say RNA sequences). Α-Anomeric nucleic acid sequences are also included.
c) Einbringung einer Speicherprotein antisense-Nukleinsäure- sequenzen kombiniert mit einem Ribozy oder einer deren Expression gewährleistenden Expressionskassettec) Introduction of a storage protein antisense nucleic acid sequences combined with a Ribozy or an expression cassette ensuring their expression
d) Einbringung von Speicherprotein sense-Nukleinsäuresequenzen zur Induktion einer Kosuppression oder einer deren Expression gewährleistenden Expressionskassette und Verminderung der Expression über eine Cosuppression, wobei die Speicherprotein sense-RNA-Nukleinsäuresequenz im wesentlichen identisch ist zu mindestens einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nukleinsäuresequenzd) introduction of storage protein sense nucleic acid sequences for inducing a cosuppression or an expression cassette ensuring their expression and reducing expression via a cosuppression, 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
f) Einbringung DNA-bindender Faktoren gege Speicherprotein-Gene oder -RNAs oder einer deren Expression gewährleistenden Expressionskassettef) Introduction of DNA-binding factors against storage protein genes or RNAs or an expression cassette ensuring their expression
g) Einbringung von den Speicherprotein RNA-Abbau bewirkende virale Nukleinsäuresequenzen und Expressionskonstrukten oder einer deren Expression gewährleistenden Expressionskassette h) • Einbringung von Konstrukten zur Induktion einer homologen Rekombination an endogenen Speicherprotein-Genen beispielsweise zur Erzeugung von Knockout-Mutanten.g) introduction of viral nucleic acid sequences and expression constructs causing the degradation of RNA in the storage protein or an expression cassette ensuring their expression h) • Introduction of constructs for the induction of a homologous recombination on endogenous storage protein genes, for example for the generation of knockout mutants.
i) Einführung von Mutationen in endogenen Speicherprotein-Gene zur Erzeugung eines Funktionsverlustes (z.B. Generierung von Stopp-Kodons , Verschiebungen im Leseraster etc.)i) Introduction of mutations in endogenous storage protein genes to generate a loss of function (e.g. generation of stop codons, shifts in the reading frame, etc.)
Dabei kann jedes einzelne dieser Verfahren eine Verminderung der Speicherprotein-Expression im Sinne der Erfindung bewirken. Auch eine kombinierte Anwendung ist denkbar. Weitere Methoden sind dem Fachmann bekannt und können die Behinderung oder Unterbindung der Prozessierung des Speicherproteins , des Transports des Speicherproteins oder dessen mRNA, Hemmung der Ribqsomen- anlagerung, Hemmung des RNA-Spleißens, Induktion eines Speicher- protein-RNA abbauenden Enzyms und/oder Hemmung der Translations- elongation oder -termination umfassen.Each of these methods can bring about a reduction in storage protein expression in the sense of the invention. A combined application is also conceivable. Other methods are known to the person skilled in the art and can include the hindrance or suppression of the processing of the storage protein, the transport of the storage protein or its mRNA, inhibition of ribsome attachment, inhibition of RNA splicing, induction of an enzyme which degrades storage protein RNA and / or inhibition translation elongation or termination.
Die einzelnen bevorzugten Verfahren seien infolge kurz beschrieben:The individual preferred methods are briefly described as a result:
a) Einbringung einer doppelsträngigen Speicherprotein RNA- Nukleinsäuresequenz (SP-dsRNA)a) Introduction of a double-stranded storage protein RNA nucleic acid sequence (SP-dsRNA)
Das Verfahren der Genregulation mittels doppelsträngiger RNA ("double-stranded RNA interference" ; dsKNAi) ist vielfach in tierischen und pflanzlichen Organismen beschrieben (z.B. Matzke MA et al . (2000) Plant Mol Biol 43:401-415; Fire A. et al (1998) Nature 391:806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364) . Auf die in den angegebenen Zitaten beschriebenen Verfahren und' Methoden wird ausdrücklich Bezug genommen. Eine effiziente Gensuppression kann auch bei transienter Expression oder nach transienter Transformation beispielsweise infolge einer biolistischen Transformation gezeigt werden (Schweizer P et al. (2000) Plant J 2000 24: 895-903). dsRNAi-Verfahren beruhen auf dem Phänomen, dass durch gleichzeitiges Einbringen von komplementären Strang- und Gegenstrang eines Gentranskriptes eine hocheffiziente Unterdrückung der Expression des entsprechenden Gens bewirkt wird bewirkt wird. Der bewirkte Phänotyp kommt dem einer entsprechenden knock-out Mutanten sehr ähnlich (Waterhouse PM et al . (1998) Proc Natl Acad Sei USA 95:13959-64) . Das dεRNAi-Verfahren hat sich bei der Verminderung der Speicherprotein-Expression als besonders effizient und vorteilhaft erwiesen. Wie u.a. in WO .99/32619 beschrieben sind dsRNAi-Ansätze klassischen antisense-Ansätzen deutlich überlegen.The method of gene regulation using double-stranded RNA ("double-stranded RNA interference"; dsKNAi) has been described many times in animal and plant organisms (for example Matzke MA et al. (2000) Plant Mol Biol 43: 401-415; Fire A. et al (1998) Nature 391: 806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). Reference is expressly made to the stated in the quotations described methods and 'methods. Efficient gene suppression can also be shown with transient expression or after transient transformation, for example as a result of a biolistic transformation (Schweizer P et al. (2000) Plant J 2000 24: 895-903). 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 dεRNAi 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.
Ein weiterer Gegenstand der Erfindung bezieht sich daher auf doppelsträngige RNA-Moleküle (dsRNA-Moleküle) , die bei Einführung in eine Pflanze (oder eine davon abgeleitete Zelle, Gewebe, Organ oder Samen) die Verminderung eines Speicherprotein bewirken.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.
Das doppelsträngiges RNA-Molekül zur Verminderung der Expression eines Speicherprotein Proteins ist dadurch gekennzeichnet, dass es enthältThe double-stranded RNA molecule for reducing the expression of a storage protein is characterized in that it contains
a) mindestens eine "sense"-Ribonukleotidsequenz, die im wesentlichen identisch ist zu mindestens einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nuklein- säuresequenz gemäß 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 oder 173 unda) 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 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 and
b) "antisense"-Ribonukleotidsequenzen, die zu den "sense"-Ribonukleotidsequenz unter a) im wesentlichen - bevorzugt vollständig - komplementären sind.b) “antisense” ribonucleotide sequences which are essentially — preferably completely — complementary to the “sense” ribonucleotide sequence under a).
"Im wesentlichen identisch" meint, dass die dsRNA Sequenz auch Insertionen, Deletionen sowie einzelne Punktmutationen im Vergleich zu- der- Speicherprotein Zielsequenz aufweisen kann und dennoch eine effizient Verminderung der Expression bewirken. Bevorzugt beträgt die Homologie nach obiger Definition mindestens 65 %, bevorzugt mindestens 75 %, ganz besonders bevorzugt mindestens 90 %, am meisten bevorzugt 95 % zwischen der "sense"-Ribonukleotidsequenz einer dsRNA und mindestens einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nukleinsäuresequenz .“Essentially identical” means that the 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.
"Teil des "sense"-RNA-Transkriptes einer Speicherprotein- Nukleinsäuresequenz" meint Fragmente einer RNA oder mRNA transkribiert von einer für ein Speicherprotein kodierenden Nukleinsäuresequenz, bevorzugt von einem Speicherprotein-Gen. Dabei haben die Fragmente bevorzugt eine Sequenzlänge von mindestens 20 Basen, bevorzugt mindestens 50 Basen, besonders bevorzugt mindestens 100 Basen, ganz besonders bevorzugt mindestens 200 Basen, am meisten bevorzugt mindestens 500 Basen. Umfasst ist auch die vollständige transkribierte RNA oder mRNA."Part of the" sense "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.
"Im wesentlichen komplementär" meint, dass die "antisense"- Ribonukleotidsequenz auch Insertionen, Deletionen sowie einzelne Punktmutationen im Vergleich zu dem Komplement der "sense"-Ribonukleotidsequenz aufweisen kann. Bevorzugt beträgt die Homologie mindestens 80 %, bevorzugt mindestens 90 %, ganz besonders bevorzugt mindestens 95 %, am meisten bevorzugt 100 % zwischen der "antisense"-Ribonukleotidsequenz und dem Komplement der
Figure imgf000026_0001
“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
Figure imgf000026_0001
Alternativ, kann eine "im wesentlichen identische" dsRNA auch als Nukleinsäuresequenz definiert werden, die befähigt ist, mit einem Teil eines Speicherprotein Gentranskriptes -zu hybridisieren (z.B. in 400 mM NaCl, 40 mM PIPES pH 6,4, 1 mM EDTA bei 50°C oder 70°C für 12 bis 16 h) .Alternatively, 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).
Eine 100%ige Sequenzidentität zwischen dsRNA und dem Speicherprotein Gentranskript ist nicht zwingend erforderlich, um eine effiziente Verminderung der Speicherprotein Expression zu bewirken. Das Verfahren ist demnach tolerant gegenüber Sequenzabweichungen, wie sie infolge genetischer Mutationen, Polymorphismen oder evolutionärer Divergenzen vorliegen können. So ist es beispielsweise möglich mit einer einzigen dsRNA, die ausgehend von einer bestimmten Speicherprotein Sequenz eines Organismus generiert wurde, die Expression weiterer homologer Speicherproteine des gleichen Organismus oder aber auch die Expression von Speicherproteinen in anderen verwandten Arten zu unterdrücken. Zu diesem Zweck umfasst die dsRNA bevorzugt Sequenz- bereich von Speicherprotein-Gentranskripten, die konservierten Bereichen der einzelnen Speicherproteinfamilien entsprechen. Besagte konservierte Bereiche können aus Sequenzvergleichen abgeleitet werden (vgl. Fig. la-7d) . Bevorzugt, enthält die dsRNA in ihrer "sense"-Ribonukleotidse- quenz mindestens eines der Sequenzmotive ausgewählt aus einer der oben definierten Gruppen von Sequenzmotiven I, II, III, IV, V, VI, VII, VIII, IX, X, XI oder XII oder den Sequenzmotiven mit der SEQ ID NO: .93 oder 129.A 100% sequence identity between dsRNA and the storage protein gene transcript is not absolutely necessary in order to effect an efficient reduction in the storage protein expression. The method is therefore tolerant of sequence deviations, such as those resulting from genetic mutations, polymorphisms or evolutionary divergences. For example, with a single dsRNA generated from a certain storage protein sequence of an organism, it is possible to suppress the expression of further homologous storage proteins of the same organism or else to suppress the expression of storage proteins in other related species. For this purpose, the 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). Preferably, 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.
Am meisten bevorzugt sind doppelsträngige RNA Moleküle beschrieben durch die Ribonukleinsäuresequenz gemäß SEQ ID NO: 106, 108 oder 110. In einer weiteren bevorzugten Ausführungsform enthält die dsRNA mehrere Sequenzabschnitte, die eine gleichzeitige Suppression mehrerer Speicherproteine, bevorzugt von Speicherproteinen aus verschiedenen Klassen - wie beispielsweise einem 2S-Albumin, 7S-Globuline, HS/12S-Globulin oder die Zein-Prolamine - bewirken. Dazu umfasst die dsRNA bevorzugtDouble-stranded RNA molecules are most preferably described by the ribonucleic acid sequence according to SEQ ID NO: 106, 108 or 110. In a further preferred embodiment, 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. For this purpose, the dsRNA preferably comprises
a) mindestens zwei "sense"-Ribonukleotidsequenzen, wobei jede dieser "sense"-Ribonukleotidsequenzen im wesentlichen identisch ist zu mindestens einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nuklein- säuresequenz gemäß 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 oder 173 und wobei zumindest zwei der Speicherprotein-Nukleinsäuresequenzen, zu deren "sense"-RNA-Transkript die besagte "sense"-Ribo- nukleotidsequenzen im wesentlichen identisch sind, untereinander eine Homologie von unter 90 %, bevorzugt unter 80 %, ganz besonders bevorzugt unter 60 % am meisten bevorzugt unter 50 % über die gesamte Länge ihrer kodierenden Nukleotidsequenz haben, unda) at least two “sense” ribonucleotide sequences, each of these “sense” ribonucleotide sequences being essentially identical 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, 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 and at least two of the storage protein nucleic acid sequences, for the "sense" RNA transcript of which the "sense" ribonucleotide sequences are essentially identical, with one another 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
b) "antisense"-Ribonukleotidsequenzen, die zu besagten "sense"-Ribonukleotidsequenzen unter a) im wesentlichen - bevorzugt vollständig - komplementären sind.b) “antisense” ribonucleotide sequences which are essentially — preferably completely — complementary to said “sense” ribonucleotide sequences under a).
Wie oben beschrieben, können die "sense"-Ribonukleotidsequen- zen und "antisense"-Ribonukleotidsequenzen als separate Moleküle oder - bevorzugt - als ein einzelnes, selbstkomplementäres RNA Molekül vorliegen, wobei im letzteren Fall die beiden Stränge bevorzugt über eine verbindende Sequenz ("Linker"), die ganz besonders bevorzugt ein Intron darstellt, miteinander verbunden sind.As described above, 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.
Am meisten bevorzugt sind doppelsträngige RNA Moleküle beschrieben durch die Ribonukleinsäuresequenz gemäß SEQ ID NO: 145 oder 147.Double-stranded RNA molecules are most preferably described by the ribonucleic acid sequence according to SEQ ID NO: 145 or 147.
Natürlich können, um den gleichen Zweck zu erreichen, auch mehrere individuelle dsRNA Moleküle, die jeweils einen der oben definierten Ribonukleotidεequenzabschnitte umfassen, in die Zelle oder den Organismus eingebracht werden. Die dsRNA kann aus einem oder mehr Strängen polymerisierter Ribonukleotide bestehen. Es können ferner Modifikationen sowohl des Zucker-Phosphat-Gerüstes als auch der Nukleoside vorliegen. Beispielsweise können die Phosphodiesterbindungen der natürlichen RNA dahingehend modifiziert sein, dass sie zumindest ein Stickstoff oder Schwefel-Heteroatom umfassen. Basen können dahingehend modifiziert werden, dass die Aktivität beispielsweise von Adenosindeaminase eingeschränkt wird. Solche und weitere Modifikationen sind weiter unten bei den Verfahren zur Stabilisierung von antisense-RNA beschrieben.Of course, in order to achieve the same purpose, several individual 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. There may also be 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.
Die dsRNA kann enzymatisch oder ganz oder teilweise chemischsynthetisch hergestellt werden.The dsRNA can be produced enzymatically or in whole or in part chemically and synthetically.
Die doppelsträngige dsRNA Struktur kann ausgehend von zwei komplementären, separaten RNA-Strängen oder - bevorzugt - ausgehend von einem einzelnen, selbstkomplementären RNA- Strang gebildet werden.The double-stranded dsRNA structure can be formed from two complementary, separate RNA strands or - preferably - from a single, self-complementary RNA strand.
Sollen die zwei separaten RNA-Stränge der dsRNA in einer Zelle oder Pflanze zusammengebracht werden, so kann dies auf verschiedene Art geschehen:If the two separate RNA strands of the dsRNA are to be brought together in a cell or plant, this can be done in different ways:
a) Transformation der Zelle oder Pflanze mit einem Vektor, der beide Expressionskassetten umfasst,a) transformation of the cell or plant with a vector which comprises both expression cassettes,
b) Kotransformation der Zelle oder Pflanze mit zwei Vektoren, wobei der eine die Expressionskassetten mit der "sense"-Ribonukleotidsequenz, der andere die Expressionskassetten mit der "antisense"-Ribonukleotidse- quenzen umfasst,b) Co-transformation of the cell or plant with two vectors, one comprising the expression cassettes with the “sense” ribonucleotide sequence, the other the expression cassettes with the “antisense” ribonucleotide sequences,
c) Kreuzung von zwei Pflanzen, die mit jeweils einem Vektor transformiert wurden, wobei der eine die Expressionskassetten mit der "sense"-Ribonukleotidsequenz, der andere die Expressionskassetten mit der "antisense"-Ribonu- kleotidsequenz umfasst.c) Crossing of two plants, each of which was transformed with a vector, one comprising the expression cassettes with the “sense” ribonucleotide sequence, the other the expression cassettes with the “antisense” ribonucleotide sequence.
In der bevorzugten Ausführungsform wird die dsRNA-Struktur durch einem einzelnen, selbstkomplementären RNA-Strang gebildet. Hier können "sense"- und "antisense"-Ribonukleotid- sequenzen durch eine verbindende Sequenz ("Linker") verknüpft sein und beispielsweise eine HaarnadelStruktur ausbilden. Bevorzugt ist die verbindende Sequenz ein Intron. Die Nukleinsäuresequenz kodierend für eine dsRNA kann weitere Elemente beinhalten, wie beispielsweise Transkriptions- terminationssignale oder Polyadenylierungssignale.In the preferred embodiment, 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.
Die Bildung der RNA Duplex kann entweder außerhalb der Zelle oder innerhalb derselben initiiert werden. Wie in WO 99/53050 kann die dsRNA auch eine HaarnadelStruktur umfassen, indem "sense"- und "antisense"-Strang durch einen "Linker" (beispielsweise ein Intron) verbunden werden. Die selbstkomplementären dsRNA-Strukturen sind bevorzugt, da sie lediglich die Expression eines Konstruktes erfordern und die komplementären Stränge stets in einem äqui olaren Verhältnis umfassen.The formation of the RNA duplex can be initiated either outside the cell or inside the cell. As in WO 99/53050, the dsRNA can also comprise a hairpin structure by connecting the “sense” and “antisense” strand by means of a “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.
Die Expressionskassetten kodierend für den "antisense"- oder "sense"-Strang einer dsRNA oder für den selbst- komplementären-Strang der dsRNA, werden bevorzugt in einen Vektor insertiert und mit den unten beschriebenen Verfahren stabil (beispielsweise unter Verwendung von Selektions- markern) in das Genom einer Pflanze insertiert, um eine dauerhafte Expression der dsRNA zu gewährleisten.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.
Die dsRNA kann unter Verwendung einer Menge eingeführt werden, die zumindest ein Kopie pro Zelle ermöglicht. Höhere Mengen (z.B. mindestens 5, 10, 100, 500 oder 1000 Kopien pro Zelle) können ggf. eine effizienter Verminderung bewirken.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.
Die dsRNA kann entweder in vivo oder in vitro synthetisiert werden. Dazu kann eine DNA-Sequenz kodierend für eine dsRNA in eine Expressionskassette unter Kontrolle mindestens eines genetischen Kontrollelementes (wie beispielsweise Promotor, Enhancer, Silencer, Splice-Donor oder -Akzeptor, Poly- adenylierungssignal) gebracht werden. Entsprechend vorteilhafte Konstruktionen sind weiter unten beschrieb. Eine Poly- adenylierung ist nicht erforderlich, ebenso müssen keine Elemente zur Initiierung einer Translation vorhanden sein.The dsRNA can be synthesized either in vivo or in vitro. For this purpose, 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). Correspondingly advantageous constructions are described below. A polyadenylation is not necessary, and there are no elements to initiate translation.
Eine dsRNA kann chemisch oder enzymatisch synthetisiert werden. Dazu können zellulare RNA Polymerasen oder Bakterio- phagen RNA Polymerasen (wie z.B. T3-, T7- oder SP6 RNA- Poly erase) verwendet werden. Entsprechende Verfahren zu in vitro Expression von RNA sind beschrieben (WO 97/32016; US 5,593,874; US 5,698,425, US 5,712,135, US 5,789,214, US 5,804,693) . Eine chemisch oder enzymatisch in vitro synthetisierte - dsRNA kann vor der Einführung in eine Zelle, Gewebe oder Organismus aus dem Reaktionsgemisch beispielsweise durch Extraktion, Präzipitation, Elektrophorese, Chromatographie oder Kombinationen dieser Verfahren ganz oder teilweise aufgereinigt werden. Die dsRNA kann unmittelbar in die Zelle eingeführt werden oder aber auch extrazellulär (z.B. in den interεtitialen Raum) appliziert werden.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 interεtitial space).
Bevorzugt wird die Pflanze jedoch stabil mit einem Expressionskonstrukt, das die Expreεεion der dεRNA realisiert, transformiert. Entsprechende Verfahren sind weiter unten beschrieben.However, the plant is preferably transformed stably with an expression construct that realizes the expression of the dεRNA. Corresponding methods are described below.
Einbringung einer Speicherprotein antisense-Nukleinsäure- sequenzIntroduction of a storage protein antisense nucleic acid sequence
Verfahren zur Suppression eineε bestimmten Proteinε durch Verhinderung der Akkumulation seiner mRNA durch die "antisenεe"-Technologie εind vielfach—- auch in Pflanzen - beεchrieben (Sheehy et al . (1988) Proc Natl Acad Sei USA 85: 8805-8809; US 4,801,340; Mol JN et al . (1990) FEBS Lett 268 (2) : 427-430) . Das antisenεe Nukleinsäure olekül hybridisiert bzw. bindet mit der zellulären mRNA und/oder genomischen DNA kodierend für das zu supprimierende Speicherprotein-Zielprotein. Dadurch wird die Transkription und/oder Translation deε Zielproteins unterdrückt. Die Hybridisierung kann auf konventionelle Art über die Bildung einer stabilen Duplex oder - im Fall von genomischer DNA - durch Bindung des antisense Nukleinsäuremoleküls mit der Duplex der genomischen DNA durch spezifische Wechselwirkung in der großen Furche der DNA-Helix entstehen.Methods for the suppression of a certain protein by preventing the accumulation of its mRNA by the "antiseise" technology are often described — including in plants (Sheehy et al. (1988) Proc Natl Acad Sei USA 85: 8805-8809; US 4,801,340; Mol JN et al. (1990) FEBS Lett 268 (2): 427-430). The antiseic nucleic acid olecule hybridizes or binds with the cellular mRNA and / or genomic DNA coding for the storage protein target protein to be suppressed. This suppresses the transcription and / or translation of the target protein. 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.
Eine antisenεe Nukleinsäuresequenz geeignet zur Verminderung eines Speicherproteins enthält einen "antisense"-RNA-Strang umfassend mindestens eine Ribonukleotidsequenz, die im wesentlichen komplementär ist zu mindestenε einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nukleinsäure- sequenz gemäß 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 oder 173.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.
"Im wesentlichen komplementär" meint, dass die antisense-RNA Sequenz auch Insertionen, Deletionen sowie einzelne Punktmutationen im Vergleich zu dem Komplement der Speicherprotein Zielεequenz aufweiεen kann. Bevorzugt beträgt die Homologie nach obiger Definition indeεtenε 75 %, bevorzugt mindestens 85 %, ganz besonders bevorzugt mindestens 95 %, am meisten bevorzugt 98 % zwischen dem "antisense"-RNA-Molekül und dem Komplement mindestenε eineε Teil des "sense"-RNA-Transkripteε einer Speicherprotein-Nukleinsäuresequenz . Das Komplement kann entεprechend den Basenpaarregeln von Watson und Crick in der dem Fachmann geläufigen Weiεe auε den entsprechenden - Sequenzen abgeleitet werden.“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 Transkripteε 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.
"Teil des "εense"-RNA-Tranεkripteε einer Speicherprotein- Nukleinsäuresequenz" meint Fragmente einer RNA oder mRNA transkribiert von einer für ein Speicherprotein kodierenden Nukleinsäure equenz , bevorzugt von einem Speicherprotein-Gen. Dabei haben die Fragmente bevorzugt eine Sequenzlänge von mindestens 20 Basen, bevorzugt mindestens 50 Basen, besonders bevorzugt mindestens 100 Basen, ganz besonderε bevorzugt mindestenε 200 Basen, am meisten bevorzugt mindestenε 500 Baεen. Umfasst ist auch die vollständige transkribierte RNA oder mRNA."Part of the" εense "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.
Die antisenεe Nukleinsäureεequenz kann zu der geεamten tranε- kribierten mRNA deε besagten Proteins komplementär sein, sich auf die kodierende Region beschränken oder nur aus einem Oligonukleotid bestehen, das zu einem Teil der kodierenden oder nicht-kodierenden Sequenz der mRNA komplementär ist. So kann das Oligonukleotid beispielsweise komplementär zu der Region sein, die den Tranεlationsstart für das besagte Protein umfasst. Antisense-Nukleinsäureεequenzen können eine Länge von zum Beispiel 5, 10, 15, 20, 25, 30, 35, 40, 45 oder 50 Nukleotide haben, können aber auch länger sein und mindestens 100, 200, 500, 1000, 2000 oder 5000 Nukleotide umfassen. Antisense-Nukleinsäuresequenzen können rekombinant exprimiert oder chemiεch bzw. enzymatisch unter Verwendung von dem Fachmann bekannten Verfahren synthetisiert werden. Bei der chemischen Syntheεe können natürlich oder modifizierte Nukleotide verwendet werden.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. For example, 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.
In einer weiteren bevorzugten Ausführungεform kann die Expreεεion eines Speicherproteins durch Nukleotidεequenzen inhibiert werden, die komplementär zu der regulatorischen Region eines Speicherprotein-Gens. (z.B. einem Speicherprotein Promoter und/oder Enhancer) sind und triple-helikale Strukturen mit der dortigen DNA-Doppelhelix auεbilden, εo dass die Transkription des Speicherprotein-Gens vermindert wird. Entsprechende Verfahren sind beschrieben (Helene CIn a further preferred embodiment, the expression of a storage protein 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. Appropriate processes are described (Helene C
(1991) Anticancer Drug Res 6 (6) : 569-84; Helene C et al .(1991) Anticancer Drug Res 6 (6): 569-84; Helene C et al.
(1992) Ann NY Acad Sei 660:27-36; Mäher LJ (1992) Bioassays 14(12) :807- 815) . In einer weiteren Ausführungsform kann das antisense Nuklein- säuremolekül eine α-anomere Nukleinsäure sein. Derartige α-anomere Nukleinsäuremoleküle bilden spezifische doppel- strängige Hybride mit komplementärer RNA in denen - im Unterschied zu den konventionellen ß-Nukleinsäuren - die beiden Stränge parallel zueinander verlaufen (Gautier C et al . (1987) Nucleic Acids Res 15:6625-6641). Das antisense Nukleinsäure olekül kann ferner auch 2 ' -O-Methylribo- nukleotide (Inoue et al. (1987) Nucleic Acids Reε 15:6131-6148) oder chimäre RNA-DNA Analoge beinhalten (Inoue et al. (1987) FEBS Lett 215:327-330).(1992) Ann NY Acad Sei 660: 27-36; Mower LJ (1992) Bioassays 14 (12): 807-815). In a further embodiment, the antisense nucleic acid molecule can be an α-anomeric nucleic acid. Such α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA in which - in contrast to the conventional β-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 Reε 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBS Lett 215 : 327-330).
Einbringung einer Speicherprotein antisenεe-Nukleinsäure- εequenz kombiniert mit einem RibozymIntroduction of a storage protein antiseise nucleic acid sequence combined with a ribozyme
Vorteilhaft kann die oben beschriebene antisense-Strategie mit einem Ribozym-Verfahren gekoppelt werden. Katalytische RNA-Moleküle oder Ribozyme können an jede beliebige Ziel-RNA angepasst werden und spalten das Phosphodiester-Gerüst an spezifischen Positionen, wodurch die Ziel-RNA funktioneil deaktiviert wird (Tanner NK (1999) FEMS Microbiol Rev 23(3) :257-275) . Das Ribozym wird dadurch nicht selber modifiziert, sondern ist in der Lage, weitere Ziel-RNA-Moleküle analog zu εpalten, wodurch eε die Eigenεchaften eineε Enzymε erhält. Der Einbau von Ribozymsequenzen in "antisense"-RNAs verleiht eben diesen "antisense"-RNAs diese enzymähnliche, RNA-spaltende Eigenεchaft und steigert so deren Effizienz bei der Inaktivierung der Ziel-RNA. Die Herεtellung und Verwendung entsprechender Ribozym-"antisenεe"-RNA-Moleküle iεt beispielsweise beschrieben bei Haseloff et al. (1988) Nature 334: 585-591.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. The incorporation of 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.
Auf diese Art können Ribozyme (z.B. "Hammerhead"-Ribozyme; Haεelhoff und Gerlach (1988) Nature 334:585-591) verwendet werden, um die mRNA eineε zu supprimierenden Enzyms - z.B. Speicherprotein - katalytisch zu spalten und die Translation zu verhindern. Die Ribozym-Technologie kann die Effizienz einer antisenεe-Strategie erhöhen. Verfahren zur Expreεsion von Ribozymen zur Verminderung bestimmter Proteine sind beschrieben in (EP 0 291 533, EP 0 321 201, EP 0 360 257). In pflanzlichen Zellen ist eine Ribozym-Expression ebenfalls beschrieben (Steinecke P et al . (1992) EMBO J 11 (4) : 1525-1530 ; de Feyter R et al. (1996) Mol Gen Genet. 250 (3) : 329-338) . Geeignete Zielsequenzen und Ribozyme können zum Beispiel wie bei "Steinecke P, Ribozymeε, Methods in Cell Biology 50, Galbraith et al . eds, Acade ic Press, Inc. (1995), S.449-460" beschrieben, durch Sekundärstrukturberechnungen von Ribozym- und Ziel-RNA sowie durch deren Interaktion bestimmt werden (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) . Beispielsweise können Derivate der Tetrahymena L-19 IVS RNA konstruiert werden, die komplementäre Bereiche zu der mRNA des zu supprimierenden Speicherprotein Proteins aufweisen (siehe auch US 4,987,071 und US 5,116,742) . Alternativ können solche Ribozyme auch über einen Selektionsprozess auε einer Bibliothek diverser Ribozyme identifiziert werden (Bartel D und Szostak JW (1993) Science 261:1411-1418).In this way, ribozymes (eg "Hammerhead"ribozymes; Haεelhoff and Gerlach (1988) Nature 334: 585-591) can be used to catalytically cleave the mRNA of an enzyme to be suppressed - eg storage protein - and to prevent translation. 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. (1996) Mol Gen Genet. 250 (3): 329- 338). 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). For example, derivatives of 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). Alternatively, such 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).
Einbringung einer Speicherprotein sense-Nukleinsäuresequenz zur Induktion eines KosuppressionIntroduction of a storage protein sense nucleic acid sequence to induce co-suppression
Die Expresεion einer Speicherprotein Nukleinεäureεequenz - oder eines teils derselben - in εense-Orientierung kann zu einer Kosuppression des entεprechenden homologen, endogenen Genε führen. Die Expression von εenεe-RNA mit Homologie zu einem endogenen Gen kann die Expreεsion desεelben vermindern oder auεεchalten , ähnlich wie eε für antisenεe Anεätze beεchrieben wurde (Jorgenεen 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. (1990) Plant Cell 2:291-99). Dabei kann daε eingeführte Konεtrukt daε zu vermindernde, homologe Gen ganz oder nur teilweise repräsentieren. Die Möglichkeit zur Translation ist nicht erforderlich. Die Anwendung dieser Technologie auf Pflanzen ist beispielsweise beschrieben bei Napoli et al . (1990) The Plant Cell 2: 279-289 und in US 5,034,323.The expression of a storage protein nucleic acid sequence - or a part thereof - in a sense orientation can lead to a co-suppression of the corresponding homologous, endogenous gene. The expression of εenεe 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 (Jorgenεen 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. (1990) Plant Cell 2: 291-99). The introduced construct can represent the homologous gene to be reduced in whole or in part. The possibility of translation is not necessary. The application of this technology to plants is described, for example, by Napoli et al. (1990) The Plant Cell 2: 279-289 and in US 5,034,323.
Eine "sense"-Nukleinsäuresequenz geeignet zur Verminderung eines Speicherproteins enthält einen "sense"-RNA-Strang umfasεend mindestens eine Ribonukleotidsequenz, die im weεentlichen identisch ist zu mindestens einem Teil des "senεe"-RNA-Tranεkript einer Speicherprotein-Nukleinεäure- sequenz gemäß 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 oder 173.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 "senεe" 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.
"Im wesentlichen identisch" meint, dass die sense-RNA Sequenz auch Inεertionen, Deletionen sowie einzelne Punktmutationen im Vergleich zu dem Komplement der Speicherprotein Zielsequenz aufweisen kann. Bevorzugt beträgt die Homologie nach obiger Definition mindeεtenε 65 %, bevorzugt mindeεtens 75 %, ganz besonders bevorzugt mindestens 90 %, am meisten bevorzugt 95 % zwischen dem "sense"-RNA-Molekül und dem "sense"-RNA-Transkript mindestens eines Teils einer Speicher- protein-Nukleinsäuresequenz .“Essentially identical” means that the sense 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.
"Teil deε "εenεe"-RNA-Tranεkriptes einer Speicherprotein- Nukleinsäureεequenz" meint Fragmente einer RNA oder mRNA tranεkribiert von einer für ein Speicherprotein kodierenden Nukleinsäuresequenz, bevorzugt von einem Speicherprotein-Gen. Dabei haben die Fragmente bevorzugt eine Sequenzlänge von mindestens 20 Basen, bevorzugt mindestens 50 Basen, besonders bevorzugt mindestens 100 Basen, ganz besonders bevorzugt mindestens 200 Basen, am meisten bevorzugt mindeεtens 500 Basen. Umfasst ist auch die vollständige transkribierte RNA oder mRNA."Part of the" εenεe "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.
Einbringung DNA-bindende Faktoren gegen Speicherprotein Gene oder RNAsIntroduction of DNA-binding factors against storage protein genes or RNAs
Eine Verminderung einer Speicherprotein Genexpression 'ist auch mit spezifischen DNA-bindenden Faktoren z.B. mit Faktoren vom Typus der Zinkfingertranskriptionsfaktoren möglich. Diese Faktoren lagern sich an die genomische Sequenz deε endogenen Zielgens, bevorzugt in den regulatorischen Bereichen, an und bewirken eine Repression des endogenen Genε . Die Verwendung eineε εolchen Verfahrens ermöglicht die Verminderung der Expresεion eines endogenen Speicherprotein Gens, ohne dass dessen Sequenz gentechnisch manipuliert werden muss . Entsprechende Verfahren zur Herstellung entsprechender Faktoren sind beschrieben (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 . (2000) Proc Natl Acad Sei USA 97 (4') : 1495-1500; Beerli RR et al . (2000) J Biol Chem 275 (42) :32617-32627; Segal DJ and Barbas CF 3rd. (2000) Curr Opin Chem Biol 4(1) : 34-39; Kang JS and Kim JS (2000) J Biol Chem 275(12) :8742-8748; Beerli RR et al . (1998) Proc Natl Acad Sei USA 95 (25) : 14628- 14633; Kim JS et al . (1997) Proc Natl Acad Sei USA 94(8):3616 -3620; Klug A (1999) J Mol Biol 293 (2) .215-218; Tsai SY et al . (1998) Adv Drug Deliv Rev 30(1-3) :23-31; Mapp AK et al . (2000) Proc Natl Acad Sei USA 97(8) :3930-3935; Sharrockε AD et al . (1997) Int J Biochem Cell Biol 29 (12) : 1371-1387 ; Zhang L et al . (2000) J Biol Chem 275(43) :33850-33860) .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. (2000) Proc Natl Acad Sei USA 97 (4 ' ): 1495-1500; Beerli RR et al. (2000) J Biol Chem 275 (42): 32617-32627; Segal DJ and Barbas CF 3rd. ( 2000) Curr Opin Chem Biol 4 (1): 34-39; Kang JS and Kim JS (2000) J Biol Chem 275 (12): 8742-8748; Beerli RR et al. (1998) Proc Natl Acad Sei USA 95 ( 25): 14628-14633; Kim JS et al. (1997) Proc Natl Acad Sei USA 94 (8): 3616-3620; Klug A (1999) J Mol Biol 293 (2) .215-218; Tsai SY et al . (1998) Adv Drug Deliv Rev 30 (1-3): 23-31; Mapp AK et al. (2000) Proc Natl Acad Sei USA 97 (8): 3930-3935; Sharrockε AD et al. (1997) Int J Biochem Cell Biol 29 (12): 1371-1387; Zhang L et al. (2000) J Biol Chem 275 (43): 33850-33860).
Die Selektion dieεer Faktoren kann unter Verwendung eines beliebigen Stückes eines Speicherprotein-Gens erfolgen. Bevorzugt liegt dieser Abschnitt in einem innerhalb der Speicherproteine konεervierten Sequenzbereichen oder im Bereich der Promotorregion. Für eine Genunterdrückung kann er aber auch im Bereich der kodierenden Exons oder Introns liegen.These factors can be selected using any piece of a storage protein gene. 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.
Die DNA-bindenden Faktoren können beiεpielsweise gegen Sequenzen gerichtet sein, die in verschiedenen Speicherprotein-Genen konserviert vorliegen. Bevorzugt enthält ist der DNA-bindende Faktor gegen ein Sequenzmotive ausgewählt aus einer der oben definierten Gruppen von Sequenzmotiven I, II, III, IV, V, VI, VII, VIII, IX, X, XI oder XII oder den Sequenzmotiven mit der SEQ ID NO: 93 oder 129 gerichtet. Ferner können aber auch Sequenzen im Promotorbereich genutzt werden, die bei vielen Speicherproteinen auftreten. Beispielhaft jedoch nicht einschränkend seinen nachfolgende Sequenzen zu nennen: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. Furthermore, 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:
i) 5 ' -CATGCATG-3 ' (SEQ ID NO: 130)i) 5 '-CATGCATG-3' (SEQ ID NO: 130)
ii) 5 ' -GCCACYTC-3 ' (SEQ ID NO: 131)ii) 5 '-GCCACYTC-3' (SEQ ID NO: 131)
Weitere geeignete Abschnitte sind für den Fachmann mittels Datenbankabfrage aus der Genbank oder - ausgehend von einer Speicherprotein cDNA, deren Gen nicht in der Genbank vorhanden iεt, durch Durchmusterung einer genomischen Bibliothek nach korreεpondierenden genomischen Klonen erhältlich.Further suitable sections are available to the person skilled in the art by means of a database query from the gene bank or - starting from a storage protein cDNA, the gene of which is not present in the gene bank, by screening a genomic library for corresponding genomic clones.
Die Genexpresεion kann auch durch maßgeschneiderte, niedermolekulare εynthetiεche Verbindungen unterdrückt werden, bei- εpielsweiεe vom Polyamid-Typ (Dervan PB und Bürli RW (1999) Current Opinion in Chemical Biology 3:688-693; Gottesfeld JM et al. (2000) Gene Expr 9 (1-2) .-77-91) . Dieεe Oligomere bestehen auε den Bauεteinen 3- (Dimethylamino)propylamin, N-Methyl-3-hydroxypyrrol, N-Methylimidazol und N-Methyl- pyrrole und können an jedeε Stück doppelεträngiger DNA so angepaεεt werden, daεε εie εequenzspezifisch in die große Furche binden und die Expresεion der dortigen Genεequenzen blockieren. Entεprechende verfahren εind beschrieben (siehe unter anderem Bremer RE et al . • (2001) Bioorg Med Chem. 9(8) :2093-103; Ansari AZ et al . (2001) Chem Biol. 8(6):583-92; Gottesfeld JM et al . (2001) J Mol Biol 309(3) : 615-29; Wurtz NR et al . (2001) Org Lett 3(8) : 1201-3; Wang CC et al . (2001) Bioorg Med Chem 9(3):653-7; Urbach AR und Dervan PB (2001) Proc Natl Acad Sei USA 98 (8) :4343-8 ; Chiang SY et al . (2000) J Biol Chem 275 (32) : 24246-54) . g) Einbringung von den Speicherprotein RNA-Abbau bewirkende virale Nukleinsäuresequenzen und ExpressionskonεtruktenThe 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. • (2001) Bioorg Med Chem. 9 (8): 2093-103; Ansari AZ et al. (2001) Chem Biol. 8 (6): 583-92; Gottesfeld JM et al. (2001) J Mol Biol 309 (3): 615-29; Wurtz NR et al. (2001) Org Lett 3 (8): 1201-3; Wang CC et al. (2001) Bioorg Med Chem 9 (3): 653-7; Urbach AR and Dervan PB (2001) Proc Natl Acad Sei USA 98 (8): 4343-8; Chiang SY et al. (2000) J Biol Chem 275 (32): 24246-54 ). g) Introduction of viral nucleic acid sequences and expression constructs causing the degradation of RNA in the storage protein
Die Speicherprotein Expression kann effektiv auch durch Induktion des εpezifischen Speicherprotein RNA-Abbaus durch die Pflanze mit Hilfe eines viralen Expresεionεεyεtems (Amplikon) (Angell SM et al . (1999) Plant J 20 (3) :357-362) realisiert werden. Diese Systeme - auch als "VIGS" (viral induced gene silencing) bezeichnet - bringen Nuklein- εäureεequenzen mit Homologie zu den zu supprimierenden Transkripten- mittels viraler Vektoren in die Pflanze ein. Die Transkription wird sodann - vermutlich vermittelt durch pflanzliche Abwehrmechaniεmen gegen Viren - abgeεchaltet . Vermutlich εind die zugrunde liegenden Mechaniεmen denen der durch doppelεträngige RNA bewirkten Effekten ähnlich. Entsprechende Techniken und Verfahren sind beschrieben (Ratcliff F et al. (2001) Plant J 25 (2) : 237-45 ; Fagard M und Vaucheret H (2000) Plant Mol Biol 43 (2-3) : 285-93 ; Anandalakεhmi R et al. (1998) Proc Natl Acad Sei USA 95 (22) : 13079-84; Ruiz MT (1998) Plant Cell 10(6): 937-46). Für die Auεwahl der entsprechenden Sequenzen gelten die gleichen Regeln wie für die Bestimmung des "sense"-Strangeε bei doppelεträngiger RNA oder der Coεuppreεε'ion (ε.o.).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). These systems - also known as "VIGS" (viral induced gene silencing) - introduce nucleic acid sequences with homology to the transcripts to be suppressed into the plant by means of viral vectors. The transcription is then switched off, presumably mediated by plant defense mechanisms against viruses. The underlying mechanisms are presumably similar to those of the effects brought about by double-stranded RNA. Appropriate techniques and processes are described (Ratcliff F et al. (2001) Plant J 25 (2): 237-45; Fagard M and Vaucheret H (2000) Plant Mol Biol 43 (2-3): 285-93; Anandalakεhmi R et al. (1998) Proc Natl Acad Sei USA 95 (22): 13079-84; Ruiz MT (1998) Plant Cell 10 (6): 937-46). Auεwahl for the corresponding sequences according to the same rules as for the determination of the "sense" RNA -Strangeε at doppelεträngiger or Coεuppreεε 'ion (ε.o.) apply.
Entsprechend geeignete Systeme zur Suppreεεion der Genexpreε- εion unter Verwendung viraler Expressionssyεteme sind beispielsweise beschrieben in WO 99/15682 und WO 98/36083.Appropriate systems for suppressing gene expression using viral expression systems are described, for example, in WO 99/15682 and WO 98/36083.
h) Einbringung von Konstrukten zur Induktion einer homologen Rekombination an endogenen Speicherprotein-Genen beiεpielε- weiεe zur Erzeugung von Knockout-Mutanten.h) Introduction of constructs for inducing a homologous recombination on endogenous storage protein genes, for example for generating knockout mutants.
Zur Herstellung eines homolog rekombinanten Organismus mit verminderter Speicherprotein-Aktivität verwendet man beispielsweise ein Nukleinsäurekonstrukt, daε zumindest einen Teil eines endogenen Speicherprotein Gens enthält, daε durch eine Deletion, Addition oder Subεtitution mindeεtenε eines Nukleotids so verändert wird, so dass die Funktionalität vermindert oder gänzlich aufgehoben wird. Die Veränderung kann auch die regulativen Elemente (z.B. den Promotor) deε Genε betreffen, so dass die kodierende Sequenz unverändert bleibt, eine Expression (Transkription und/oder Translation) jedoch unterbleibt und vermindert wird.To produce a homologous recombinant organism with reduced storage protein activity, use is made, for example, of a 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.
Bei der konventionellen homologen Rekombination ist die veränderte Region an ihrem 5'- und 3 '-Ende von weiteren Nuklein- εäureεequenzen flankiert, die eine ausreichende Länge für die Ermöglichung der Rekombination aufweisen müsεen. Die Länge liegt in der Regel in einem Bereich von mehreren einhundert Basen bis zu mehreren Kilobaεen (Thomaε KR und Capecchi MR (1987) Cell 51:503; Strepp et al . (1998) Proc Natl Acad Sei USA 95(8) :4368-4373) . Für die homologe Rekombination wird der WirtsOrganismus - zum Beispiel eine Pflanze - mit dem Rekombinationskonεtrukt unter Verwendung der unten beschriebenen Verfahren transformiert und erfolgreich rekombinierte Klone unter Verwendung zum Beispiel einer Antibiotika- oder Herbizidresiεtenz selektioniert .In conventional homologous recombination, 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 (Thomaε KR and Capecchi MR (1987) Cell 51: 503; Strepp et al. (1998) Proc Natl Acad Sei USA 95 (8): 4368-4373) , For the homologous recombination, 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.
Homologe Rekombination ist ein relativ selteneε Ereignis in höheren Eukaryoten, vor allem in Pflanzen. Zufällige Integrationen in das Wirtsgenom überwiegen. Eine Möglichkeit die zufällig integrierten Sequenzen zu entfernen und so Zeilklone mit einer korrekten homologen Rekombination anzureichern, besteht in der Verwendung eines sequenzspezifischen Rekombinationsεystems wie in US 6,110,736 beschrieben, durch welche unspezifiεch integrierte Sequenzen wieder deletiert werden können, was die Selektion erfolgreich über homologe Rekombination integrierter Ereignisεe erleichtert. Eine Vielzahl von εequenzspezifischen Rekombi- nationssyεte en kann verwendet werden, beiεpielhaft εind das Cre/lox-System deε Bacteriophagen Pl, daε FLP/FRT System der Hefe, die Gin Rekombinase deε Mu Phagen, die Pin Rekombinase aus E. coli und das R/RS System des pSRl Plasmids genannt. Bevorzugt εind daε Bacteriophagen Pl Cre/lox und das Hefe FLP/FRT System. Das FLP/FRT und cre/lox Rekombinaseεyεtem wurde bereits in pflanzlichen Systemen angewendet (Odell et al. (1990) Mol Gen Genet 223: 369-378)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)
Einführung von Mutationen in endogenen Speicherprotein Gene zur Erzeugung eineε Funktionεverlustes (z.B. Generierung von Stopp-Kodons , Verschiebungen im Leseraster etc.)Introduction of mutations in endogenous storage protein genes to produce a loss of function (e.g. generation of stop codons, shifts in the reading frame, etc.)
Weitere geeignete Methoden zur Verminderung der Speicherprotein-Aktivität εind die Einführung von Nonεenεe-Mutationen in endogene Speicherprotein Gene zum Beiεpiel mittels Einführung von RNA/DNA-Oligonukleotiden in die Pflanze (Zhu et al. (2000) Nat Biotechnol 18 (5) : 555-558) sowie die Generierung von Knockout-Mutanten mit Hilfe von z.B. T-DNA-Mutagenese (Koncz et al . (1992) Plant Mol Biol 20(5) :963-976) , ENU- (N-Ethyl-N-nitrosoharnstoff) - Mutagenese oder homoiger Rekombination (Hohn B und Puchta (1999) H Proc Natl Acad Sei USA 96:8321-8323.). Punktmutationen können auch mittels DNA-RNA Hybriden erzeugt werden, die auch als "chimeraplasty" bekannt sind (Cole-Strauss et al . (1999) Nucl Acidε Reε 27 (5) : 1323-1330; K iec (1999) Gene therapy American Scientiεt 87 (3) :240-247) .Other suitable methods for reducing the storage protein activity are the introduction of nonseen mutations into endogenous storage protein genes, for example by introducing RNA / DNA oligonucleotides into the plant (Zhu et al. (2000) Nat Biotechnol 18 (5): 555- 558) and the generation of 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 Acidε Reε 27 (5): 1323-1330; K iec (1999) Gene therapy American Science 87 (3): 240-247).
Die Methoden der dεRNAi , der Kosuppression mittels senεe-RNA und der "VIGS" ("viruε induced gene silencing") werden auch als "post-transcriptional gene silencing" (PTGS) bezeichnet. PTGS- Verfahren sind besonders vorteilhaft, weil die Anforderungen an die Homologie zwischen dem zu supprimierenden endogenem Gen und der tranεgen exprimierten εenεe- oder dsRNA-Nukleinsäuresequenz (bzw. zwischen dem endogenen Gen und seiner dominant-negativen Variante) geringer sind alε beiεpielsweise bei einem klasεischen antisenεe-Anεatz . Entsprechende Homologie-Kriterien εind bei der Beεchreibung deε dεRNAI-Verfahrens genannt und allgemein für PTGS-Verfahren oder dominant-negative Ansätze übertragbar. Aufgrund der hohen Homologie zwischen den pflanzlichen Speicherproteinen (s. Fig la bis 7d) kann man voraussichtlich unter Verwendung einer bestimmten Speicherprotein-Nukleinsäuresequenzen auch die Expression von homologen Speicherproteinen in' der gleichen oder anderen Arten effektiv supprimieren, ohne dasε die Iεolierung, Strukturaufklärung und Konεtruktion entεprechender Suppreεεionεkonstrukte für dort vorkommenden Speicherprotein- Homologen zwingend erforderlich wäre. Dies erleichtert erheblich den Arbeitsaufwand.The methods of dεRNAi, cosuppression using senεe RNA and "VIGS"("viruε induced gene silencing") are also referred to as "post-transcriptional gene silencing" (PTGS). PTGS methods are particularly advantageous because the requirements on the homology between the endogenous gene to be suppressed and the transgenically expressed εenεe or dsRNA nucleic acid sequence (or between the endogenous gene and its dominant-negative variant) are lower than, for example, in the case of a classic one antiseise approach. Corresponding homology criteria are mentioned in the description of the dRNAI method and are generally transferable for PTGS methods or dominant-negative approaches. Due to the high homology between the plant storage proteins (see Fig. 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 dasε the Iεolierung, structure elucidation and Konεtruktion Corresponding suppression constructs for storage protein homologs occurring there would be absolutely necessary. This greatly simplifies the workload.
Alle Subεtanzen und Verbindungen die direkt oder indirekt eine Verminderung der Proteinmenge, RNA-Menge oder Genaktivität zumindest eines Speicherproteins bewirken, und so direkt oder indirekt eine Verminderung der Proteinmenge zumindest eines Speicherproteinε bewirken, εeien infolge unter der Bezeichnung "anti-SP"-Verbindungen zuεammengefasst. Der Begriff "anti-SP"-Verbindung εchließt explizit die in den oben beεchriebenen Verfahren zum Einεatz kommenden Nuklein- εäuresequenzen, Peptide, Proteine oder andere Faktoren ein.All substances and 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.
"Einbringung" umfaεεt im Rahmen der Erfindung alle Verfahren, die dazu geeignet eine "anti-SP"-Verbindung, direkt oder indirekt, in eine Pflanze oder eine Zelle, Kompartiment, Gewebe, Organ oder Samen derεelben einzuführen oder dort zu generieren. Direkte und indirekte Verfahren εind umfaεεt. Die Einbringung kann zu einer vorübergehenden (tranεienten) Präεenz einer "anti-SP"-Verbindung (beispielsweise einer dsRNA) führen oder aber auch zu einer dauerhaften (εtabilen) .In the context of the invention, "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.
Gemäß der unterschiedlichen Natur der oben beschriebenen Ansätze kann die "anti-SP"-Verbindung ihre Funktion direkt ausüben (zum Beispiel durch Insertion in ein endogenes Speicherprotein Gen) . Die Funktion kann aber auch indirekt nach Transkription in eine RNA (zum Beispiel bei antisense Ansätzen) oder nach Transkription und Translation in ein Protein (zum Beispiel bei Bindungsfaktoren) ausgeübt werden. Sowohl direkte als auch indirekt wirkende "anti-SP"-Verbindungen sind erfindungsgemäß umfasst.According to the different nature of the approaches described above, 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.
Einführen umfasεt beispielsweiεe Verfahren wie Tranεfektion, Tranεduktion oder Tranεformation.Introducing includes, for example, methods such as transfection, transduction or transformation.
"anti-SP" Verbindungen umfaεεt εomit beiεpielsweiεe auch rekombinante Expreεεionεkonεtrukte, die eine Expression (d.h. Transkription und ggf. Translation) beispielsweiεe einer Speicherprotein-dsRNA oder einer Speicherprotein "antisense"- RNA - bevorzugt in einer Pflanze oder einem Teil, Gewebe, Organ oder Samen derselben - bedingen."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.
In besagten Expressionskonεtrukten steht ein Nukleinsäuremolekül, deεεen Expreεsion (Transkription und ggf. Translation) eine "anti-SP"-Verbindung generiert, bevorzugt in funktioneller Verknüpfung mit mindestenε einem genetiεchen Kontrollelement (beiεpielsweise einem Promotor) , das eine Expression in einem Organismus, bevorzugt in Pflanzen, gewährleiεtet . Soll das Expresεionskonstrukt direkt in die Pflanze eingeführt und die "anti-SP"-Verbindung (beispielsweise die Speicherprotein dsRNA) dort in plantae erzeugt werden, so εind pflanzenspezifiεche genetische Kontrollelemente (beispielsweise Promotoren) bevorzugt. Die "anti-SP"-Verbindung kann jedoch auch in anderen Organismen oder in vitro erzeugt und dann in die Pflanze eingebracht werden. In diesem sind all prokaryotisehen oder eukaryotisehen genetischen Kontrollelemente (beispielsweise Promotoren) bevorzugt, die die Expression in den jeweils für die Herstellung gewählten Organiεmus erlauben.In said expression constructs there is a 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. If the expression construct is to be introduced directly into the plant and the “anti-SP” compound (for example the storage protein dsRNA) is to be generated there in plantae, plant-specific genetic control elements (for example promoters) are preferred. However, 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.
Unter einer funktionellen Verknüpfung versteht man zum Beispiel die εequentielle Anordnung eines Promotorε mit der zu expri ierenden Nukleinsäuresequenz (zum Beispiel einer "anti-SP"-Verbindung) und ggf. weiterer regulativer Elemente wie zum Beispiel einem Terminator derart, dasε jedes der regulativen Elemente seine Funktion bei der transgenen Expression der Nukleinsäureεequenz, je nach Anordnung der Nukleinsäure- εequenzen zu senεe oder anti-sense RNA, erfüllen kann. Dazu ist nicht unbedingt eine direkte Verknüpfung im chemischen Sinne erforderlich. Genetische Kontrollsequenzen, wie zum Beispiel Enhancer-Sequenzen, können ihre Funktion auch von weiter entfernten Positionen oder gar von anderen DNA-Molekülen aus auf die Zielsequenz ausüben. Bevorzugt sind Anordnungen, in denen die tranεgen zu exprimierende Nukleinsäuresequenz hinter der als Promoter fungierenden Sequenz positioniert wird, so dass beide Sequenzen kovalent miteinander verbunden εind. Bevorzugt ist dabei der Abstand zwiεchen der Promotorεequenz und der tranεgen zu exprimierende Nukleinεäureεequenz geringer alε 200 Baεenpaare, beεonderε bevorzugt kleiner als 100 Baεenpaare, ganz besonders bevorzugt kleiner als 50 Baεenpaare.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. Preferably, 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.
Die Herεtellung einer funktionellen Verknüpfung alε auch die Herεtellung einer Expreεεionεkaεεette kann mittelε gängiger Rekombinations- und Klönierungstechniken realiεiert werden, wie εie beispielsweise in Maniatis T, Fritsch EF und Sambrook JThe production of a functional link as well as the production of an expression cassette can be realized using common recombination and cloning techniques, such as those in Maniatis T, Fritsch EF and Sambrook J
(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Silhavy TJ, Berman ML und Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Ausubel FM et al .(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Ausubel FM et al.
(1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience und bei Gelvin et al . (1990) In: Plant Molecular" Bi'ology Manual beεchrieben sind. Zwischen beide Sequenzen können aber auch weitere Sequenzen positioniert werden, die zum Beispiel die Funktion eines Linkers mit beεtimmten Restriktionsenzymεchnittεtellen oder eines Signalpeptides haben. Auch kann die Insertion von Sequenzen zur Expresεion von Fuεionε- proteinen führen. Bevorzugt kann die Expreεεionskaεεette, be- εtehend auε einer Verknüpfung von Promoter und zu exprimierender Nukleinsäuresequenz, integriert in einem Vektor vorliegen und durch zum Beispiel Transformation in ein pflanzlicheε Genom insertiert werden.(1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and Gelvin et al. (1990) In: Plant Molecular " Bi'ology Manual. Other sequences can also be positioned between the two sequences, which, for example, have the function of a linker with specific restriction enzyme interfaces or a signal peptide. The insertion of 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.
Unter einer Expresεionεkaεεette sind aber auch solche Konstruktionen zu verstehen, bei denen ein Promoter - zum Beispiel durch eine homologe Rekombination - hinter ein endogenes Speicherprotein-Gen platziert wird, und durch Expression einer antisenεe Speicherprotein-RNA die erfindungεgemäße Verminderung eineε Speicherproteinε bewirkt wird. Analog kann auch eine "anti-SP" Verbindung (zum Beispiel eine Nukleinsäuresequenz kodierend für eines Speicherprotein dεRNA oder eine Speicherprotein antisense RNA) derart hinter einen endogenen Promotor platziert werden, dass der gleiche Effekt auftritt. Beide Ansätze führen zu Expreεsionskaεεetten im Sinne der Erfindung.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. Analogously, an "anti-SP" compound (for example a nucleic acid sequence coding for a storage protein dεRNA or a storage protein antisense RNA) can also be placed behind an endogenous promoter in such a way that the same effect occurs. Both approaches lead to expression cassettes in the sense of the invention.
Pflanzenεpezifiεche Promotoren meint grundsätzlich jeden Promotor, der die Expression von Genen, insbesondere Fremdgenen, in Pflanzen oder Pflanzenteilen, -zellen, -geweben, -kulturen steuern kann. Dabei kann die Expresεion beiεpielεweise konstitu- tiv, induzierbar oder entwicklungεabhängig sein.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.
Bevorzugt sind: a) Konstitutive PromotorenPreferred are: a) Constitutive promoters
"Konstitutive" Promotoren meint solche Promotoren, die eine Expression in zahlreichen, bevorzugt allen, Geweben über einen größeren Zeitraum der Pflanzenentwicklung, bevorzugt zu allen Zeitpunkten der Pflanzenentwicklung, gewährleisten“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
(Benfey et al.(1989) EMBO J 8:2195-2202). Vorzugsweise verwendet man insbesondere einen pflanzlichen Promotor oder einen Promotor, der einem Pflanzenvirus entεtammt. Ins- besondere bevorzugt ist der Promotor deε 35S-Tranεkriptes des CaMV Blumenkohlmosaikviruε (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) oder der 19S CaMV Promotor (US 5,352,605; WO 84/02913; Benfey et al . (1989) EMBO J 8:2195-2202). Ein weiterer geeigneter konεtitutiver Promotor iεt der "Rubisco small subunit (SSU) "-Promotor (US 4,962,028), der LeguminB-Promotor (GenBank Acc.-Nr. X03677) , der Promotor der .Nopalinsynthase auε Agrobacterium, der TR- Doppelpromotor, der OCS (Octopin Synthase) Promotor aus Agrobacterium, der Ubiquitin Promotor (Holtorf S et al . (1995) Plant Mol Biol "29 : 637-649) , den Ubiquitin 1 Promotor (Chri- stensen et al . (1992) Plant Mol Biol 18:675-689; Bruce et al .(Benfey et al. (1989) EMBO J 8: 2195-2202). 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). Another suitable constitutive promoter is the "Rubisco small subunit (SSU)" promoter (US 4,962,028), the LeguminB promoter (GenBank Acc. No. X03677), the promoter of .Nopalinsynthase from Agrobacterium, the TR double promoter, the OCS (Octopin Synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf 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.
(1989) Proc Natl Acad Sei USA 86:9692-9696), den Smas Promotor, den Cinnamylalkoholdehydrogenaεe-Promotor (US 5,683,439), die Promotoren der vakuolärer ATPase Untereinheiten oder der Promotor eineε prolinreichen Proteinε auε Weizen(1989) Proc Natl Acad Sei USA 86: 9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (US 5,683,439), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat
(WO 91/13991) , εowie weitere Promotoren von Genen, deren kon- εtitutive Expression in Pflanzen dem Fachmann bekannt ist.(WO 91/13991), as well as further promoters of genes, the connective expression of which in plants is known to the person skilled in the art.
b) Gewebespezifiεche Promotorenb) tissue-specific promoters
Bevorzugt εind ferner Promotoren mit Spezifitäten für Samen, wie zum Beiεpiel der Promotor des Phaseolins (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9) : 839-53) , des 2S Albumingens (Joεeffεon LG et al . (1987) J Biol Chem 262:12196-12201), deε Leguminε (Shirεat A et al . (1989) Mol Gen Genet 215(2): 326-331), deε USP (unknown seed protein; Bäumiein H et al . (1991) Mol Gen Genet 22'5 (3 ): 459-67) , des Napin Gens (US 5,608,152; Stalberg K et al . (1996) L Planta 199:515-519), des Saccharosebindeproteins (WO 00/26388) oder der Legumin B4-Promotor (LeB4; Bäumlein H et al . (1991) Mol Gen Genet 225: 121-128; Baeumlein et al . (1992) Plant Journal 2(2):233-9; Fiedler ü et al . (1995) Biotechnology (NY) 13 (10) :1090f) , der Oleosin-Promoter aus Arabidopsis (WO 98/45461) , der Bce4-Promoter aus Brassica (WO 91/13980) . Weitere geeignete samenspezifische Promotoren sind die der Gene kodierend für das "High Molecular Weight Glutenin" (HMWG) , Gliadin, Verzweigungsenzym, ADP Glucose Pyro- phosphatase (AGPase) oder die Stärkesynthase . Bevorzugt sind ferner Promotoren, die eine samenspezifische Expression in Monokotyledonen wie Mais, Gerste, Weizen, Roggen, Reis etc. erlauben. Vorteilhaft eingesetzt werden können der Promoter des lpt2 oder lptl-Gen (WO 95/15389, WO 95/23230) oder die Promotoren beschrieben in WO 99/16890 (Promotoren des Hordein-Gens, des Glutelin-Gens , des Oryzin-Genε, des Prola in-Gens , des Gliadin-Gens, des Glutelin-Gens, des Zein-Gens., des Kaεirin-Gens oder des Secalin-Gens) .Also preferred are 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 (Joεeffεon LG et al. ( 1987) J Biol Chem 262: 12196-12201), deε Leguminε (Shirεat A et al. (1989) Mol Gen Genet 215 (2): 326-331), deε 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. (1996) L Planta 199: 515-519), the sucrose binding protein (WO 00/26388) or the Legumin B4 promoter (LeB4; Bäumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baeumlein et al. (1992) Plant Journal 2 (2): 233-9; Fiedler ü et al. (1995) Biotechnology (NY) 13 (10): 1090f), the oleosin promoter from Arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980). Other suitable 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. 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).
c) Chemiεch induzierbare Promotorenc) Chemically inducible promoters
Die Expreεεionεkasεetten können auch einen chemiεch induzierbaren Promotor enthalten (Überεichtsartikel : Gatz et al .The expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al.
(1997) A nu Rev Plant Physiol Plant Mol Biol 48:89-108), durch den die Expresεion deε exogenen Genε in der Pflanze zu einem bestimmten Zeitpunkt geεteuert werden kann. Derartige Promotoren, wie z.B. der PRP1 Promotor (Ward et al . (1993) Plant Mol Biol 22:361-366), durch Salicylsäure induzierbarer Promotor (WO 95/19443), ein durch Benzolsulfonamid-induzier- barer Promotor (EP 0 388 186) , ein durch Tetrazyklin- induzierbarer Promotor (Gatz et al . (1992) Plant J 2:397-404), ein durch Abscisinsäure induzierbarer Promotor(1997) A nu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a specific point in time. Such 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
(EP 0 335 528) bzw. ein durch Ethanol- oder Cyclo exanon- induzierbarer Promotor (WO 93/21334) können ebenfallε verwendet werden.(EP 0 335 528) or a promoter inducible by ethanol or cyclo exanone (WO 93/21334) can also be used.
Besonders bevorzugt sind konstitutive sowie sa enεpezifiεche Promotoren.Constitutive and seed-specific promoters are particularly preferred.
Eε können ferner weitere Promotoren funktioneil mit der zu expri ierenden Nukleinsäuresequenz verknüpft sein, die eine Expresεion in weiteren Pflanzengeweben oder in anderen Organismen, wie zum Beispiel E. coli Bakterien ermöglichen. Als Pflanzen Promotoren kommen im Prinzip alle oben beschriebenen Promotoren in Frage .Furthermore, other 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. In principle, all promoters described above can be used as plant promoters.
Die in den erfindungsgemäßen Expressionεkasεetten oder Vektoren enthaltenen Nukleinsäuresequenzen können mit weiteren genetischen Kontrollsequenzen neben einem Promoter funktioneil verknüpft sein. Der Begriff der genetischen Kontrollsequenzen ist breit zu verstehen und meint all solche Sequenzen, die einen Ein- fluεε auf das Zustandekommen oder die Funktion der erfindungsgemäßen Expresεionεkaεεette haben. Genetiεche Kontrollεequenzen modifizieren zum Beispiel die Transkription und Tranεlation in prokaryotiεchen oder eukaryotiεchen Organiεmen. Vorzugsweise umfasεen die erfindungεgemäßen Expreεεionskassetten 5 '-stromaufwärts von der jeweiligen transgen zu exprimierenden Nuklein- säuresequenz einen pflanzenspezifischen Promoter und 3 '-stromabwärts eine Terminatorsequenz als zusätzliche genetische Kontroll- εequenz, sowie gegebenenfalls weitere übliche regulative Elemente, und zwar jeweils funktioneil verknüpft mit der transgen zu exprimierenden Nukleinsäureεequenz .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.
Genetische Kontrollsequenzen umfassen auch weitere Promotoren, Promotorelemente oder Minimalpromotoren, die die expression- εteuernden Eigenschaften modifizieren können. So kann durch genetische Kontrollsequenzen zum Beispiel die gewebespezifiεche Expreεsion zusätzlich abhängig von bestimmten Stressfaktoren erfolgen. Entsprechende Elemente εind zum Beiεpiel für Wasser- stresε, Abεciεinεäure (Lam. E und Chua NH, J Biol Chem 1991; 266(26): 17131 -17135) und Hitzestress (Schoffl F et al . (1989) Mol Gen Genetics 217 (2-3) :246-53) beεchrieben.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).
Weitere vorteilhafte Kontrollsequenzen sind beispielsweise in den gram-positiven Promotoren amy und SP02 , in den Hefe- oder Pilzpromotoren ADC1, MFa, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH.Further advantageous 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.
Prinzipiell können alle natürlichen Promotoren mit ihren Regulationsεequenzen wie die oben genannten für daε erfindungε- gemäße Verfahren verwendet werden. Darüberhinauε können auch synthetische Promotoren vorteilhaft verwendet werden.In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used advantageously.
Genetische Kontrollsequenzen umfaεεen ferner auch die 5'-untranε- latierte Regionen, Intronε oder nichtkodierende 3 '-Region von Genen wie beipielεweise das Actin-1 Intron, oder die Adhl-S Introns 1, 2 und 6 (allgemein: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). Es ist gezeigt worden, daεs dieεe eine signifikante Funktionen bei der Regulation der Genexpreεεion εpielen können. So wurde gezeigt, daεs 5 '-untranslatierte Sequenzen die transiente Expression heterologer Gene verstärken können. Beispielhaft für Translationsverstärker sei die 5 ' -Leadersequenz aus dem Tabak- Mosaik-Virus zu nennen (Gallie et al . (1987) Nucl Acids Res 15:8693-8711) und dergleichen. Sie können ferner die Gewebs- εpezifität fördern (Rouster J et al . (1998) Plant J 15:435-440).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).
Die Expressionskassette kann vorteilhafterweiεe eine oder mehrere εogenannte "enhancer Sequenzen" funktioneil verknüpft mit dem Promoter enthalten, die eine erhöhte tranεgene Expreεεion der Nukleinεäuresequenz ermöglichen. Auch am 3 '-Ende der transgen zu exprimierenden Nukleinsäuresequenzen können zuεätzliche vorteil- hafte Sequenzen inεeriert werden, wie weitere regulatoriεche Elemente oder Terminatoren. Die tranεgen zu exprimierenden Nukleinεäuresequenzen können in einer oder mehreren Kopien im Genkonεtrukt enthalten sein.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. At the 3 'end of the nucleic acid sequences to be expressed transgenically, 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.
Als Kontrollsequenzen geeignete Polyadenylierungssignale sind pflanzliche Polyadenylierungssignale, vorzugsweiεe solche, die im wesentlichen T-DNA Polyadenylierungssignale aus Agrobacterium tumefaciens, insbesondere des Genε 3 der T-DNA (Octopin Synthase) deε Ti-Plasmidε pTiACHS entεprechen (Gielen et al . (1984) EMBO J 3:835 ff) oder funktionelle Äquivalente davon. Beispiele für besonders geeignete Terminatorsequenzen sind der OCS (Octopin- Synthaεe) -Terminator und der NOS (Nopalin-Synthase) -Terminator.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.
Als KontrollSequenzen sind weiterhin solche zu verεtehen, die eine homologe Rekombination bzw. Insertion in das Genom eines Wirtsorganismuε ermöglichen oder die Entfernung auε dem Genom erlauben. Bei der homologen Rekombination kann zum Beiεpi.el die kodierende Sequenz eines bestimmten endogenen Gens gegen die für eine dsRNA kodierende Sequenz gezielt ausgetauscht werden. Methoden wie die cre/lox-Technologie erlauben eine gewebespezifiεche, unter Umständen induzierbare Entfernung der Expreεεionskassette aus dem Genom des WirtsOrganismus (Sauer B (1998) Methods. 14(4) :381-92) . Hier werden beεtimmte flankierende Sequenzen dem Zielgen angefügt (lox-Sequenzen) , die später eine Entfernung mittels der cre-Rekombinase ermöglichen.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. In homologous recombination, for example, 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). Here certain flanking sequences are added to the target gene (lox sequences), which later enable removal by means of the cre recombinase.
Eine Expressionskassetten und die von ihr abgeleiteten Vektoren können weitere Funktionselemente enthalten. Der Begriff Funktionselernent ist breit zu verεtehen und meint all εolche Elemente, die einen Einfluεs auf Herstellung, Vermehrung oder Funktion der erfindungsgemäßen Expressionskassetten, Vektoren oder transgenen Organismen haben. Beispielhaft aber nicht einschränkend seien zu nennen: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:
a) Selektionsmarker, die eine Resistenz gegen einena) Selection markers showing resistance to one
Metabolismusinhibitor wie 2-Desoxyglucoεe-6-phosphat (WO 98/45456) , Antibiotika oder Biozide, bevorzugt Herbizide, wie zum Beispiel Kanamycin, G 418, Bleomycin, Hygromycin, oder Phosphinotricin etc. verleihen. Besonders bevorzugte Selektionsmarker sind solche die eine Reεiεtenz gegen Herbizide verleihen. Beispielhaft εeien genannt: DNA Sequenzen, die für Phosphinothricinacetyltransferasen (PAT) kodieren und Glutaminsynthaεeinhibitoren inaktivieren (bar und pat Gen) , 5-Enolpyruvylεhikimat-3-phoεphatεynthaεegene (EPSP Synthaεegene) , die eine Resistenz gegen Glyphosat® (N- (phosphonomethyl) glycin) verleihen, das für daε Glyphosat® degradierende Enzyme kodierende gox Gen (Glyphosatoxido- reduktaεe) , das deh Gen (kodierend für eine Dehalogenase, die Dalapon inaktiviert) , Sulfonylurea- und Imidazolinon inaktivierende Acetolactatsynthasen sowie bxn Gene, die für Bromoxynil degradierende Nitrilaseenzyme kodieren, daε aaεa-Gen, daε eine Resistenz gegen das Antibiotikum Apectino- mycin verleih, das Streptomycinphosphotransferase (SPT) Gen, das eine Reεistenz gegen Streptomycin gewährt, daε Neomycin- phosphotransferas (NPTII) Gen, das eine Resistenz gegen Kana- mycin oder Geneticidin verleiht, das Hygromycinphosphotrans- ferase (HPT) Gen, das eine Reεistenz gegen Hygromycin vermittelt, das Acetolactatεynthaε Gen (ALS) , das eine Resistenz gegen Sulfonylhamstoff-Herbizide verleiht (z.B. mutierte ALS-Varianten mit z.B. der S4 und/oder Hra Mutation) .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. Particularly preferred selection markers are those which confer resistance to herbicides. Examples include εeien: DNA sequences which encode phosphinothricin (PAT) and Glutaminsynthaεeinhibitoren inactivate (bar and pat genes), 5-Enolpyruvylεhikimat-3-phoεphatεynthaεegene (EPSP Synthaεegene), which confer resistance to glyphosate ® (N- (phosphonomethyl) glycine) confer that for daε 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 (SPT) gene, which confers resistance to streptomycin, the neomycin phosphotransferas (NPTII) gene, which confers resistance to canaminomine or geneticidin, the hygromycin phosphotransferase (HPT) gene , which mediates resistance to hygromycin, the acetolactate synthase (ALS) gene, which confers resistance to sulfonylurea herbicides (for example mutated ALS variants with, for example, the S4 and / or Hra mutation).
b) Reportergene, die für leicht quantifizierbare Proteine kodieren und über Eigenfarbe oder Enzymaktivität eine Bewertung der Tranεformationεeffizienz oder deε Expressionε- ortes oder -Zeitpunktes gewährleisten. Ganz besonders bevorzugt sind dabei Reporter-Proteine (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(l):29-44) wie daε "green fluorescence protein" (GFP) (Sheen et al.(1995) Plant Journal 8 (5) : 777-784) , die Chloramphenicoltransferaεe, eine Luziferase (Ow et al . (1986) Science 234:856-859), das Aequorin-Gen (Prasher et al. (1985) Biochem Biophyε Res Com- mun 126 (3) :1259-1268) , die ß-Galactosidase, ganz besonders bevorzugt ist die ß-Glucuronidase (Jefferson et al. (1987) EMBO J 6:3901-3907) .b) 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 (Schenborn 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. (1985) Biochem Biophyε Res Communun 126 ( 3): 1259-1268), the β-galactosidase, the β-glucuronidase is very particularly preferred (Jefferson et al. (1987) EMBO J 6: 3901-3907).
c) Replikationsurεprünge, die eine Vermehrung der erfindungsgemäßen Expressionskassetten oder Vektoren in zum Beispiel E.coli gewährleisten. Beispielhaft seien genannt OR (origin of DNA replication) , der pBR322 ori oder der P15A ori (Sam- brook et al . : Molecular Cloning. A Laboratory Manual, 2 d ed. Cold Spring Harbor Laboratory Presε, Cold Spring Harbor, NY, 1989) .c) 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 ).
d) Elemente, die für eine Agrobakterium vermittelte Pflanzentransformation erforderlich, sind, wie zum Beiεpiel die rechte oder linke Begrenzung der T-DNA oder die vir-Region.d) Elements which mediated for Agrobacterium plant transformation are required, such Beiεpiel the right or left border of the T-DNA or the vir region.
Zur Selektion erfolgreich homolog rekombinierter oder auch tranεformierter Zellen iεt eε in der Regel erforderlich, einen selektionierbaren Marker zusätzlich einzuführen, der den erfolgreich rekombinierten Zellen eine Resistenz gegen ein Biozid (zum Beispiel ein Herbizid) , einen Metaboliεmuεinhibitor wie 2-Deεoxy- glucose-6-phosphat (WO 98/45456) oder ein Antibiotikum verleiht. Der Selektionsmarker erlaubt die Selektion der transformierten Zellen von untransformierten (McCormick et al . (1986) Plant Cell Reports 5:81-84) .For the selection of successfully homologously recombined or also transformed cells, it is generally necessary to additionally introduce 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).
Zusätzlich kann besagte transgene Expreεεionεkaεsette oder Expressionsvektoren Nukleinsäuresequenzen enthalten, die nicht zu einer Verminderung mindestens eines Speicherproteins führen, und deren transgene Expresεion zu einer zusätzlichen Steigerung der Fettsäure-Biosynthese führt (infolge proOIL) . Diese zusätzlich transgen exprimierte proOIL Nukleinsäuresequenz kann beispielhaft aber nicht einschränkend ausgewählt sein aus Nukleinsäuren kodierend für Acetyl-CoA-Carboxylase (ACCase) , Glycerol- 3-Phoεphat Acyltranεferaεe (GPAT) , Lyεophosphatidat-Acyl- tranεferase (LPAT) , Diacylglycerol-Acyltransferase (DAGAT) und Phospholipid:diacylglycerol-acyltranεferase (PDAT) . Entsprechende Sequenzen εind dem Fachmann bekannt' und auε Datenbanken oder entsprechenden cDNA-Banken der jeweiligen Pflanzen leicht zugänglich.In addition, said 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). Corresponding sequences εind known in the art 'and auε databases or corresponding cDNA libraries of the respective plants easily accessible.
Die Einführung einer erfindungsgemäßen Expressionskaεsette in einen Organismus oder Zellen, Geweben, Organe, Teile bzw. Samen desselben (bevorzugt in Pflanzen bzw. pflanzliche Zellen, Gewebe, Organe, Teile oder Samen) , kann vorteilhaft unter Verwendung von Vektoren realisiert werden, in denen die Expressionskassetten enthalten sind. Vektoren können beispielhaft Plasmide, Cosmide, Phagen, Viren oder auch Agrobacterien εein. Die Expreεεionεkaε- εette kann in den Vektor (bevorzugt ein Plasmidvektor) über eine geeignete Restriktionsεchnittεtelle eingeführt werden. Der entstandene Vektor wird zunächst in E.coli eingeführt. Korrekt transformierte E.coli werden selektioniert, gezüchtet und der re- kombinante Vektor mit dem Fachmann geläufigen Methoden gewonnen. Reεtriktionεanalyse und Sequenzierung können dazu dienen, den Klonierungsεchritt zu prüfen. Bevorzugt sind solche Vektoren, die eine εtabile Integration der Expreεsionskasεette in daε Wirtεge- nom ermöglichen.The introduction of 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.
Die Herεtellung eineε' transformierten Organismus (bzw. einer transformierten Zelle oder Gewebes) erfordert, dass die entsprechende DNA (z.B. der Expressionsvektor), RNA oder Protein in die entsprechende Wirtεzelle eingebracht wird. Für diesen Vorgang, der als Transformation (oder Transduktion bzw. Transfek- tion) bezeichnet wird, εteht eine Vielzahl von Methoden zur Verfügung (Keown et al . (1990) Methods in Enzymology 185:527-537). So kann die DNA oder RNA beispielhaft direkt durch Mikroinjektion oder durch Bombardierung mit DNA-beschichteten Mikropartikeln eingeführt werden. Auch kann die Zelle chemisch, zum Beispiel mit Polyethylenglycol, permeabilisiert werden, so dass die DNA durch Diffusion in die Zelle gelangen kann. Die DNA kann auch durch Protoplastenfusion mit anderen DNA-enthaltenden Einheiten wie Mi- nicells, Zellen, Lysoεomen oder Lipoεomen erfolgen. Elektropora- tion iεt eine weitere geeignete Methode zur Einführung von DNA, bei der die Zellen reverεibel durch einen elektrischen Impuls permeabilisert werden. Entsprechende Verfahren εind beschrieben (beispielsweise bei 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 . (1980) Science 208:1265; Horεch et al.(1985) Science 227:1229-1231; DeBlock et al . (1989) Plant Physiology 91:694-701; Methodε for Plant Molecular Biology (Weiεsbach and Weissbach, eds . ) Academic Preεs Inc. (1988); and Methods in Plant Molecular Biology (Sch ler and Zielinεki, ed . ) Academic Press Inc. (1989) ) .The production of a transformed organism (or a transformed cell or tissue) requires that the corresponding DNA (for example the expression vector), RNA or protein be introduced into the corresponding host cell. A large number of methods are available for this process, which is referred to as transformation (or transduction or transfection) (Keown et al. (1990) Methods in Enzymology 185: 527-537). For example, 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. (1980) Science 208: 1265; Horεch et al. (1985 ) Science 227: 1229-1231; DeBlock et al. (1989) Plant Physiology 91: 694-701; Methodε for Plant Molecular Biology (Weiεsbach and Weissbach, eds.) Academic Preεs Inc. (1988); and Methods in Plant Molecular Biology (Sch ler and Zielinεki, ed.) Academic Press Inc. (1989)).
Bei Pflanzen werden dabei die beschriebenen Methoden zur Transformation und Regeneration von Pflanzen auε Pflanzengeweben oder Pflanzenzellen zur tranεienten oder stabilen Transformation genutzt. Geeignete Methoden sind vor allem die Protoplastentransformation durch Polyethylenglykol-induzierte DNA-Aufnähme, das bioliεtiεche Verfahren mit der Genkanone, die sogenannte "particle bo bardment" Methode, die Elektroporation, die Inkubation trockener Embryonen in DNA-haltiger Lösung und die Mikroinjektion.In plants, the methods described for transforming and regenerating plants from plant tissues or plant cells for transient or stable transformation are used. 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.
Neben diesen "direkten" Tranεformationstechniken kann eine Transformation auch durch bakterielle Infektion mittels Agro- bacterium tumefaciens oder Agrobacterium rhizogenes durchgeführt werden.' Die Agrobacteriύm-ver ittelte Transformation ist am besten für dicotyledone Pflanzenzellen geeignet. Die Verfahren sind beispielsweise beschrieben bei Horsch RB et al . (1985) Science 225: 1229f) .In addition to these "direct" transformation techniques, a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes. ' The Agrobacteriύm-mediated transformation is best suited for dicotyledonous plant cells. The methods are described, for example, by Horsch RB et al. (1985) Science 225: 1229f).
Werden Agrobacterien verwendet, so iεt die Expresεionεkassette in spezielle Plasmide zu integrieren, entweder in einen Zwischenvektor (englisch: Shuttle or intermediate vector) oder einen binären Vektor. Wird ein Ti oder Ri Plasmid zur Transformation verwendet werden soll, iεt zumindest die rechte Begrenzung, meistenε jedoch die rechte und die linke Begrenzung der Ti oder Ri Plasmid T-DNA als flankierende Region mit der einzuführenden Expreεεionεkaεεette verbunden.If Agrobacteria are used, 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.
Bevorzugt werden binäre Vektoren verwendet. Binäre Vektoren , können εowohl in E.coli als auch in Agrobacterium replizieren. Sie enthalten in der Regel ein Selektionsmarkergen und einen Linker oder Polylinker flankiert von der rechten und linken T-DNA Begrenzungssequenz . Sie können direkt in Agrobacterium transformiert werden (Holsters et al . (1978) Mol Gen Genet 163:181-187). Das Selektionsmarkergen erlaubt eine Selektion transformierter Agrobakteria und ist zum Beispiel das nptll Gen, das eine Resistenz gegen Kanamycin verleiht. Das in diesem Fall als Wirtsorganiε uε fungierende Agrobacterium sollte bereits ein Plasmid mit der vir-Region enthalten. Diese ist für die Übertragung der T-DNA auf die pflanzliche Zelle erforderlich. Ein so transformiertes Agrobacterium. kann zur Transformation pflanzlicher Zellen verwendet werden. Die Verwendung von T-DNA zur Transformation pflanzlicher Zellen ist intensiv untersucht und beschrieben (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanterε B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287) . Verschiedene binäre Vektoren sind bekannt und teilweise kommerziell erhältlich wie zum Beiεpiel pBHOl.2 oder pBIN19 (Clontech Laboratorieε , Inc. USA) .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. The use of 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 Kanterε 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 Laboratorieε, Inc. USA).
Weitere zur Expression in Pflanzen geeignet Promotoren sind beschrieben (Rogers et al . (1987) Meth in Enzymol 153:253-277; Schardl et al . (1987)' Gene 61:1-11; Berger et al . (1989) Proc Natl Acad Sei USA 86:8402-8406).Other promoters suitable for expression in plants are described (Rogers et al. (1987) Meth in Enzymol 153: 253-277; Schardl et al. (1987) ' Gene 61: 1-11; Berger et al. (1989) Proc Natl Acad Sei USA 86: 8402-8406).
Direkte Transformationstechniken eignen sich für jeden Organismus und Zeiltyp. Im Falle von Injektion oder Elektroporation von DNA bzw. RNA in pflanzliche Zellen sind keine besonderen Anforderungen an das verwendete Plasmid gestellt. Einfache Plasmide wie die der pUC-Reihe können verwendet werden. Sollen vollεtändige Pflanzen auε den tranεformierten Zellen regeneriert werden, so iεt er erforderlich, das sich auf dem Plasmid ein zusatz-lich.es selektionierbares Markergen befindet.Direct transformation techniques are suitable for every organism and cell type. In the case of injection or electroporation of DNA or RNA into plant cells, there are no special requirements for the plasmid used. 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.
Stabil transformierte Zellen d.h. solche, die die eingeführte DNA integriert in die DNA der Wirtszelle enthalten, können von untransformierten selektioniert werden, wenn ein selektionier- barer Marker Bestandteil der eingeführten DNA ist. Alε Marker kann beispielhaft jedes Gen fungieren, daεs eine Resiεtenz gegen Antibiotika oder Herbizide (wie Kanamycin, G 418, Bleomycin, Hygromycin oder Phoεphinotricin etc.) zu verleihen vermag (s.o.). Transformierte Zellen, die ein solches Markergen exprimieren, sind in der Lage, in der Gegenwart von Konzentrationen eines entsprechenden Antibiotikums oder Herbizides zu überleben, die einen untransformierten Wildtyp abtöten. Beispiel sind oben genannt und umfassen bevorzugt das bar Gen, dass Resistenz gegen das Herbizid Phosphinotricin verleiht (Rathore KS et al . (1993) Plant Mol Biol 21 (5) : 871-884) , das nptll Gen, dass Resistenz gegen Kanamycin verleiht, das hpt Gen, das Reεiεtenz gegen Hygromycin verleiht, oder daε EPSP-Gen, das Resistenz gegen das Herbizid Glyphoεat verleiht. Der Selektionεmarker erlaubt die Selektion von trans- formierten Zellen von untranεformierten (McCormick et al . (1986) Plant Cell Reportε 5:81-84). Die erhaltenen Pflanzen können in üblicher Weise gezüchtet und gekreuzt werden. Zwei oder mehr Generationen sollten kultiviert werden, um sicherzustellen, dasε die genomische Integration stabil und vererblich ist.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. As an example, 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. (1993) Plant Mol Biol 21 (5): 871-884), the nptll gene which confers resistance to kanamycin, the hpt gene which confers resistance to hygromycin, or the EPSP gene which confers resistance to the herbicide glyphosate. 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.
Die oben genannten Verfahren εind beispielsweise beschrieben in Jeneε B et al.(1993) Techniqueε for Gene Tranεfer, in: T.ranεgenic Plantε, Vol. 1, Engineering and Utilization, herauεgegeben von SD Kung und R Wu, Academic Preεs, S.128-143 sowie in Potrykus (1991) Annu.Rev Plant Physiol Plant Molec Biol 42:205-225). Vor- zugεweiεe wird daε zu exprimierende Konstrukt in einen Vektor kloniert, der geeignet ist, Agrobacterium tumefaciens zu transformieren, beispielεweise pBinl9 (Bevan et al. (1984) Nucl Acids Reε 12:8711f) .The above-mentioned methods are described, for example, in Jeneε B et al. (1993) Techniqueε for Gene Transfer, in: T.ranεgenic Plantε, Vol. 1, Engineering and Utilization, edited by SD Kung and R Wu, Academic Preεs, 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 Reε 12: 8711f).
Sobald eine transformierte Pflanzenzelle hergestellt wurde, kann eine vollständige Pflanze unter Verwendung von dem Fachmann bekannten Verfahren erhalten werden. Hierbei geht man beispielhaft von Kalluskulturen aus. Auε dieεen noch undifferenzierten Zellmassen kann die Bildung von Sprosε und Wurzel in bekannter Weiεe induziert werden. Die erhaltenen Spröεεlinge können ausgepflanzt und gezüchtet werden.Once a transformed plant cell has been made, 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.
Dem Fachmann sind such Verfahren bekannt, um aus Pflanzenzellen, Pflanzenteile und ganze Pflanzen zu regenerieren. Beiεpielsweise werden hierzu Verfahren beschrieben von Fennell et al . (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14:273-278; Jahne et al . (1994) Theor Appl Genet 89:525-533 verwendet .Methods are known to the person skilled in the art to regenerate from plant cells, plant parts and whole plants. For example, methods for this are described by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14: 273-278; Jahne et al. (1994) Theor Appl Genet 89: 525-533.
"Transgen" meint bezüglich zum Beiεpiel einer Nukleinsäuresequenz, einer Expressionskassette oder einem Vektor enthaltend besagte Nukleinsäuresequenz oder einem Organismuε tranεformiert mit beεagter Nukleinεäureεequenz, Expreεεionεkaεεette oder Vektor alle εolche durch gentechniεche Methoden zustandegekommene Konstruktionen, in denen εich entwederWith regard to, for example, a nucleic acid sequence, an expression cassette or a vector containing said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector, “transgene” means all such constructions, either in which the genetic engineering methods are used, in which
a) die Speicherprotein Nukleinsäuresequenz , odera) the storage protein nucleic acid sequence, or
b) eine mit der Speicherprotein Nukleinsäuresequenz funktioneil verknüpfte genetische Kontrollsequenz, zum Beispiel ein Promotor, oder c ) (a) und (b)b) a genetic control sequence functionally linked to the storage protein nucleic acid sequence, for example a promoter, or c) (a) and (b)
sich nicht in ihrer natürlichen, genetischen Umgebung befinden Dder durch gentechnische Methoden modifiziert wurden, wobei äie Modifikation beispielhaft eine Substitutionen, Additionen, Deletionen, Inversion oder Insertionen eines oder mehrerer • STukleotidreste sein kann. Natürliche genetische Umgebung meint ien natürlichen chromosomalen Locus in dem Herkunftsorganismus oder das Vorliegen in einer genomischen Bibliothek. Im Fall einer genomischen Bibliothek ist die natürliche, genetische Umgebung der Nukleinsäuresequenz bevorzugt zumindest noch teilweise erhalten. Die Umgebung flankiert die Nukleinsäuresequenz zumindest an einer Seite und hat eine Sequenzlänge von mindestens 50 bp, bevorzugt mindestenε 500 bp, besonders bevorzugt mindestens 1000 bp, ganz besonderε bevorzugt mindeεtens 5000 bp. Eine natürlich vorkommende Expresεionskassette - beispielsweise die natürlich vorkommende Kombination des Speicherprotein-Promotors mit dem entsprechenden Speicherprotein-Gen - wird zu einer transgenen Expressionskasεette, wenn diese durch nicht-natürliche, synthetische ("künstliche") Verfahren wie beispielεweise einer Mutageniεierung geändert wird. Entsprechende Verfahren sind beschrieben (US 5,565,350; WO 00/15815; siehe auch oben).are not in their natural, genetic environment, which have been modified by genetic engineering methods, such modification being, for example, substitution, addition, deletion, inversion or insertion of one or more STucleotide residues. Natural genetic environment means a natural chromosomal locus in the organism of origin or the presence in a genomic library. In the case of 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. A naturally occurring expression cassette - for example the naturally occurring combination of the storage protein promoter with the corresponding storage protein gene - becomes a transgenic expression cassette if this is changed by non-natural, synthetic ("artificial") methods such as, for example, mutagenization. Corresponding methods are described (US 5,565,350; WO 00/15815; see also above).
Alε tranεgene Organiεmen bevorzugte Wirts- oder Ausgangsorganismen sind vor allem Pflanzen gemäß der oben genannten Definition. Eingeεchlosεen sind im Rahmen der Erfindung alle Gattungen und Arten höherer und niedrigerer Pflanzen des Pflanzenreiches, insbesondere Pflanzen, die für die Gewinnung von Ölen verwendet werden wie beispielsweise Raps, Sonnenblume, Sesam, Färberdistel, Ölbaum,' Soja, Mais, Weizen und Nussarten. Eingeschlossen sind ferner die reifen Pflanzen, Saatgut, Sprossen und Keimlinge, sowie davon abgeleitete Teile, Vermehrungsgut und Kulturen, zum Beispiel Zellkulturen. Reife Pflanzen meint Pflanzen zu jedem beliebigen Entwicklungεεtadium jenεeits deε Keimlingε. Keimling meint eine junge, unreife Pflanze in einem frühen Entwicklungεstadium.Preferred host or starting organisms as transgenic organisms are, above all, plants as defined above. All genera and species are Eingeεchlosεen 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.
Die Herstellung der transgenen Organismen kann mit den oben beschriebenen Verfahren zur Transformation oder Transfektion von Organismen realiεiert werden.The production of the transgenic organisms can be carried out using the processes described above for transforming or transfecting organisms.
Ein weiterer Gegenεtand der Erfindung betrifft die Verwendung der erfindungεgemäßen, tranεgenen Organismen und der von ihnen abgeleitete Zellen, Zellkulturen, Teile - wie zum Beispiel bei transgenen pflanzlichen Organismen Wurzeln, Blätter etc.- , und trans- genes Vermehrungsgut wie Saaten oder Früchte, zur Herstellung von Nahrungs- oder Futtermitteln, Pharmazeutika oder Feinchemikalien, inεbesondere von Ölen, Fetten, Fettsäuren oder Derivaten der vorgenannten. 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.
Sequenzensequences
1. SEQ ID NO: 11. SEQ ID NO: 1
Nukleinsäuresequenz kodierend für A.thaliana Albumin 2S subunit 1 (GenBank Acc . -No . : M22032 )Nucleic acid sequence coding for A.thaliana albumin 2S subunit 1 (GenBank Acc. -No.: M22032)
2. SEQ ID NO: 22. SEQ ID NO: 2
Proteinsequenz kodierend für A.thaliana Albumin 2S subunit 1Protein sequence coding for A.thaliana Albumin 2S subunit 1
3. SEQ ID NO: 33. SEQ ID NO: 3
Nukleinsäureεequenz kodierend für A.thaliana Albumin 2S subunit 3 (GenBank Acc.-No.: M22035)Nucleic acid sequence coding for A.thaliana albumin 2S subunit 3 (GenBank Acc.-No .: M22035)
4. SEQ ID NO: 44. SEQ ID NO: 4
Proteinsequenz kodierend für A.thaliana Albumin 2S subunit 3Protein sequence coding for A.thaliana Albumin 2S subunit 3
5. SEQ ID NO: 55. SEQ ID NO: 5
Nukleinsäureεequenz kodierend für A.thaliana Albumin.2S subunit 2 (GenBank Acc.-No.: M22034)Nucleic acid sequence coding for A.thaliana Albumin.2S subunit 2 (GenBank Acc.-No .: M22034)
6. SEQ ID NO: 66. SEQ ID NO: 6
Proteinsequenz kodierend für A.thaliana Albumin 2S subunit 2Protein sequence coding for A.thaliana Albumin 2S subunit 2
7. SEQ ID NO: 77. SEQ ID NO: 7
Nukleinsäureεequenz kodierend für A.thaliana Albumin 2S εubunit 4 (GenBank Acc.-No.: M22033)Nucleic acid sequence coding for A.thaliana albumin 2S εubunit 4 (GenBank Acc.-No .: M22033)
8. ■ SEQ ID NO : 88. ■ SEQ ID NO: 8
Proteinεequenz kodierend für A.thaliana Albumin 2S εubunit 4Protein sequence coding for A.thaliana albumin 2S ububit 4
9. SEQ ID NO: 99. SEQ ID NO: 9
Nukleinεäureεequenz kodierend für B.napuε Cruciferin Speicherprotein (GenBank Acc.-No.: X59294)Nucleic acid sequence coding for B.napuε cruciferin storage protein (GenBank Acc.-No .: X59294)
10. SEQ ID NO: 1010. SEQ ID NO: 10
Proteinεequenz kodierend für B.napuε Cruciferin SpeicherproteinProtein sequence coding for B.napuε cruciferin storage protein
11. SEQ ID NO: 1111. SEQ ID NO: 11
Nukleinεäuresequenz kodierend für Brassica napuε Cruciferin (GenBank Acc.-No.: X14555)Nucleic acid sequence coding for Brassica napuε cruciferin (GenBank Acc.-No .: X14555)
12. SEQ ID NO: 1212. SEQ ID NO: 12
Proteinεequenz kodierend für Braεεica napuε Cruciferin 13 . SEQ ID NO : 13Protein sequence coding for Braεεica napuε cruciferin 13. SEQ ID NO: 13
Nukleinsäureεequenz kodierend für B.napuε BnC2 Cruciferin Speicherprotein (GenBank Acc.-No.: X59295)Nucleic acid sequence coding for B.napuε BnC2 cruciferin storage protein (GenBank Acc.-No .: X59295)
14. SEQ ID NO: 1414. SEQ ID NO: 14
Proteinsequenz kodierend für B.napus BnC2 Cruciferin SpeicherproteinProtein sequence coding for B.napus BnC2 cruciferin storage protein
15. SEQ ID NO: 15 partielle Nukleinsäureεequenz kodierend für B.napuε Cruciferin cru4 εubunit (GenBank Acc.-No.: X57848)15. SEQ ID NO: 15 partial nucleic acid sequence coding for B.napuε cruciferin cru4 ububit (GenBank Acc.-No .: X57848)
16. SEQ ID NO: 16 partielle Proteinεequenz kodierend für B.napus Cruciferin cru4 subunit16. SEQ ID NO: 16 partial protein sequence coding for B.napus cruciferin cru4 subunit
17. SEQ ID NO: 1717. SEQ ID NO: 17
Nukleinsäuresequenz kodierend für B.napus crul Cruciferin εubunit (GenBank Acc.-No. : X62120)Nucleic acid sequence coding for B.napus crul cruciferin εubunit (GenBank Acc.-No .: X62120)
18. SEQ ID NO: 1818. SEQ ID NO: 18
Proteinsequenz kodierend für B.napuε crul Cruciferin subunitProtein sequence coding for B.napuε crul cruciferin subunit
19. SEQ ID NO: 1919. SEQ ID NO: 19
Nukleinsäuresequenz kodierend für Glycinin A-la-B-x subunit aus des Sojabohne (GenBank Acc.-No.: M36686)Nucleic acid sequence coding for glycinin A-la-B-x subunit from the soybean (GenBank Acc.-No .: M36686)
20. SEQ ID NO: 2020. SEQ ID NO: 20
Proteinsequenz kodierend für Glycinin A-la-B-x subunit aus des SojabohneProtein sequence coding for glycinin A-la-B-x subunit from the soybean
21. SEQ ID NO: 2121. SEQ ID NO: 21
Nukleinsäuresequenz kodierend für Sojabohne Glycinin subunit G2 (GenBank Acc.-No. : X15122)Nucleic acid sequence coding for soybean glycinin subunit G2 (GenBank Acc.-No .: X15122)
22. SEQ ID NO: 2222. SEQ ID NO: 22
Proteinsequenz kodierend für Sojabohne Glycinin subunit G2Protein sequence coding for soybean glycinin subunit G2
23. SEQ ID NO: 2323. SEQ ID NO: 23
Nukleinsäuresequenz kodierend für Sojabohne A5A4B3 Glycinin subunits (GenBank Acc . -No . : X02626)Nucleic acid sequence coding for soybean A5A4B3 glycinin subunits (GenBank Acc. -No.: X02626)
24. SEQ ID NO: 2424. SEQ ID NO: 24
Proteinsequenz kodierend für Sojabohne A5A4B3 Glycinin subunits 25 . SEQ ID NO : 25Protein sequence coding for soybean A5A4B3 glycinin subunits 25th SEQ ID NO: 25
Nukleinsäureεequenz kodierend für Sojabohne (G.max) Glycinin Speicherprotein subunit A3-B4 (GenBank Acc.-No.: M10962)Nucleic acid sequence coding for soybean (G.max) glycinin storage protein subunit A3-B4 (GenBank Acc.-No .: M10962)
26. SEQ ID NO: 2626. SEQ ID NO: 26
Proteinsequenz kodierend für Sojabohne (G.max) Glycinin Speicherprotein subunit A3-B4Protein sequence coding for soybean (G.max) glycinin storage protein subunit A3-B4
27. SEQ ID NO: 2727. SEQ ID NO: 27
Nukleinsäuresequenz kodierend für Sojabohne Glycinin subunit G3 (GenBank Acc.-No. : X15123)Nucleic acid sequence coding for soybean glycinin subunit G3 (GenBank Acc.-No .: X15123)
28. SEQ ID NO: 2828. SEQ ID NO: 28
Proteinsequenz kodierend für Sojabohne Glycinin εubunit G3Protein sequence coding for soybean glycinin εubunit G3
29. SEQ ID NO: 2929. SEQ ID NO: 29
Nukleinεäureεequenz kodierend für Sonnenblume IIS Speicherprotein (G3-D1) (GenBank Acc.-No. : M28832)Nucleic acid sequence coding for sunflower IIS storage protein (G3-D1) (GenBank Acc.-No .: M28832)
30. SEQ ID NO: 3030. SEQ ID NO: 30
Proteinεequenz kodierend für Sonnenblume IIS Speicherprotein (G3-D1)Protein sequence coding for sunflower IIS storage protein (G3-D1)
31. SEQ ID NO: 3131. SEQ ID NO: 31
Nukleinεäuresequenz kodierend für Raps (B.napus) -Napin (GenBank Acc.-No. : J02586)Nucleic acid sequence coding for rapeseed (B.napus) napin (GenBank Acc.-No.: J02586)
32. SEQ ID NO: 3232. SEQ ID NO: 32
Proteinsequenz kodierend für Raps (B.napuε) NapinProtein sequence coding for rapeseed (B.napuε) napin
33. SEQ ID NO: 3333. SEQ ID NO: 33
Nukleinεäuresequenz kodierend für Brasεica juncea 2S Speicherprotein (GenBank Acc.-No.: X65040)Nucleic acid sequence coding for Brasεica juncea 2S storage protein (GenBank Acc.-No .: X65040)
34. SEQ ID NO: 3434. SEQ ID NO: 34
Proteinεequenz kodierend für Braεεica juncea 2S SpeicherproteinProtein sequence coding for Braεεica juncea 2S storage protein
35. SEQ ID NO: 3535. SEQ ID NO: 35
Nukleinεäureεequenz kodierend für Brassica oleracea 2S Speicherprotein (GenBank Acc.-No.: X65038)Nucleic acid sequence coding for Brassica oleracea 2S storage protein (GenBank Acc.-No .: X65038)
36. SEQ ID NO: 3636. SEQ ID NO: 36
Proteinsequenz kodierend für Brasεica oleracea 2S Speicherprotein 37 . SEQ ID NO : 37Protein sequence coding for Brasεica oleracea 2S storage protein 37. SEQ ID NO: 37
Nukleinsäuresequenz kodierend für Brassica napus cv. Topaε Napin (GenBank Acc . -No . : U04945)Nucleic acid sequence coding for Brassica napus cv. Topaε Napin (GenBank Acc. -No.: U04945)
38. SEQ ID NO: 3838. SEQ ID NO: 38
Proteinsequenz kodierend für Brassica napus cv. Topas NapinProtein sequence coding for Brassica napus cv. Topaz Napin
39. SEQ ID NO: 39 partielle Nukleinsäuresequenz kodierend für Sinapis alba sinl Speicherprotein (GenBank Acc. -No. : X91799)39. SEQ ID NO: 39 partial nucleic acid sequence coding for Sinapis alba sinl storage protein (GenBank Acc. -No.: X91799)
40. SEQ ID NO: 40 partielle Proteinsequenz kodierend für Sinapis alba sinl •Speicherprotein40. SEQ ID NO: 40 partial protein sequence coding for Sinapis alba sinl • storage protein
41. SEQ ID NO: 4141. SEQ ID NO: 41
Nukleinεäureεequenz. kodierend für Sojabohne (Glycine max) napin-type 2S Albumin 1 (GenBank Acc.-No.: U71194)Nukleinεäureεequenz. coding for soybean (Glycine max) napin-type 2S Albumin 1 (GenBank Acc.-No .: U71194)
42. SEQ ID NO: 4242. SEQ ID NO: 42
Proteinsequenz kodierend für Sojabohne (Glycine max) napin- type 2S Albumin 1Protein sequence coding for soybean (Glycine max) napin-type 2S albumin 1
43. SEQ ID NO: 4343. SEQ ID NO: 43
Nukleinsäureεequenz kodierend für Sojabohne (Glycine max) 2S Albumin (GenBank Acc . -No. : AF005030)Nucleic acid sequence coding for soybean (Glycine max) 2S albumin (GenBank Acc. -No.: AF005030)
44. SEQ ID NO: 4444. SEQ ID NO: 44
Proteinεequenz kodierend für Sojabohne (Glycine max) 2S AlbuminProtein sequence coding for soybean (Glycine max) 2S albumin
45. SEQ ID NO: 4545. SEQ ID NO: 45
Nukleinεäureεequenz kodierend für Braεsica nigra 2S Speicherprotein (GenBank Acc.-No. : X65039)Nucleic acid sequence coding for Braεsica nigra 2S storage protein (GenBank Acc.-No .: X65039)
46. SEQ ID NO: 4646. SEQ ID NO: 46
Proteinsequenz kodierend für Brassica nigra 2S SpeicherproteinProtein sequence coding for Brassica nigra 2S storage protein
47. SEQ ID NO: 4747. SEQ ID NO: 47
Nukleinsäuresequenz kodierend für Sinapis alba sin5 Speicherprotein (GenBank Acc.-No. : X91798)Nucleic acid sequence coding for Sinapis alba sin5 storage protein (GenBank Acc.-No .: X91798)
48. SEQ ID NO: 4848. SEQ ID NO: 48
Proteinεequenz kodierend für Sinapiε alba εin5 Speicherprotein 49 . SEQ ID NO : 49Protein sequence coding for Sinapiε alba εin5 storage protein 49. SEQ ID NO: 49
Nukleinsäureεequenz kodierend für Sonnenblume HaG5 2 S Albumin (GenBank Acc . -No . : X06410)Nucleic acid sequence coding for sunflower HaG5 2 S albumin (GenBank Acc. -No.: X06410)
50. SEQ ID NO: 50 proteinεequenz kodierend für Sonnenblume HaG5 2 S Albumin50th SEQ ID NO: 50 protein sequence coding for sunflower HaG5 2 S albumin
51. SEQ ID NO: 51 partielle Nukleinsäuresequenz kodierend für Sonnenblume (Helianthus annuus) 2S Albumin (GenBank Acc.-No. : X76101)51. SEQ ID NO: 51 partial nucleic acid sequence coding for sunflower (Helianthus annuus) 2S albumin (GenBank Acc.-No.: X76101)
52. SEQ ID NO: 52 partielle Proteinsequenz kodierend für Sonnenblume (Helianthus annuus) 2S Albumin52. SEQ ID NO: 52 partial protein sequence coding for sunflower (Helianthus annuus) 2S albumin
53.-104:53.-104:
SEQ ID NO: 51 - 104 Sequenzmotive aus verschiedenen Speicher- proteinklaεεenSEQ ID NO: 51-104 sequence motifs from different storage protein classes
105. SEQ ID NO: 105105. SEQ ID NO: 105
Nukleinεäureεequenz kodierend für dsRNA zur Suppresεion von Arabidopsis thaliana 12S Speicherprotein AtCru3 (Insert von Vektor pCR2. l-AtCRU3-RNAi)Nucleic acid sequence coding for dsRNA for the suppression of Arabidopsis thaliana 12S storage protein AtCru3 (insert of vector pCR2.1-AtCRU3-RNAi)
106. SEQ ID NO: 106106. SEQ ID NO: 106
Ribonukleinsäuresequenz kodierend für dsRNA zur Suppreεεion von Arabidopsis thaliana 12S Speicherprotein AtCru3Ribonucleic acid sequence coding for dsRNA for the suppression of Arabidopsis thaliana 12S storage protein AtCru3
107. SEQ ID NO: 107107. SEQ ID NO: 107
Nukleinsäuresequenz kodierend für dsRNA zur Suppression von Arabidopsis thaliana 12S Speicherprotein AtCralNucleic acid sequence coding for dsRNA for the suppression of Arabidopsis thaliana 12S storage protein AtCral
108. SEQ ID NO: 108108. SEQ ID NO: 108
Ribonukleinsäuresequenz kodierend für dsRNA zur Suppreεεion von Arabidopεis thaliana 12S Speicherprotein AtCralRibonucleic acid sequence coding for dsRNA for the suppression of Arabidopεis thaliana 12S storage protein AtCral
109. SEQ ID NO: 109109. SEQ ID NO: 109
Nukleinsäuresequenz kodierend für dsRNA zur Suppression von Arabidopsis thaliana 2S Speicherprotein At2S2Nucleic acid sequence coding for dsRNA for the suppression of Arabidopsis thaliana 2S storage protein At2S2
110. SEQ ID NO: 110110. SEQ ID NO: 110
Ribonukleinsäuresequenz kodierend für dsRNA zur Suppression von Arabidopsis thaliana 2S Speicherprotein At2S2 111 . SEQ ID NO : 111Ribonucleic acid sequence coding for dsRNA for the suppression of Arabidopsis thaliana 2S storage protein At2S2 111. SEQ ID NO: 111
Nukleinεäureεequenz kodierend für Arabidopεiε thaliana 12S Cruciferin Speicherprotein (ATCRU3 ; GenBank Acc . -No. : U66916)Nucleic acid sequence coding for Arabidopsis thaliana 12S cruciferin storage protein (ATCRU3; GenBank Acc. -No.: U66916)
.12. SEQ ID NO: 112.12. SEQ ID NO: 112
Proteinsequenz kodierend für Arabidopsis thaliana 12S Cruciferin Speicherprotein (ATCRU3)Protein sequence coding for Arabidopsis thaliana 12S cruciferin storage protein (ATCRU3)
L13.SEQ ID. NO: 113L13.SEQ ID. NO: 113
Nukleinsäuresequenz kodierend für A.thaliana 12S Speicherprotein (CRA1; GenBank Acc . -No . : M37247)Nucleic acid sequence coding for A.thaliana 12S storage protein (CRA1; GenBank Acc. -No.: M37247)
L14.SEQ ID NO: 114L14.SEQ ID NO: 114
Proteinεequenz kodierend für A.thaliana 12S Speicherprotein (CRA1)Protein sequence coding for A.thaliana 12S storage protein (CRA1)
115. SEQ ID NO: 115115. SEQ ID NO: 115
Nukleinεäureεequenz kodierend für Arabidopεiε thaliana 12S Speicherprotein AT5g44120/MLNl_4 (GenBank Acc .-No. : AY070730)Nucleic acid sequence coding for Arabidopεialian thaliana 12S storage protein AT5g44120 / MLNl_4 (GenBank Acc. No .: AY070730)
116. SEQ ID NO: 116116. SEQ ID NO: 116
Proteinεequenz kodierend für Arabidopsis thaliana 12S Speicherprotein AT5g44120/MLNl_4Protein sequence coding for Arabidopsis thaliana 12S storage protein AT5g44120 / MLNl_4
117. SEQ ID NO: 117117. SEQ ID NO: 117
Nukleinsäuresequenz kodierend für Arabidopsis 12S Speicherprotein (CRB; GenBank Acc.-No. : X14313; M37248)Nucleic acid sequence coding for Arabidopsis 12S storage protein (CRB; GenBank Acc.-No .: X14313; M37248)
118. SEQ ID NO: 118118. SEQ ID NO: 118
Proteinsequenz kodierend für Arabidopsis 12S Speicherprotein (CRB)Protein sequence coding for Arabidopsis 12S storage protein (CRB)
119. SEQ ID NO: 119119. SEQ ID NO: 119
Nukleinsäuresequenz kodierend für Arabidopsis thaliana putatives 12S Speicherprotein (aus GenBank. Acc . -No . : AC003027)Nucleic acid sequence coding for Arabidopsis thaliana putative 12S storage protein (from GenBank. Acc. -No.: AC003027)
120. SEQ ID NO: 120120. SEQ ID NO: 120
Proteinsequenz kodierend für Arabidopsiε thaliana putativeε Speicherprotein (Protein_id="AAD10679.1)Protein sequence coding for Arabidopsiε thaliana putativeε storage protein (Protein_id = "AAD10679.1)
121. SEQ ID NO: 121121. SEQ ID NO: 121
Nukleinsäuresequenz kodierend für Arabidopsiε thaliana Cruciferin 12S Spwicherprotein (Atlg03890) (GenBank Acc.-No.: AY065432) 122 . SEQ ID NO : 122Nucleic acid sequence coding for Arabidopsiε thaliana cruciferin 12S Spwicherprotein (Atlg03890) (GenBank Acc.-No .: AY065432) 122. SEQ ID NO: 122
Proteinsequenz kodierend für Arabidopεiε thaliana Cruciferin 12S Speicherprotein (Atlg03890)Protein sequence coding for Arabidopεiε thaliana cruciferin 12S storage protein (Atlg03890)
123.-131:123.-131:
SEQ ID NO : 123-131 Sequenzmotive aus Nuklemsäuresequenzen verschiedener SpeicherproteinklassenSEQ ID NO: 123-131 Sequence motifs from nuclear acid sequences of different storage protein classes
132 . SEQ ID NO : 132132. SEQ ID NO: 132
Nukleinsäuresequenz kodierend für Arabidopsis thaliana Prohibitin 1 (Atphbl) (GenBank Acc.-No.: U66594)Nucleic acid sequence coding for Arabidopsis thaliana prohibitin 1 (Atphbl) (GenBank Acc.-No .: U66594)
133. SEQ ID NO: 133133. SEQ ID NO: 133
Proteinsequenz kodierend für Arabidopsiε thaliana Prohibitin 1 (Atphbl)Protein sequence coding for Arabidopsiε thaliana prohibitin 1 (Atphbl)
134. SEQ ID NO: 134 Oligonukleotidprimer OPN1134. SEQ ID NO: 134 oligonucleotide primer OPN1
135. SEQ ID NO: 135 Oligonukleotidprimer 0PN2135. SEQ ID NO: 135 oligonucleotide primer 0PN2
136. SEQ ID NO: 136 Oligonukleotidprimer OPN3136. SEQ ID NO: 136 oligonucleotide primer OPN3
137. SEQ ID NO: 137 Oligonukleotidprimer OPN4137. SEQ ID NO: 137 oligonucleotide primer OPN4
138. SEQ ID NO: 138 Oligonukleotidprimer OPN5138. SEQ ID NO: 138 oligonucleotide primer OPN5
139. SEQ ID NO: 139 Oligonukleotidprimer OPN6139. SEQ ID NO: 139 oligonucleotide primer OPN6
140. SEQ ID NO: 140 Oligonukleotidprimer OPN7140. SEQ ID NO: 140 oligonucleotide primer OPN7
141. SEQ ID NO: 141 , Oligonukleotidprimer OPN8141. SEQ ID NO: 141, oligonucleotide primer OPN8
142. SEQ ID NO: 142 Oligonukleotidprimer OPN9142. SEQ ID NO: 142 oligonucleotide primer OPN9
143. SEQ ID NO: 143 Oligonukleotidprimer OPN10143. SEQ ID NO: 143 oligonucleotide primer OPN10
144. SEQ ID NO: 144144. SEQ ID NO: 144
Nukleinεäuresequenz kodierend für sRNAi4-dsRNA zur Suppression mehrerer SpeicherproteineNucleic acid sequence coding for sRNAi4-dsRNA for the suppression of several storage proteins
145. SEQ ID NO: 145145. SEQ ID NO: 145
Ribonukleinsäuresequenz kodierend für sRNAi4-dsRNA zur Suppreεεion mehrerer Speicherproteine 146 . SEQ ID NO : 146Ribonucleic acid sequence coding for sRNAi4-dsRNA for the suppression of several storage proteins 146. SEQ ID NO: 146
Nukleinsäuresequenz kodierend für sRNAi8-dsRNA zur Suppreεsion mehrerer SpeicherproteineNucleic acid sequence coding for sRNAi8-dsRNA for the suppression of several storage proteins
147. SEQ ID NO: 147147. SEQ ID NO: 147
Ribonukleinsäureεequenz kodierend für εRNAiδ-dsRNA zur Suppression mehrerer SpeicherproteineRibonucleic acid sequence coding for εRNAiδ-dsRNA for the suppression of several storage proteins
148. SEQ ID NO: 148 Oligonukleotidprimer OPN11148. SEQ ID NO: 148 oligonucleotide primer OPN11
149..SEQ ID NO: 149 Oligonukleotidprimer OP12149..SEQ ID NO: 149 oligonucleotide primer OP12
150. SEQ ID NO: 150 Oligonukleotidprimer OPN13150. SEQ ID NO: 150 oligonucleotide primer OPN13
151. SEQ ID NO: 151 Oligonukleotidprimer OPN15151. SEQ ID NO: 151 oligonucleotide primer OPN15
152. SEQ ID NO: 152 Oligonukleotidprimer OPN16152. SEQ ID NO: 152 oligonucleotide primer OPN16
153. SEQ ID NO: 153 Oligonukleotidprimer OPN17153. SEQ ID NO: 153 oligonucleotide primer OPN17
154. SEQ ID NO: 154154. SEQ ID NO: 154
Nukleinsäuresequenz kodierend für Arabidopsis thaliana "globulin-like protein" (GenBank Acc.-No.: NM_119834)Nucleic acid sequence coding for Arabidopsis thaliana "globulin-like protein" (GenBank Acc.-No .: NM_119834)
155. SEQ ID NO: 155155. SEQ ID NO: 155
Proteinsequenz kodierend für Arabidopsiε thaliana "globulin- like protein" (Protein_id="NP_195388.1)Protein sequence coding for Arabidopsiε thaliana "globulin-like protein" (Protein_id = "NP_195388.1)
156. SEQ ID NO: 156156. SEQ ID NO: 156
Nukleinεäuresequenz kodierend für Glycine max 7S Samenglobulm (GenBank Acc.-No. : U59425)Nucleic acid sequence coding for Glycine max 7S seed globulm (GenBank Acc.-No .: U59425)
157. SEQ ID NO: 157157. SEQ ID NO: 157
Proteinsequenz kodierend für für Glycine max 7S SamenglobulinProtein sequence coding for Glycine max 7S seed globulin
158. SEQ ID NO: 158158. SEQ ID NO: 158
Nukleinsäureεequenz kodierend für Zea mayε 19kD Zein (GenBank Acc.-No. : E01144)Nucleic acid sequence coding for Zea may 19kD Zein (GenBank Acc.-No .: E01144)
159. SEQ ID NO: 159159. SEQ ID NO: 159
Proteinεequenz kodierend für Zea mayε 19kD ZeinProtein sequence coding for zea may 19kD zein
160. SEQ ID NO: 160160. SEQ ID NO: 160
Nukleinεäureεequenz kodierend für Zea mays 19kD alpha Zein Bl (GenBank Acc.-No. : AF371269) 161 . SEQ ID NO : 161Nucleic acid sequence coding for Zea mays 19kD alpha Zein Bl (GenBank Acc.-No .: AF371269) 161. SEQ ID NO: 161
Proteinsequenz kodierend für Zea mays 19kD alpha Zein BlProtein sequence coding for Zea mays 19kD alpha Zein Bl
162. SEQ ID NO: 162162. SEQ ID NO: 162
Nukleinsäuresequenz kodierend für Zea mays 19kD alpha Zein B2 (GenBank Acc.-No. : AF371270)Nucleic acid sequence coding for Zea mays 19kD alpha Zein B2 (GenBank Acc.-No .: AF371270)
163. SEQ ID NO: 163163. SEQ ID NO: 163
Proteinsequenz kodierend für Zea mays 19kD alpha Zein B2Protein sequence coding for Zea mays 19kD alpha Zein B2
164. SEQ ID NO: 164164. SEQ ID NO: 164
Nukleinsäuresequenz kodierend für Zea mays 22kD alpha-zein (GenBank Acc.-No. : X61085)Nucleic acid sequence coding for Zea mays 22kD alpha-zein (GenBank Acc.-No .: X61085)
165. SEQ ID NO: 165165. SEQ ID NO: 165
Proteinsequenz kodierend für Zea mays 22kD alpha-zeinProtein sequence coding for Zea mays 22kD alpha-zein
166. SEQ ID NO: 166166. SEQ ID NO: 166
Nukleinsäureεequenz kodierend für Oryza εativa Prolamin (GenBank Acc.-No. : AB016503)Nucleic acid sequence coding for Oryza εativa prolamin (GenBank Acc.-No .: AB016503)
167. SEQ ID NO: 167167. SEQ ID NO: 167
Proteinsequenz kodierend für Oryza sativa ProlaminProtein sequence coding for Oryza sativa prolamin
168. SEQ ID NO: 168168. SEQ ID NO: 168
Nukleinεäuresequenz kodierend für A. sativa Avenin (GenBank Acc.-No. : M38446)Nucleic acid sequence coding for A. sativa Avenin (GenBank Acc.-No.: M38446)
169. SEQ ID NO: 169169. SEQ ID NO: 169
Proteinsequenz kodierend für A. sativa AveninProtein sequence coding for A. sativa Avenin
170. SEQ ID NO: 170170. SEQ ID NO: 170
Nukleinsäuresequenz kodierend für Hordeum vulgäre C-Hordein (GenBank Acc.-No. : M36941)Nucleic acid sequence coding for Hordeum vulgar C-hordein (GenBank Acc.-No .: M36941)
171. SEQ ID NO: 171171. SEQ ID NO: 171
Proteinsequenz Teil 1 kodierend für Hordeum vulgäre C-HordeinProtein sequence part 1 coding for Hordeum vulgar C-hordein
172. SEQ ID NO: 172172. SEQ ID NO: 172
Proteinεequenz Teil 2 kodierend für Hordeum vulgäre C-HordeinProtein sequence part 2 coding for Hordeum vulgar C-hordein
173. SEQ ID NO: 173173. SEQ ID NO: 173
Nukleinεäureεequenz kodierend für Triticum aeεtivum LMW Glutenin-lDl (GenBank Acc. -No. : X13306) 174 . SEQ ID NO : 174Nucleic acid sequence coding for Triticum aetivum LMW Glutenin-IDL (GenBank Acc. -No.: X13306) 174. SEQ ID NO: 174
Proteinsequenz kodierend für Triticum aestivum LMW Glutenin-lDlProtein sequence coding for Triticum aestivum LMW Glutenin-IDL
175. SEQ ID NO: 175175. SEQ ID NO: 175
Expreεsionskassette für Arabidosis ' thaliana ACCase (GenBank Acc.-No.: D34630) alε Fuεionεprotein mit der plaεtidären Signalεequenz der Tranεketolaεe aus Tabak unter Kontrolle des Napin-PromotorsExpression cassette for Arabidosis ' thaliana ACCase (GenBank Acc.-No .: D34630) as a fusion protein with the plausoidal signal sequence of the tranεketolase from tobacco under the control of the napin promoter
176. SEQ ID NO: 176176. SEQ ID NO: 176
Proteinsequenz kodierend ein Fusionsprotein aus der Arabidosis thaliana ACCase (GenBank Acc.-No.: D34630) und der plastidären Signalsequenz der Transketolase aus TabakProtein sequence encoding a fusion protein from the Arabidosis thaliana ACCase (GenBank Acc.-No .: D34630) and the plastid signal sequence of the transketolase from tobacco
177. SEQ ID NO: 177177. SEQ ID NO: 177
Nukleotidεequenz kodierend für Arabidoεiε thaliana ACCase (GenBank Acc.-No.: D34630) alε Fuεionεprotein mit der plaεtidären Signalεequenz der Transketolase aus Tabak unter Kontrolle des Napin-PromotorsNucleotide sequence coding for Arabidoεiε 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
178. SEQ ID NO: 178178. SEQ ID NO: 178
Binärer Expreεεionεvektor für Agrobakterium vermittelte Pflanzentranεformation pSUN2-USPl ,2,3.Binary expression vector for agrobacterium-mediated plant transformation pSUN2-USPl, 2,3.
179. SEQ ID NO: 179179. SEQ ID NO: 179
Binärer Expreεεionsvektor für Agrobakterium vermittelte Pflanzentransformation pSUN2-USP.Binary expression vector for Agrobacterium-mediated plant transformation pSUN2-USP.
Abbildungenpictures
1. Fig. la-b: Alignment von Brassica 2S Albuminen1. Fig. La-b: Alignment of Brassica 2S albumins
Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nuklemsäuresequenzen mit ihren jeweiligen GenBank Acc . -No . :The nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
J02586 Brasεica napus 2S Albumin (SEQ ID NO: 31) X65040 Brassica juncea 2S Albumin (SEQ ID NO: 33) X65038 Brassica oleracea 2S Albumin (SEQ ID NO: 35) U04945 Brasεica napus cv. Topas 2S Albumin (SEQ ID NO: 37) X91799 Sinapis alba 2S Albumin (SEQ ID NO: 39) 2. Fig. 2: Alignment von Soja (Glycine max) 2S AlbuminenJ02586 Brasεica napus 2S Albumin (SEQ ID NO: 31) X65040 Brassica juncea 2S Albumin (SEQ ID NO: 33) X65038 Brassica oleracea 2S Albumin (SEQ ID NO: 35) U04945 Brasεica napus cv. Topas 2S Albumin (SEQ ID NO: 37) X91799 Sinapis alba 2S Albumin (SEQ ID NO: 39) 2. Fig. 2: Alignment of soy (Glycine max) 2S albumins
Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nuklemsäuresequenzen mit ihren jeweiligen GenBank Acc . -No . :The nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
U71194 Glycine max 2S Albumin 1 (SEQ ID NO: 41)U71194 Glycine max 2S Albumin 1 (SEQ ID NO: 41)
AF005030 Glycine max 2S Albumin (SEQ ID NO: 43)AF005030 Glycine max 2S Albumin (SEQ ID NO: 43)
3. Fig. 3a-b: Alignment von Brassica nigra, Sinapis alba und3. Fig. 3a-b: Alignment of Brassica nigra, Sinapis alba and
Helianthus annuus 2S Albuminen.Helianthus annuus 2S albumins.
Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nukleinsäureεequenzen mit ihren jeweiligen GenBank Acc . -No . :The nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
X65039 Brasεica napus 2S Albumin (SEQ ID NO: 45)X65039 Brasεica napus 2S Albumin (SEQ ID NO: 45)
X91798 Sinapis alba 2S Albumin (SEQ ID NO: 47)X91798 Sinapis alba 2S Albumin (SEQ ID NO: 47)
X06410 Helianthus annuus 2S Albumin HaG5 (SEQ ID NO: 49) X76101 Helianthus annuus 2S Albumin (SEQ ID NO: 51)X06410 Helianthus annuus 2S Albumin HaG5 (SEQ ID NO: 49) X76101 Helianthus annuus 2S Albumin (SEQ ID NO: 51)
4. Fig. 4a-b: Alignment von Helianthus annuus 2S Albuminen.4. Fig. 4a-b: Alignment of Helianthus annuus 2S albumins.
Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nukleinsäureεequenzen mit ihren jeweiligen GenBank Acc . -No . :The nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
X06410 Helianthuε annuuε 2S Albumin HaG5 (SEQ ID NO: 49) X76101 Helianthuε annuus 2S Albumin (SEQ ID NO: 51)X06410 Helianthuε annuuε 2S Albumin HaG5 (SEQ ID NO: 49) X76101 Helianthuε annuus 2S Albumin (SEQ ID NO: 51)
5. Fig. 5: Alignment von Arabidopsis thaliana 2S Albuminen.5. Fig. 5: Alignment of Arabidopsis thaliana 2S albumins.
Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nuklemsäuresequenzen mit ihren jeweiligen GenBank Acc . -No . : gi 1.166609: 951-1445 (M22032) At2Sl (SEQ ID.NO: 1) gijl66611:212-706 (M22035) At2S3 (SEQ ID NO: 3) gi|l66610:375-887 (M22034) At2S2 (SEQ ID NO: 5) gi 166612:500-1000 (M22033) At2S4 (SEQ ID NO: 7) 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 | l66610: 375-887 (M22034) At2S2 (SEQ ID NO: 5 ) gi 166612: 500-1000 (M22033) At2S4 (SEQ ID NO: 7)
6. Fig. 6a-c : DNA-Alignment von Brasεica napus IIS Speicher6. Fig. 6a-c: DNA alignment of Brasεica napus IIS memory
Proteinenproteins
Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nuklemsäuresequenzen mit ihren jeweiligen GenBank Acc . -No . :The nucleic acid sequences coding for the individual storage proteins are indicated with their respective GenBank Acc. -No. :
X59294 CRUl_Bn (SEQ ID NO: 9)X59294 CRUl_Bn (SEQ ID NO: 9)
X14555 CRUA_Bn (SEQ ID NO: 11)X14555 CRUA_Bn (SEQ ID NO: 11)
X59295 CRU2_Bn (SEQ ID NO: 13)X59295 CRU2_Bn (SEQ ID NO: 13)
X57848 CRU4_Bn (SEQ ID NO: 15)X57848 CRU4_Bn (SEQ ID NO: 15)
X62120 CRU3_BN (SEQ ID NO: 17)X62120 CRU3_BN (SEQ ID NO: 17)
(sequence ID from soybean IIS εtorage proteinε after alignement)(sequence ID from soybean IIS εtorage proteinε after alignement)
7. Fig.7a-d:7. Fig.7a-d:
DNA-Alignment von Soja IIS Speicherproteinen Angegeben sind die für die einzelnen Speicherproteinen kodierenden Nuklemsäuresequenzen mit ihren jeweiligen GenBank Acc . -No . :DNA alignment of soybean IIS storage proteins. The nucleic acid sequences coding for the individual storage proteins with their respective GenBank Acc. -No. :
GLC1: M36686; D00566 GMGLYBSU_2 (SEQ ID NO: 19)GLC1: M36686; D00566 GMGLYBSU_2 (SEQ ID NO: 19)
GLC2: X15122 GMGY2_8 (SEQ ID NO: 21)GLC2: X15122 GMGY2_8 (SEQ ID NO: 21)
GLC3: X02626 GMGLYRl_3 (SEQ ID NO: 23)GLC3: X02626 GMGLYRl_3 (SEQ ID NO: 23)
GLC4: Ml0962 GMGLYAB_4 (SEQ ID NO: 25)GLC4: Ml0962 GMGLYAB_4 (SEQ ID NO: 25)
GLC5: X15123; S44896 GMGY3_7 (SEQ ID NO: 27)GLC5: X15123; S44896 GMGY3_7 (SEQ ID NO: 27)
8. Fig. 8:8. Fig. 8:
Ergebnisse der Meεεung des Lipidgehalts (TAG) in T3 Samen tranεgener Arabidopsis Pflanzen, die mit dem Konstrukt Napin-TK-AtACCase transformiert wurden. Samen von jeweils 10 individuellen T2 Pflanzen pro unabhängiger transgener Linie wurden wie in Beispiel 9 beschrieben gemessen. Die Menge an TAG in Prozent entspricht dem Anteil an der Gesamtmasse des Trockengewichts der Samen. Wt bezeichnet die nicht-transformierte Kontrolle, 5L, 3L, 12L und IL sind die unabhängigen transgenen Linien. Die Federbalken beschreiben den Standardfehler aus allen gemessenen Werten. 9 . Fig . 9 :Results of the measurement of the lipid content (TAG) in T3 seeds of transgenic Arabidopsis plants which were transformed with the construct Napin-TK-AtACCase. Seeds from 10 individual T2 plants per independent transgenic line were measured as described in Example 9. The amount of TAG in percent corresponds to the proportion of the total mass of the dry weight of the seeds. Wt denotes the non-transformed control, 5L, 3L, 12L and IL are the independent transgenic lines. The cantilevers describe the standard error from all measured values. 9. Fig. 9:
Schematische Darstellung' der Speicherprotein-Suppressions- konstrukte .Schematic representation 'of the storage protein suppression- constructs.
A: Insert (1155 bp) auε Vektor pCR2. l-AtCRU3-RNAi (5025 bp) kodierend für eine die AtCru3-Expreεsion supprimierende dsRNA.A: Insert (1155 bp) from vector pCR2. l-AtCRU3-RNAi (5025 bp) coding for a dsRNA which suppresses the AtCru3 expression.
B: Insert aus Vektor pCR2. l-εRNAi4 (1) und pCR2. l-εRNAi8 (2) kodierend für eine die AtCru3-, AtCRB und At2S3-Expreεεion εupprimierende dεRNA. In den beiden Konεtrukten εind die "sense"-RNA-Stränge und "anti- sense"-RNA-Stränge für die einzelnen zu supprimierenden Ziele unterεchiedlich angeordnet. Schraffierte Bereiche stellen Intronsequenzen (Linker) dar.B: Insert from vector pCR2. l-εRNAi4 (1) and pCR2. l-εRNAi8 (2) coding for a dεRNA which suppresses the AtCru3, AtCRB and At2S3 expression. In the two constructs, the "sense" RNA strands and "anti-sense" RNA strands are arranged differently for the individual targets to be suppressed. Hatched areas represent intron sequences (linkers).
10. -Fig.l0: Ergebnis der quantitativen Ölbestimmung.10. -Fig.l0: Result of the quantitative oil determination.
Samen von unabhängigen tranεgenen Pflanzen der TI Generation wurden analysiert. Angegeben ist der Fettsäuregehalt in Gewichtsprozent des Samens (%FA [w/w] ) . Mehrere unabhängige Linien (2,1; 3-1; 5-1; 12-1; 14-1; 16-1; 17-1) zeigten einen signifikant erhöhten Ölgehalt (biε zu 38 % erhöhten Ölgehal- ten für Linie 16-1) verglichen zu Kontrollpflanzen (C) .Seeds from independent transgenic plants of the TI generation were analyzed. The fatty acid content is given in percent by weight of the seed (% FA [w / w]). Several independent lines (2.1; 3-1; 5-1; 12-1; 14-1; 16-1; 17-1) showed a significantly increased oil content (up to 38% higher oil content for line 16- 1) compared to control plants (C).
BeiεpieleBeiεpiele
Allgemeine Methoden:General methods:
Alle Chemikalien, wenn nicht anders erwähnt, stammen von den Firmen Fluka (Buchs), Merck (Darmstadt), Roth (Karlsruhe), ServaUnless otherwise stated, all chemicals come from the companies Fluka (Buchs), Merck (Darmstadt), Roth (Karlsruhe), Serva
(Heidelberg) und Sigma (Deisenhofen) . Restriktionεenzyme, DNA- modifizierende Enzyme und Molekularbiologie-Kitε wurden von den Firmen Amerεham-Pharmacia (Freiburg) , Biometra (Göttingen) , Röche(Heidelberg) and Sigma (Deisenhofen). Restriction enzymes, DNA-modifying enzymes and molecular biology kits were obtained from Amerεham-Pharmacia (Freiburg), Biometra (Göttingen), Röche
(Mannheim) , New England Biolabε (Schwalbach) , Novagen (Madiεon, Wiεconεin, USA) , Perkin-Elmer (Weiterstadt) , Qiag'en (Hilden) , Stratagen (Amsterdam, Niederlande) , Invitrogen (Karlsruhe) und Ambion (Cambridgeshire, United Kingdom) . Die verwendeten Reagenzien wurden entsprechend der Angaben des Herstellerε eingeεetzt .(Mannheim), New England Biolabε (Schwalbach), Novagen (Madiεon, Wiεconεin, USA), Perkin-Elmer (Weiterstadt), Qiag ' en (Hilden), Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom) . The reagents used were used in accordance with the manufacturer's instructions.
Die chemiεche Syntheεe von Oligonukleotiden kann beispielsweiεe, in bekannter Weise, nach der Phoεphoamiditmethode (Voet, Voet, 2. Auflage, Wiley Press New York, Seite 896-897) erfolgen. Die im Rahmen der vorliegenden Erfindung durchgeführten Klonierungs- εchritte wie z.B. Restriktionεεpaltungen, Agaroεegelelektro- phoreεe, Reinigung von DNA-Fragmenten, Tranεfer von Nukleinsäuren auf Nitrozellulose und Nylonmembranen, Verknüpfen von DNA-Fragmenten, Tranεfor ation von E. coli Zellen, Anzucht von Bakterien, Vermehrung von Phagen und Sequenzanalyεe rekombinanter DNA werden wie bei Sambrook et al . (1989) Cold Spring Harbor Laboratory Preεε; ISBN 0-87969-309-6 beεchrieben durchgeführt. Die Sequenzierung rekombinanter DNA-Moleküle erfolgt mit einem Laεerfluoreεzenz-DNA-Sequenzierer der Firma ABI nach der Methode von Sanger (Sanger et al . (1977) Proc Natl Acad Sei USA 74:5463-5467).The chemical synthesis of 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 Preεε; 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).
Beispiel 1: Allgemeine VerfahrenExample 1: General procedures
Die Pflanze Arabidopsis thaliana repräsentiert ein Mitglied der höheren Pflanzen (Samenpflanzen) . Diese Pflanze ist eng verwandt mit anderen Pflanzenarten aus der Familie der Cruciferen wie z.B. Brasεica napus, aber auch mit anderen Pflanzenfamilien der Dikotyledonen. Aufgrund des hohen Grades an Homologie ihrer DNA-Sequenzen bzw. Polypeptldεequenzen kann Arabidopsis thaliana als Modellpflanze für andere Pflanzenarten eingesetzt werden.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 Brasεica 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.
a) Anzucht von Arabidopsiε Pflanzena) Cultivation of Arabidopsiε plants
Die Pflanzen werden entweder auf Murashige-Skoog Medium mit ,5 % Saccharose (Ogas et al. (1997) Science 277:91-94) oder auf Erde gezogen (Focks & Benning (1998) Plant Physiol 118:91-101) . Um einheitliche Keimungs- und Blühzeiten zu erreichen, werden die Samen nach Ausplattieren bzw. Aus- εtreuen auf Erde zwei Tage bei 4°C stratifiziert. Nach der Blüte werden die Schoten markiert . Entsprechend der Markierungen werden dann Schoten mit einem Alter von 6 bis 20 Tagen nach der Blüte geerntet.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). In order to achieve uniform germination and flowering times, the seeds are stratified at 4 ° C for two days after plating or sprinkling on soil. After flowering, the pods are marked. According to the markings, pods are harvested 6 to 20 days after flowering.
b) Isolierung von total RNA und poly-A+ RNA aus Pflanzenb) Isolation of total RNA and poly-A + RNA from plants
Für die Herstellung von Suppressionskonεtrukten wird RNA bzw. polyA+ RNA isoliert. RNA wurde aus Schoten von Arabidopsis Pflanzen nach folgender Vorschrift isoliert: Schotenmaterial im Alter von 6 bis 20 Tage nach Blühte wurde geerntet und .in flüssigem Stickstoff schockgefroren. Daε Material wurde vor der weiteren Verwendung bei -80°C gelagert. 75 mg des Materials wurde im gekühlten Mörεer zu einem feinem Pulver gemahlen und mit 200 μL deε Lysis-Pufferε auε dem Ambion RNA- queoε-Kit verεetzt. Die Isolierung der totalen RNA wurde dann nach Herstellerangaben durchgeführt. Die RNA wurde mit 50 μL Elutionspuffer (Ambion) eluiert und die Konzentration durch Absorption einer 1 zu 100 verdünnten Lösung am Photometer (Eppendorf) bei 260 nm bestimmt. 40 μg/ml RNA entspricht dabei einer Absorption von 1. Die RNA-Lösungen wurden mit RNAεe freiem Wasser auf eine Konzentration von 1 μg/μL eingestellt. Die Konzentrationen wurden durch Agarosegelelektrophorese überprüft. Zur Isolierung von polyA+ RNA wurde oligo(dT)- Zelluloεe von Amerεham Pharmacia nach Herεtellerangaben verwendet. RNA bzw. polyA+ RNA wurde bei -70°C gelagert.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 μL of the lysis buffer from the Ambion RNAqueoε kit. The isolation of the total RNA was then carried out according to the manufacturer's instructions. The RNA was eluted with 50 μL elution buffer (Ambion) and the concentration was determined by absorbing a solution diluted 1 to 100 on a photometer (Eppendorf) at 260 nm. 40 μg / ml RNA corresponds to an absorption of 1. The RNA solutions were with RNAεe free water adjusted to a concentration of 1 μg / μL. The concentrations were checked by agarose gel electrophoresis. For the isolation of polyA + RNA, oligo (dT) cellulose from Amerεham Pharmacia according to the manufacturer's instructions was used. RNA or polyA + RNA was stored at -70 ° C.
c) Konstruktion der cDNA-Bankc) Construction of the cDNA library
Zur Konstruktion der cDNA-Bank aus Arabidopsis Schoten-RNA wurde die Erststrangsynthese unter Verwendung von Reverser Transkriptase aus Maus-Leukämie-Virus (Clontech) und Oligo- d(T) -Primern, die Zweitstrangεyntheεe durch Inkubation mit DNA-Polymerase I, Klenow-Enzym und RNAse H-Spaltung bei 12°C (2 Std.), 16°C (1 Std.) und 22°C (1 Std.) erzielt. Die Reak- tion wurde durch Inkubation bei 65°C (10 min) gestoppt und anschließend auf Eis überführt. Doppelsträngige DNA-Moleküle wurde mit T4-DNA-Polymerase (Röche, Mannheim) bei 37°C (30 min) mit glatten Enden versehen. Die Nukleotide wurden durch Phenol/Chloroform-Extraktion und Sephadex-G50-Zentrifugier- εäulen entfernt. EcoRI/XhoI-Adapter (Pharmacia, Freiburg, Deutεchland) wurden mittelε T4-DNA-Ligaεe (Röche, 12°C, über Nacht) an die cDNA-Enden ligiert, mit Xhol nachgeεchnitten und durch Inkubation mit Polynukleotidkinaεe (Röche, 37°C, 30 min) phoεphoryliert. Dieεes Gemisch wurde der Trennung auf einem Low-Melting-Agarose-Gel unterworfen. DNA-Moleküle über 300 Basenpaaren wurden aus dem Gel eluiert, Phenol-extra- hiert, auf Elutip-D-Säulen (Schleicher und Schüll, Dassel, Deutschland) konzentriert und an Vektorarme ligiert und in la bda-ZAPII-Phagen oder lambda-ZAP-Expreεs-Phagen unter Verwendung deε Gigapack Gold-Kits (Stratagene, Amsterdam, Niederlande) verpackt, wobei Material des Herstellerε verwendet und εeine Anweiεungen befolgt wurden.To construct the cDNA library from Arabidopsis pod RNA, the first strand synthesis using reverse transcriptase from mouse leukemia virus (Clontech) and oligod (T) primers, the second strand synthesis by incubation with DNA polymerase I, Klenow- Enzyme and RNAse H cleavage achieved at 12 ° C (2 hrs), 16 ° C (1 hrs) and 22 ° C (1 hrs). The reaction was stopped by incubation at 65 ° C. (10 min) and then transferred to ice. Double-stranded DNA molecules were blunt-ended with T4-DNA polymerase (Röche, Mannheim) at 37 ° C (30 min). 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-Ligaεe (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 -Expreεs phages packaged using the Gigapack Gold Kit (Stratagene, Amsterdam, the Netherlands), using the manufacturer's material and following its instructions.
d) Isolierung von genomischer DNA aus Pflanzen wie Arabidopsis thaliana oder Brassica napus (CTAB-Methode)d) isolation of genomic DNA from plants such as Arabidopsis thaliana or Brassica napus (CTAB method)
Zur Isolierung genomischer DNA aus Pflanzen wie Arabidopsis thaliana oder Brassica napus werden ca. 0,25 g Blattmaterial junger Pflanzen im vegetativen Stadium in flüssigem Stickstoff zu feinem Pulver gemörsert. Das pulveriεierte Pflanzenmaterial wird zusammen mit 1 ml 65°C-warmem CTAB I-Puffer (CTAB: Hexadecyltrimethylammoniumbromid, auch genannt Cetyl- trimethylammonivimbromid; Sigma Kat.-Nr.: H6269) und 20 μl ß-Mercaptoethanol in einen vorgewärmten zweiten Mörser gegeben und nach vollεtändiger Ho ogeniεierung wird der Extrakt in ein 2 ml Eppendorf-Gefäß überführt und für 1 h - bei 65°C unter regelmäßiger, vorεichtiger Durchmischung inkubiert. Nach Abkühlung auf Raumtemperatur wird der Ansatz mit 1 ml Chlorofor /Octanol (24:1, mit IM Tris/HCl, pH 8,0 ausgeschüttelt) durch langsameε Invertieren extrahiert und zur Phaεentrennung für 5 min bei 8,500 rpm (7,500 x g) und Raumtemperatur zentrifugiert . Anεchließend wird die wäεεrige Phase erneut mit 1 ml Chloroform/Octanol extrahiert, zentrifugiert und durch Invertieren mit 1/10 Volumen auf 65°C vorgewärmtem CTAB II-Puffer sorgfältig gemiεcht. Anεchließend wird der Anεatz durch vorsichtiges Schwenken mit 1 ml Chloro- form/Octanol-Gemiεch (siehe oben) versetzt und zur erneuten Phasentrennung für 5 min bei 8,500 rpm (7,500 x g) und Raumtemperatur zentrifugiert. Die wässrige untere Phase wird in ein friεcheε Eppendorf-Gefäß überführt und die obere organiεche Phase wird in einem frischen Eppendorf-Gefäß erneut für 15 min bei 8,500 rpm (7,500 x g) und Raumtemperatur zentrifugiert . Die hieraus resultierende wäsεrige Phaεe wird mit der wäεsrigen Phaεe deε vorherigen Zentri- fugationsschrittes vereinigt und der gesamte Ansatz mit exakt demεelben Volumen vorgewärmtem CTAB III-Puffer versetzt. Es folgt eine Inkubation bei 65°C, bis die DNA in Flocken ausfällt. Dies kann bis zu 1 h dauern oder durch Inkubation bei 37°C über Nacht erfolgen. Das aus dem anschließenden Zentrifugationsschritt (5 min, 2000 rpm (500 x g) , 4°C) resultierende Sediment wird mit 250 μl auf 65°C vorgewärmtem CTAB IV-Puffer versetzt und für mindeεtenε 30 min bzw. bis zur vollständigen Auflöεung deε Sedimentε bei 65°C inkubiert. Anεchließend wird die Löεung zur Fällung der DNA mit 2,5 Volumina eiεkaltem Ethanol vermiεcht und für lh bei -20°C inkubiert. Alternativ kann der Ansatz mit 0.6 Volumina Iso- propanol vermischt und ohne weitere Inkubation sofort für 15 min bei 8,500 rpm (7,500 x g) und 4°C zentrifugiert werden. Die sedimentierte DNA wird durch Invertieren deε Eppendorf-Gefäßes zweimal mit je 1 ml 80%igem eiskaltem Ethanol gewaεchen, nach jedem Waεchschritt erneut zentrifugiert (5 min, 8,500 rpm (7,500 x g) , 4°C) und anschließend für ca. 15 min luftgetrocknet. Abschließend wird die DNA in 100 μl TE mit 100 μg/ml RNase resuεpendiert und für 30 min bei Raumtemperatur inkubiert. Die DNA Lösung ist nach einer weiteren Inkubationsphase über Nacht bei 4°C homogen und kann für weiterführende Experimente verwendet werden.In order to isolate genomic DNA from plants such as Arabidopsis thaliana or Brassica napus, approx. 0.25 g of leaf material from young plants in the vegetative stage is ground into fine powder in liquid nitrogen. The powdered plant material is added to a preheated second mortar together with 1 ml of 65 ° C warm CTAB I buffer (CTAB: hexadecyltrimethylammonium bromide, also called cetyltrimethylammonivimbromide; Sigma cat. No .: H6269) and 20 μl ß-mercaptoethanol after complete hoogenization, the extract is transferred to a 2 ml Eppendorf tube and for 1 h - at 65 ° C with regular, careful mixing incubated. After cooling to room temperature, 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. Subsequently, 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. This can take up to 1 h or can be done by incubation at 37 ° C overnight. The sediment resulting from the subsequent centrifugation step (5 min, 2000 rpm (500 × g), 4 ° C.) is mixed with 250 μl 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. Alternatively, 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 μl TE with 100 μg / 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.
Lösungen für CTAB:Solutions for CTAB:
Lösung I (für 200 ml) :Solution I (for 200 ml):
100- mM Tris/HCl pH 8,0 (2,42 g)100 mM Tris / HCl pH 8.0 (2.42 g)
1,4 M NaCl (16,36 g)1.4 M NaCl (16.36 g)
20 mM EDTA (8,0 ml von 0,5 M Stammlösung) 2 % (w/v) CTAB ( 4 , 0 g)20 mM EDTA (8.0 ml of 0.5 M stock solution) 2% (w / v) CTAB (4.0 g)
Jeweils vor der Verwendung werden frisch zugesetzt: 2 % ß-Mercaptoethanol (20 μl für 1 ml Lösung I) .Before each use, freshly added: 2% ß-mercaptoethanol (20 ul for 1 ml of solution I).
Lösung II (für 200 ml) : 0,7 M NaCl (8,18 g) 10 % (w/v) CTAB (20 g)Solution II (for 200 ml): 0.7 M NaCl (8.18 g) 10% (w / v) CTAB (20 g)
Lösung III (für 200 ml) :Solution III (for 200 ml):
50 mM Tris/HCl pH 8,0 (1,21 g)50 mM Tris / HCl pH 8.0 (1.21 g)
10 mM EDTA (4 ml 0,5 M von 0,5 M Stammlösung)10 mM EDTA (4 ml 0.5 M of 0.5 M stock solution)
1 % (w/v) CTAB (2,0 g)1% (w / v) CTAB (2.0 g)
Lösung IV (High-salt TE) (für 200 ml) : 10 mM Tris/ HC1 pH 8,0 (0,242 g) 0,1 mM EDTA (40 μl 0.5 M Stammlösung) 1 M NaCl (11, 69 g)Solution IV (high-salt TE) (for 200 ml): 10 mM Tris / HC1 pH 8.0 (0.242 g) 0.1 mM EDTA (40 μl 0.5 M stock solution) 1 M NaCl (11.69 g)
Chloroform/Octanol (24:1) (für 200 ml):Chloroform / octanol (24: 1) (for 200 ml):
192 ml Chloroform192 ml chloroform
8 ml Octanol Die Mischung wird 2x mit 1 M TriεHCl pH 8 , 0 auεgeεchüttelt und vor Licht geschützt gelagert.8 ml octanol The mixture is shaken twice with 1 M TriεHCl pH 8.0 and stored protected from light.
Beispiel 2 : Herstellung von SuppresεionεkonstruktenExample 2: Production of suppression constructs
Ausgehend von der genomiεcher Arabidopsis thaliana DNA oder cDNA wurden über PCR mittels der aufgeführten Oligonukleotide folgende Fragmente von Speicherproteinsequenzen amplifiziert . Dabei kam . nachfolgendes PCR Protokoll zum Einεatz : -Starting from the genomic Arabidopsis thaliana DNA or cDNA, the following fragments of storage protein sequences were amplified by PCR using the oligonucleotides listed. It came. following PCR protocol for use: -
Zusammensetzung des PCR-Ansatzes (50 μL) :Composition of the PCR approach (50 μL):
5,00 μL Template cDNA oder genomische DNA (ca. 1 μg)5.00 μL template cDNA or genomic DNA (approx. 1 μg)
5,00 μL lOx Puffer (Advantage-Polymerase) + 25 mM MgCl2 5.00 μL 10X buffer (Advantage polymerase) + 25 mM MgCl 2
5,00 μL 2mM dNTP5.00 µL 2mM dNTP
1,25 μL je Primer (10 pmol/μL)1.25 μL per primer (10 pmol / μL)
0,50 μL Advantage-Polymeraεe (Clontech)0.50 μL Advantage polymerase (Clontech)
PCR-Programm: Anfangsdenaturierung für 2 min bei 95°C, dann 35 Zyklen mit 45 sec 95°C, 45 sec 55°C und 2 min 72°C. Abschließende Extension von 5 min bei 72°C. Suppreεsionskonstrukt für Arabidopεis thaliana 12S Speicherprotein AtCRU3 (SEQ ID NO: 111 bzw. 112; GenBank Acc.-No: U66916) :PCR program: initial denaturation for 2 min at 95 ° C, then 35 cycles with 45 sec 95 ° C, 45 sec 55 ° C and 2 min 72 ° C. Final extension of 5 min at 72 ° C. Suppression construct for Arabidopεis thaliana 12S storage protein AtCRU3 (SEQ ID NO: 111 or 112; GenBank Acc.-No: U66916):
Für die den senεe-Strang der dsRNA und den Linker kodierende Sequenz wurde aus genomischer Arabidopsis thaliana DNA mit nachfolgendem Oligonukleotid-Primerpaar ein Exonbereich mit dem vollständigen anschließenden Intron einεchließlich der an daε Intron anεchließenden Spleiß-Akzeptorεequenz (Basenpaar 1947 bis 2603 der Sequenz mit der GenBank Acc.-No: U66916) amplifiziert :For the sequence encoding the senεe strand of the dsRNA and the linker, 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:
0NP1 (SEQ ID NO: 134) :0NP1 (SEQ ID NO: 134):
5 -ATAAGAATGCGGCCGCGTGTTCCATTTGGCCGGAAACAAC-3 '5 -ATAAGAATGCGGCCGCGTGTTCCATTTGGCCGGAAACAAC-3 '
0NP2 (SEQ ID NO: 135) :0NP2 (SEQ ID NO: 135):
5 Λ -CCCGGATCCTTCTGTAACATTTGACAAAACATG-3 '5 Λ -CCCGGATCCTTCTGTAACATTTGACAAAACATG-3 '
Daε PCR-Produkt wurden in den pCR2.1-TOPO Vektor (Invitrogen) gemäß Herεtellerangaben kloniert, resultierend in dem pCR2.1-1 Vektor und die Sequenz überprüft.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.
Für die den antisense-Strang der dεRNA kodierende Sequenz wurde auε Arabidopεis thaliana cDNA lediglich daε gleiche Exon wie oben (Baεenpaar 1947 biε 2384) mit dem nachfolgenden Primerpaar amplifiziert:For the sequence encoding the antisense strand of the dεRNA, only the same exon as above (base pair 1947 to 2384) was amplified with the following primer pair from Arabidopεis thaliana cDNA:
0NP3 (SEQ ID NO: 136) :0NP3 (SEQ ID NO: 136):
5 'ATAAGAATGCGGCCGCGTGTTCCATTTGGCCGGAAACAAC-S '5 'ATAAGAATGCGGCCGCGTGTTCCATTTGGCCGGAAACAAC-S'
0NP4 (SEQ ID NO: 137) :0NP4 (SEQ ID NO: 137):
5 λ ATAAGAATGCGGCCGCGGATCCACCCTGGAGAACGCCACGAGTG-3 '5 λ ATAAGAATGCGGCCGCGGATCCACCCTGGAGAACGCCACGAGTG-3 '
Daε PCR-Produkt wurden in den pCR2.1-TOPO Vektor (Invitrogen) gemäß Herεtellerangaben kloniert, reεultierend in dem pCR2.1-2 Vektor und die Sequenz überprüft.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.
0,5 μg von Vektor pCR2.1-l wurden mit dem Reεtriktionsenzym BamHI (New England Biolabs) für 2 Stunden nach Herstellerangaben inkubiert und dann für 15 min mit alkalischer Phosphatase (New England Biolabs) dephosphoryliert . Der so präparierte Vektor (1 μL) wurde dann mit dem aus Vektor pCR2.1-2 gewonnenen Fragment ligiert . Dazu wurden 0,5 μg von Vektor pCR2.1-2 2 Stunden mit BamHI (New England Biolabε) verdaut und die DNA-Fragmente per Gelelektorphorese aufgetrennt. Das neben dem Vektor (3,9 kb) entstandene 489 bp große Stück wurde aus dem Gel ausgeschnitten und mit dem "Gelpurification"-Kit (Qiagen) nach Herεtellerangaben aufgereinigt und mit 50 μL Elutionspuffer eluiert. 10 μL des Eluatε wurden mit Vektor pCR2.1-l (s.o.) über Nacht bei 16°C ligiert (T4 Ligase, New England Biolabs) . Die Ligations- produkte wurden dann in TOP10 Zellen (Stratagene) nach Herstellerangaben transformiert und entsprechend selektioniert . Positive Klone wurden mit dem Primerpaar ONP1 und 0NP2 durch PCR verifiziert. Der erhaltene Vektor pCR2.l-AtCRU3-RNAi • wurde dann für 2 Stunden mit Notl (New England Biolabs) inkubiert, mit Klenow-Fragment "geblunted" und die DNA-Fragmente über Gelelektorphorese analyεiert. Daε 1155 bp große Fragment wurde dann in den mit StuI geεchnittenen, dephosphorylierten binären Vektor pSUN2-USPl, 2 ,3 (SEQ ID NO: 178; siehe Beispiel 5) ligiert. Bei dem Vektor pSUN2-USPl,2 , 3 handelt eε sich um ein Derivat des Vektors pPZPlll ( (Hajdukiewicz, P et al . 1994 Plant Mol Biol 25:989-994), in dem drei Expresεionskassetten, jeweils unter Kontrolle des USP-Promotor , vorliegen. Vektoren mit der gewünschten Orientierung des Inserts wurden mittels Restriktionsverdau und Sequenzierung ermittelt. Daε entstehende Konstrukt trägt die Bezeichnung pSUN2-USP-RNAi-a. Die für die dεRNA kodierende Nukleinsäuresequenz ist durch SEQ ID NO: 105 beschrieben.0.5 μg 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 μL) was then ligated with the fragment obtained from vector pCR2.1-2. For this purpose, 0.5 μg of vector pCR2.1-2 was digested with BamHI (New England Biolabε) for 2 hours and the 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 μL elution buffer. 10 μL 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. 1994 Plant Mol Biol 25: 989-994), in which three expression cassettes, each under the control of the USP promoter Vectors with the desired orientation of the insert were determined by restriction digestion and sequencing, and the resulting construct is called pSUN2-USP-RNAi-a. The nucleic acid sequence coding for the dεRNA is described by SEQ ID NO: 105.
b) Suppressionεkonεtrukt für Arabidopεiε thaliana 12S Speicherprotein AtCRB (SEQ ID NO: 117 bzw. 118; GenBank Acc.-No: X14313; M37248) :b) Suppression construct for Arabidopεi thaliana 12S storage protein AtCRB (SEQ ID NO: 117 or 118; GenBank Acc.-No: X14313; M37248):
Für die den εenεe-Strang und antiεenεe-Strang der dsRNA wird mit nachfolgendem Oligonukleotid-Primerpaar ein Exonbereich (Basenpaar 601 bis 1874 der Sequenz mit der GenBank Acc.-No: M37248) auε Arabidopεiε thaliana cDNA amplifiziert :For the εenεe strand and antiεenεe strand of the dsRNA, an exon region (base pair 601 to 1874 of the sequence with GenBank Acc.-No: M37248) from Arabidopεiε thaliana cDNA is amplified with the following oligonucleotide primer pair:
ONP5 (SEQ ID NO: 138) :ONP5 (SEQ ID NO: 138):
5 λ ATAAGAATGCGGCCGCGGATCCCTCAGGGTCTTTTCTTGCCCACT-3 '5 λ ATAAGAATGCGGCCGCGGATCCCTCAGGGTCTTTTCTTGCCCACT-3 '
ONP6 (SEQ ID NO: 139) :ONP6 (SEQ ID NO: 139):
5 -CCGCTCGAGTTTACGGATGGAGCCACGAAG-3 '5 -CCGCTCGAGTTTACGGATGGAGCCACGAAG-3 '
Daε PCR-Produkt wird in den pCR2.1-T0P0 Vektor (Invitrogen) gemäß Herεtellerangaben kloniert, resultierend in dem pCR2.1-3 Vektor und die Sequenz überprüft.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.
Für den als Linker fungierenden Bereich wird auε Arabidopsis thaliana genomischer DNA ein Intron mit den entsprechenden Spliceakzeptor und -donorSequenzen der flankierenden Exons (Baεenpaar 1874 biε 2117 der Sequenz mit der GenBank Acc.-No: M37248) mit dem nachfolgenden Primerpaar amplifiziert :For the region functioning as a linker, 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:
0NP7 (SEQ ID NO: 140) :0NP7 (SEQ ID NO: 140):
5 *-CCGCTCGAGGTAAGCTCAACAAATCTTTAG-3 '5 * -CCGCTCGAGGTAAGCTCAACAAATCTTTAG-3 '
0NP8 (SEQ ID NO: 141) :0NP8 (SEQ ID NO: 141):
5 λ -ACGCGTCGACGCGTTCTGCGTGCAAGATATT-3 '5 λ -ACGCGTCGACGCGTTCTGCGTGCAAGATATT-3 '
Daε PCR-Produkt wird in den pCR2.1-T0P0 Vektor (Invitrogen) gemäß Herεtellerangaben kloniert, resultierend in dem pCR2.1-4 Vektor und die Sequenz überprüft.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.
Das Konεtrukt für AtCRB wirde in einer ähnlichen Strategie wie für AtCRU3 erläutert, erstellt. Vektor pCR2.1-3 wird mit mit Xhol (New England Biolabs) für 2 Stunden inkubiert und dephosphoryliert (alkalische Phosphataεe, New England Bio- labε) . Vektor pCR2.1-4 wird ebenfallε mit Xhol in derεelben Weiεe inkubiert und die Gelfragmente per Gelelektrophoreεe aufgetrennt . Die entsprechenden Fragmente werden in der unter AtCRU3 beschriebenen Art und Weise aufgereinigt und ligiert, resultierend nach Bakterientransformation in dem Vektor pCR2.1-AtCRB Exon/Intron. Dieser Vektor wird für 2 Stunden mit Xbal (NEB) , anschließend für 15 min mit Klenow-Fragment (NEB) , dann für 2 Stunden mit Sall inkubiert und zuletzt 15 min mit alkaliεcher Phosphatase (NEB) behandelt. Parallel wird der Vektor pCR2.1-3 mit BamHI (NEB), dann 15 min mit Klenow-Fragment und anschließend 2 Stunden mit Xhol (NEB) inkubiert. Das Exon-Fragment von AtCRB wird nach Gelelektrophorese isoliert, gereinigt und zur Ligation eingeεetzt. Beide Fragmente werden dann ligiert und der Vektor pCR2.1-AtCRB-RNAi resultiert .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. In parallel, 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). 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.
Der erhaltene Vektor pCR2.1-AtCRB-RNAi wird dann für 2 Stunden mit HindiII und Pvul verdaut und 15 min mit Klenow- Fragment inkubiert (blunten) . Das auεgeεchnittene Fragment wird über Gelelektorphorese isoliert und dann für die Ligation eingesetzt. Daε entεprechende Fragment wird dann in den mit EcoRV geεchnittenen, dephoεphorylierten Vektor pSUN2-USP-RNAi-a (εiehe oben) ligiert. Vektoren mit der gewünschten Orientierung des Inεertε werden mittelε Restriktionsverdau und Sequenzierung ermittelt. Daε entstehende Konstrukt trägt die Bezeichnung pSUN2-USP-RNAi-b. Die für die dsRNA kodierende Nukleinsäuresequenz ist durch SEQ ID NO: 107 beεehrieben. Suppresεionεkonstrukt für Arabidopsiε thaliana 2S Speicherprotein At2S3 (SEQ ID NO: 3 bzw. 4; GenBank Acc.-No: M22035) :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. Suppressεionεstrukt for Arabidopsiε thaliana 2S storage protein At2S3 (SEQ ID NO: 3 or 4; GenBank Acc.-No: M22035):
Für die den εenεe-Strang und antiεense-Strang der dsRNA wird auε Arabidopεiε thaliana cDNA mit nachfolgendem Oligo- nukleotid-Primerpaar ein Exonbereich (Basenpaar 212 bis 706 der Sequenz mit der GenBank Acc.-No: M22035) amplifiziert :For the εenεe strand and antiεense strand of the dsRNA, an exon region (base pair 212 to 706 of the sequence with the GenBank Acc.-No: M22035) is amplified from Arabidopεiε thaliana cDNA with the subsequent pair of oligonucleotides and primers:
0NP9 (SEQ ID NO: 142) :0NP9 (SEQ ID NO: 142):
5 λ -ATAAGAATGCGGCCGCGGATCCATGGCTAACAAGCTCTTCCTCGTC-3 '5 λ -ATAAGAATGCGGCCGCGGATCCATGGCTAACAAGCTCTTCCTCGTC-3 '
ONP10 (SEQ ID NO: 143):ONP10 (SEQ ID NO: 143):
5 -ATAAGAATGCGGCCGCGGATCCCTAGTAGTAAGGAGGGAAGAAAG-3 '5 -ATAAGAATGCGGCCGCGGATCCCTAGTAGTAAGGAGGGAAGAAAG-3 '
Das PCR-Produkt wird in den pCR2.1-TOPO Vektor (Invitrogen) gemäß Herstellerangaben kloniert, resultierend in dem pCR2.1-5 Vektor und die Sequenz überprüft .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.
Für den als Linker fungierenden Bereich wird das gleiche Intron wie unter b) mit den Primern OPN 7 und OPN 8 apli- fiziert eingesetzt.The same intron as used under b) with the primers OPN 7 and OPN 8 is used for the area functioning as a linker.
Das Konstrukt für At2S3 wird in einer ähnlichen Strategie wie für AtCRU3 erläutert, erstellt. Vektor pCR2.1-5 wird mit mit Xhol (New England Biolabs) für 2 Stunden inkubiert und de- phoεphoryliert (alkalische Phoεphataεe, New England Biolabε) . Vektor pCR2.1-3 wird ebenfallε mit Xhol in derselben Weise inkubiert und die Gelfragmente per Gelelektrophorese aufgetrennt. Die entsprechenden Fragmente werden in der unter At- CRU3 beschriebenen Art und Weise aufgereinigt und ligiert, resultierend nach Bakterientransformation in dem Vektor pCR2.1-At2S3 Exon/Intron. Dieser Vektor wird für 2 Stunden mit Sall (NEB) , anschließend für 15 min mit Klenow-Fragment (NEB) inkubiert und zuletzt 15 min mit alkalischer Phosphataεe (NEB) behandelt. Parallel wird der Vektor pCR2.1-5 mit BamHI (NEB) und dann 15 min mit Klenow-Fragment inkubiert. Daε Exon-Fragment von At2S3 wird nach Gelelektorphorese isoliert, gereinigt und zur Ligation eingesetzt. Beide Fragmente werden dann ligiert und der Vektor pCR2. l-At2S3-RNAi resultierte.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). In parallel, the vector pCR2.1-5 is incubated with BamHI (NEB) and then for 15 min with Klenow fragment. 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.
Der erhaltene Vektor pCR2. l-At2S3-RNAi wird dann für 2 Stunden mit Hindlll und Xbal (New England Biolabs) verdaut und 15 min mit Klenow-Fragment inkubiert (blunten) . Das ausgeschnittene Fragment wird über Gelelektorphorese isoliert und dann für die Ligation eingesetzt. Das entsprechende Fragment wird dann in den mit Smal verdauten und dephoεphory- lierten Vektor pSUN2-USP-RNAi-b (siehe oben) ligiert. Vekto- ren mit der gewünschten Orientierung des Inεertε werden mittels Restriktionεverdau und Sequenzierung ermittelt. Das entstehende Konstrukt trägt die Bezeichnung pSUN2-USP-RNAil . Die für die dsRNA kodierende Nukleinsäuresequenz iεt durch SEQ ID NO: 109 beschrieben.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.
Durch die oben erwähnten Klonierungεεchritte befinden εich alle drei Konεtrukte AtCRU3 , AtCRB und At2S3 im binären Vektor pSUN2-USP 1,2,3 jeweils unter der Kontrolle des USP-Promotors (Bäumlein et al . 1991, Mol Gen Genet 225 (3) : 459-67) .Due to the above-mentioned cloning steps, all three constructs AtCRU3, AtCRB and At2S3 in the binary vector pSUN2-USP 1,2,3 are each under the control of the USP promoter (Bäumlein et al. 1991, Mol Gen Genet 225 (3): 459 -67).
Beispiel 3 : Plasmide für die PflanzentranεformationExample 3: Plasmids for plant transformation
Zur Pflanzentranεformation können binäre Vektoren, wie pBinAR verwendet werden (Höfgen und Willmitzer (1990) Plant Science 66: 221-230) . Die Konstruktion der binären Vektoren kann durch Ligation der cDNA in Sense- oder Antiεense-Orientierung in T-DNA erfolgen. 5' der cDNA aktiviert ein Pflanzenpromotor die Transkription der cDNA. Eine Polyadenylierungssequenz befindet sich 3' von der cDNA.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.
Die gewebespezifiεche Expreεεion lässt sich unter Verwendung eines gewebespezifiεchen Promotorε erzielen. Beispielεweiεe kann die samenspezifische Expression erreicht werden, indem der Napin- oder der LeB4- oder der USP-Promotor 5 ' der cDNA einkloniert wird. Auch jedes andere samenεpezifische Promotorelement kann verwendet werden. Zur konstitutiven Expression in der ganzen Pflanzen läsεt εich der CaMV-35S-Promotor verwenden.The tissue-specific expression can be achieved using a tissue-specific promoter. For example, 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.
Ein weitereε Beispiel für einen binären Vektor ist der Vektor pSUN2-USPl, 2 , 3 , in welchen die Fragmente aus Beispiel 2 kloniert wurden, sowie pSUN2-USP. Der Vektor pSUN2-USP enthält den USP- Promotor sowie den OCS Terminator. pSUN2-USPl, 2, 3, enthält dreimal den USP-Promotor. Die Fragmente aus Beispiel 2 wurden in die multiple Klonierungsstelle der Vektors pSUN2-USPl, 2, 3 kloniert, um die εamenspezifische Expreεεion der Suppreεsionskonεtrukte zu ermöglichen.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.
Beiεpiel 4: Tranεformation von AgrobacteriumExample 4: Transformation of Agrobacterium
Die Agrobacterium-vermittelte Pflanzentransformation kann zum Beispiel unter Verwendung der Agrobacterium tumefacienε-Stämme GV3101 (pMP90) (Koncz und Schell (1986) Mol Gen Genet 204: -383- 396) oder LBA4404 (Clontech) durchgeführt werden. Die Tranε- formation kann durch Standard-Transformationstechniken durchgeführt werden (Deblaere et al.(1984) Nucl Acids Res 13:4777-4788). Beispiel 5 : PflanzentransformationThe Agrobacterium -mediated plant transformation can be carried out, for example, using the Agrobacterium tumefacienε 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
Die Agrobacterium-vermittelte Pflanzentransformation kann unter Verwendung von Standard-Transformations- und Regenerations- techniken durchgeführt werden (Gelvin, Stanton B., Schilperoort, Robert A. , Plant Molecular Biology Manual, 2 . Aufl., Dordrecht: Kluwer Academic Publ . , 1995, in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R. , Thompεon, John E. , Methodε in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).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., Thompεon, John E., Methodε in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).
Die Transformation mittels Agrobacterium von Arabidopsis thaliana wurde durch die Methode nach Bechthold et al . , 1993 (C.R. Acad. Sei. Ser. III Sei. Vie., 316, 1194-1199) durchgeführt. Beispielsweise kann Raps mittels Kotyledonen- oder Hypokotyl- tranεformation transformiert werden (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al . , Plant Physiol. 91 (1989) 694-701) . Die Verwendung von Antibiotika für die Agrobacterium- und Pflanzenselektion hängt von dem für die Transformation verwendeten binären Vektor und Agrobacterium-Stamm ab. Die Rapsselektion wird gewöhnlich unter Verwendung von Kanamycin als selektierbarem Pflanzenmarker durchgeführt.The transformation using Agrobacterium from Arabidopsis thaliana was carried out by the Bechthold et al. , 1993 (C.R. Acad. Sci. Ser. III Sci. Vie., 316, 1194-1199). For example, 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.
Der Agrobacterium-vermittelte Gentransfer in Lein (Linum usitatissimum) lässt sich unter Verwendung von beispielsweiεe einer von Mlynarova et al . (1994) Plant Cell Report 13:282-285 beεchriebenen Technik durchführen.The Agrobacterium -mediated gene transfer in linseed (Linum usitatissimum) can be carried out using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 perform the technique described.
Die Tranεformation von Soja kann unter Verwendung von beispielsweise einer in EP-A-0 0424 047 (Pioneer Hi-Bred International) oder in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University Toledo) beschriebenen Technik durchgeführt werden.The transformation of 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).
Die Pflanzentransformation unter Verwendung von Teilchen- beεchuεε, Polyethylenglycol-vermittelter DNA-Aufnahme oder über die Siliziumcarbonatfaεer-Technik ist beispielsweise beschrieben von Freeling und Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York) .Plant transformation using particle detection, polyethylene glycol-mediated DNA recording or using silicon carbon fiber technology is described, for example, by Freeling and Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York) ,
Beispiel 6: Untersuchung der Expresεion eines rekombinanten Genproduktes in einem transformierten OrganismuεExample 6: Examination of the expression of a recombinant gene product in a transformed organism
Die Aktivität eines rekombinanten Genprodukteε im transformierten Wirtsorganiεmuε wurde auf der Transkriptionε- und/oder der Tranεlationεebene gemessen. Ein geeigneteε Verfahren zur Beεtimmung der Menge an Tranεkription deε Gens (ein Hinweis auf die Menge an RNA, die für die Tranεlation des Genproduktes zur Verfügung steht) ist die Durchführung eines Northern-Blots wie unten ausgeführt (als Bezugsstelle siehe Auεubel et al . (1988) Current Protocolε in Molecular Biology, Wiley: New York, oder den oben erwähnten Beispielteil) , wobei ein Primer, der so gestaltet ist, dass er an das Gen von Interesse bindet, mit einer nachweisbaren Markierung (gewöhnlich radioaktiv oder chemilumineεzent) markiert wird, εo daεs, wenn die Gesamt-RNA einer Kultur deε Organiεmuε extrahiert, auf einem Gel aufgetrennt, auf eine stabile Matrix transferiert und mit dieεer Sonde inkubiert wird, die Bindung und das Ausmaß der Bindung der Sonde das Vorliegen und auch die Menge der mRNA für dieseε Gen anzeigt. Dieεe Information zeigt den Grad der Tranεkription deε transformierten Gens an. Zelluläre Gesamt-RNA kann aus Zellen, Geweben oder Organen mit mehreren Verfahren, die alle im- Fachgebiet bekannt εind, wie zum Beiεpiel daε von Bormann, E.R., et al . (1992) Mol. Microbiol. 6:317-326 beschriebene, präpariert werden.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 (an indication of the amount of RNA available for the translation of the gene product) is to carry out a Northern blot as explained below (for reference see Auεubel et al. (1988 ) Current Protocol in Molecular Biology, Wiley: New York, or the example section mentioned above), a primer which is designed in such a way that it binds to the gene of interest is labeled with a detectable label (usually radioactive or chemiluminescent), So when the total 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.
Northern-Hybridisierung:Northern hybridization:
Für die RNA-Hybridisierung wurden 20 μg Gesamt-RNA oder 1 μg poly(A)+-RNA mittels Gelelektrophoreεe in Agarosegelen mit einer Stärke von 1,25 % unter Verwendung von Formaldehyd, wie beschrieben in A asino (1986, Anal. Biochem. 152, 304) aufgetrennt, mittels Kapillaranziehung unter Verwendung von 10 x SSC auf positiv geladene Nylonmembranen (Hybond N+, A ersham, Braunschweig) übertragen, mittels UV-Licht immobiliεiert und 3 Stunden bei 68°C unter Verwendung von Hybridiεierungεpuffer (10 % Dextransulfat Gew. /Vol., 1 M NaCl, 1 % SDS, 100 mg Herings- εperma-DNA) vorhybridiεiert. Die Markierung der DNA-Sonde mit dem Highprime DNA labeling-Kit (Röche, Mannheim, Deutεchland) erfolgte während der Vorhybridisierung unter Verwendung von alpha-32P-dCTP (Amersham Pharmacia, Braunschweig, Deutschland) . Die Hybridisierung wurde nach Zugabe der markierten DNA-Sonde im gleichen Puffer bei 68°C über Nacht durchgeführt. Die Waεch- εchritte wurden zweimal für 15 min unter Verwendung von 2 X SSC und zweimal für 30 min unter Verwendung von 1 X SSC, 1 % SDS, bei 68°C durchgeführt. Die Expoεition der verschlosεenen Filter wurde bei -70°C für einen Zeitraum von 1 biε 14 T durchgeführt.For the RNA hybridization, 20 μg of total RNA or 1 μg 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 εperma DNA) prehybridized. 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.
Zur Unterεuchung deε Vorliegenε oder der relativen Menge an von dieεer mRNA tranεlatiertem Protein können Standärdtechniken, wie ein Weεtern-Blot, eingesetzt werden (siehe beispielεweiεe Auεubel et al. (1988) Current Protocolε in Molecular Biology, Wiley: New York) . Bei dieεem Verfahren werden die zellulären Gesamt- Proteine extrahiert, mittels Gelelektrophoreεe aufgetrennt, auf eine Matrix, wie Nitrozelluloεe, übertragen und mit einer Sonde, wie einem Antikörper, der spezifisch an das gewünεchte Protein bindet, inkubiert. Diese Sonde ist gewöhnlich mit einer i chemilumineszenten oder kolorimetrischen Markierung versehen, die sich leicht nachweisen läεst. Das Vorliegen und die Menge der beobachteten Markierung zeigt das Vorliegen und die Menge des gewünschten, in der Zelle vorliegenden mutierten Proteins an.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). With this method the total cellular Proteins extracted, separated by means of gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. 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.
ι Beispiel 7: Analyse der Auswirkung der rekombinanten Proteine auf die Produktion des gewünschten ProduktesExample 7: Analysis of the effect of the recombinant proteins on the production of the desired product
Die Auswirkung der genetischen Modifikation in Pflanzen, Pilzen, Algen, Ciliaten oder auf die Produktion einer gewünschten Verbindung (wie einer Fettsäure) kann bestimmt werden, indem die modifizierten Mikroorganismen oder die modifizierte Pflanze unter geeigneten Bedingungen (wie den vorstehend beschriebenen) gezüchtet werden und das Medium und/oder die zellulären Komponenten auf die erhöhte Produktion des gewünschten Produktes (d.h. von Lipiden oder einer Fettsäure) untersucht wird. Diese Analysetechniken sind dem Fachmann bekannt und umfassen Spektroskopie, Dünnschichtchromatographie, Färbeverfahren verschiedener Art, enzymatische und mikrobiologische Verfahren sowie analytiεche Chromatographie, wie Hochleistungs-Flüεεigkeitschromatographie (siehe beiεpielεweise Ullman, Eneyclopedia of Industrial Chemistry, Bd. A2 , S. 89-90 und S. 443-613, VCH: Weinheim (1985); Fallon, A. , et al . , (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17; Rehm et al . (1993) Biotechnology, Bd. 3, Kapitel III: "Product recovery and purification" , S. 469-714, VCH: Weinheim; Belter, P.A., et al. (1988) Bioεeparationε: downstream proceεsing for Biotechnology, John Wiley and Sons; Kennedy, J.F., und Cabral, J.M.S. (1992) Recovery processeε for biological Materials, John Wiley and Sons; Shaeiwitz, J.A., und Henry, J.D. (1988) Biochemical Separationε, in: Ullmann'ε Eneyclopedia of Induεtrial Chemiεtry, Bd. B3 ; Kapitel 11, S. 1-27, VCH: Weinheim; und Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publicationε) .The effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound (such as a fatty acid) 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. 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. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993 ) Biotechnology, Vol. 3, Chapter III: "Product recovery and purification", pp. 469-714, VCH: Weinheim; Belter, PA, et al. (1988) Bioεeparationε: downstream processes for Biotechnology, John Wiley and Sons; Kennedy , JF, and Cabral, JMS (1992) Recovery processeε for biological Materials, John Wiley and Sons; Shaeiwitz, JA, and Henry, JD (1988) Biochemical Separationε, in: Ullmann'ε Eneyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publicationε).
Neben den oben erwähnten Verfahren werden Pflanzenlipide auε Pflanzenmaterial wie von Cahoon et al. (1999) Proc. Natl. Acad. Sei. USA 96 (22) : 12935-12940 , und Browse et al . (1986) Analytic Biochemiεtry 152:141-145, beεchrieben extrahiert. Die qualitative und quantitative Lipid- oder Fettεäureanalyse ist beschrieben bei 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 Presε Lipid Library; 1) ,- "Progreεε i-n Lipid Research, Oxford: Pergamon Presε, 1 (1952) - 16 (1977) u.d.T.: Progress in the Chemistry of Fatε and Other Lipidε CODEN.In addition to the methods mentioned above, 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 Presε Lipid Library; 1), - "Progreεε in Lipid Research, Oxford: Pergamon Presε, 1 (1952) - 16 (1977) udT: Progress in the Chemistry of Fatε and Other Lipidε CODEN.
Zusätzlich zur Messung des Endproduktes der Fermentation ist es auch möglich, andere Komponenten der Stoffwechselwege zu analyεieren, die zur Produktion der gewünεchten Verbindung -verwendet werden, wie Zwischen- und Nebenprodukte, um die Gesamteffizienz der Produktion der Verbindung zu beεtimmen. Die Analyεeverfahren umfaεεen Messungen der Nährεtoffmengen im Medium (z . B . Zucker, Kohlenwaεεerεtoffe, Stickεtoff uellen, Phosphat und andere Ionen) , Mesεungen der Biomassezuεammen- εetzung und des Wachstums, Analyse der Produktion üblicher Meta- bolite von Biosynthesewegen und Messungen von Gasen, die während der Fermentation erzeugt werden. Standardverfahren für diese Mesεungen sind in Applied Microbial Physiology';, "A Practical Approach, P.M. Rhodes und P.F. Stanbury, Hrsgb., IRL Presε, S. 103-129; 131-163 und 165-192 (ISBN: 0199635773) und darin angegebenen Literaturεteilen beschrieben.In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways that are used to produce the desired compound, such as intermediates and by-products, in order to determine the overall efficiency of the production of the compound. 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. Standard methods for these are described in Applied Microbial Physiology Mesεungen '; 131-163 and 165-192; "A Practical Approach, Rhodes PM and Stanbury PF, eds, IRL Presε, pp 103-129: specified therein and (ISBN 0199635773). Literature parts described.
Ein Beispiel ist die Analyse von Fettsäuren (Abkürzungen: FAME, Fettsäuremethyleεter; GC-MS, Gas-Flüεεigkeitεchromatographie- Maεεenεpektro etrie; TAG, Triacylglycerin; TLC, Dünnschichtchromatographie) .One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid chromatography / mass spectrometry; TAG, triacylglycerol; TLC, thin layer chromatography).
Der unzweideutige Nachweis für das Vorliegen von Fettsäureprodukten kann mittels Analyεe rekombinanter Organiεmen nach Standard-Analyseverfahren erhalten werden: GC, GC-MS oder TLC, wie verεchiedentlich beschrieben von Christie und den Literaturstellen darin (1997, in: Advances on Lipid Methodology, Vierte Aufl.: Christie, Oily Presε, Dundee, 119-169; 1998, Gaεchromato- graphie-Massenspektrometrie-Verfahren, Lipide 33:343-353).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 Presε, Dundee, 119-169; 1998, gas chromatography-mass spectrometry method, lipids 33: 343-353).
Das zu analysierende Material kann durch Ultraschallbehandlung, Mahlen in der Glasmühle, flüsεigen Stickεtoff und Mahlen oder über andere anwendbare Verfahren aufgebrochen werden. Daε Material muss nach dem Aufbrechen zentrifugiert werden. Daε Sediment wird in Aqua deεt. reεuεpendiert, 10 min bei 100°C erhitzt, auf Eiε abgekühlt und erneut zentrifugiert, gefolgt von Extraktion in 0,5 M Schwefelεäure in Methanol mit 2 % Dimethoxy- propan für 1 Std. bei 90°C, waε zu hydrolyεierten Öl- und Lipid- verbindungen führt, die tranεmethylierte Lipide ergeben. Diese Fettsäuremethyleεter werden in Petrolether extrahiert und schließlich einer GC-Analyse unter Verwendung einer Kapillarsäule (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 mikrom, 0,32 mm) bei einem Temperaturgradienten zwischen 170°C und 240°C für 20 min und 5 min bei 240°C unterworfen. Die Identität der erhaltenen Fettsäuremethylester musε unter Verwendung von Standardε, die auε kommerziellen Quellen erhältlich εind (d.h. Sigma) , definiert werden.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. These 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).
Für die Öl-Analyse der mit den Suppressionskonεtrukten transformierten Arabidopsiε Pflanzen wurde folgendes Protokoll angewendet :The following protocol was used for the oil analysis of the Arabidopsia plants transformed with the suppression constructs:
Die Extraktion der Lipide aus Samen wird nach der Methode von Bligh & Dyer (1959) Can J Biochem Physiol 37:911 durchgeführt. Dazu werden 5 mg Arabidopsiε Samen in 1,2 ml Qiagen-Microtubes (Qiagen, Hilden) auf einer Sartorius (Göttingen) Mikrowaage abgewogen. Das Samenmaterial wird mit 500 uL Chloroform/Methanol (2:1; enthält Mono-C17-glycerin von Sigma als internen Standard) in der Rätεchmühle MM300 der Firma Retsch (Ηaan) homogenisiert und 20 min bei RT" inkubiert . Nach Zugabe von 500 uL 50 mM Kalium- phoεphatpuffer pH 7,5 erfolgt die Phasentrennung. Von der organischen Phase werden 50 μL abgenommen, mit 1500 uL Chloroform verdünnt und 5 μL auf die Kapillaren Chromarodε SIII der Firma Iatroscan (SKS, Bechenheim) aufgetragen. Nach Auftrag der Proben werden diese für 15 min in einer Dünnschichtkammer, die gesättigt iεt mit 6:2:2 Chloroform: Methanol: Toluol in einem ersten Schritt aufgetrennt. Nach Ablauf der Zeit werden die Kapillaren 4 min bei Raumtemperatur getrocknet und dann für 22 min in eine Dünnschichtkammer, die geεättigt iεt mit 7:3 n-Hexan:Dieethyl- ether gestellt. Nach einem weiteren Trocknungεεchritt für 4 min bei Raumtemperatur werden die Proben in einem Iatroεcan MK-5 (SKS, Bechenheim) entεprechend Fraεer & Taggart, 1988 J. Chromatogr. 439:404 analysiert. Folgende Parameter wurden für die Mesεungen eingestellt: Slice width 50 msec, Treεhold 20 mV, Noiεe 30, Skim ratio 0. Die Quantifizierung der Daten erfolgte anhand des internen Standards Mono-C17-glycerin (Sigma) sowie einer erstellten Eichkurve mit Tri-C17-glycerin (Sigma) mittels des Programms ChromStar (SKS, Beichenheim) .The extraction of lipids from seeds is carried out according to the method of Bligh & Dyer (1959) Can J Biochem Physiol 37: 911. For this purpose, 5 mg Arabidopsiε 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 Rätεchmühle MM300 from Retsch (Ηaan) 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 μL are removed from the organic phase, diluted with 1500 μL chloroform and 5 μL applied to the capillaries Chromarodε 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 Iatroεcan MK-5 (SKS, Bechenheim) in accordance with F raεer & Taggart, 1988 J. Chromatogr. 439: 404 analyzed. The following parameters were set for the measurements: Slice width 50 msec, Treεhold 20 mV, Noiεe 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).
Für die quantitative Bestimmung der Ölgehalte wurden Samen von unabhängigen tranεgenen Pflanzen der TI Generation analyεiert. Die geernteten Samen wurde entεprechend der beschriebenen Analysemethoden aufgearbeitet und die Ölgehalte quantitativ bestimmt (Fig. 10) . Dabei konnten mehrere unabhängige Linien identifiziert werden, die einen signifikant erhöhten Ölgehalt verglichen zu Kontrollpflanzen zeigen, die mit dem Vektor ohne das Supressions- konεtrukt transformiert wurden. So ergeben sich für die transgenen Linien Samen mit einem um biε zu 38 % erhöhten Ölgehalten (Linie 16-1 zeigt die höchste Ölmenge) . Der Vergleich mit den parallel gewachsenen Kontrollpflanzen verdeutlicht die Spezifität und damit die Funktionalität des eingesetzten Konstrukteε . Damit konnte gezeigt werden, dass eine spezifiεche Verringerung einer Speicherproteinklaεεe zu einer Erhöhung deε Ölgehaltes in Samen führt .For the quantitative determination of the oil contents, seeds from independent transgenic plants of the TI generation were analyzed. The harvested seeds were processed in accordance with the analysis methods described and the oil contents were determined quantitatively (FIG. 10). Several independent lines could be identified which show a significantly increased oil content compared to control plants which were transformed with the vector without the suppression construct. This results in seeds with up to 38% higher oil content for the transgenic lines (line 16-1 shows the highest oil quantity). The comparison with the control plants grown in parallel shows the specificity and thus the functionality of the construct used. It could thus be shown that a specific reduction in a storage protein class leads to an increase in the oil content in seeds.
Beispiel 8 : Klonierung der Super-RNAi Konstrukte für dieExample 8: Cloning of the super-RNAi constructs for the
Suppression von Speicherproteinen aus verschiedenen KlassenSuppression of storage proteins from different classes
a) Herstellung von Super-Suppressionskonstrukt 1a) Production of super suppression construct 1
Die Vektoren pCR2. l-AtCRU3-RNAi und pCR2.1-4 (εiehe Beiεpiel 2) werden mit den Reεtriktionεenzymen Xhol und Sall für 2 Stunden bei 37°C inkubiert, die DNA-Fragmente durch Agarose-Gelelektrophorese aufgetrennt und sowohl der Vektor als auch daε PCR-Inεert aus pCR2.1-4 ausgeεchnitten und mit dem "Gelpurification"-Kit von Qiagen nach Herεtellerangaben aufgereinigt und mit 50 μL Elutionεpuffer eluiert. Vom Vektor wird 1 μL, vom PCR-Inεert auε pCR2.1-4 8 μL der Eluate für die Ligation eingeεetzt, reεultierend in dem Konstrukt pCR2.1-sRNAil . Dieser Vektor wird für 2 Stunden mit dem Reεtriktionεenzym Xhol und dann für 15 min mit Klenow-Fragment inkubiert. Der Vektor pCR2.1-AtCRB-RNAi (εiehe Beiεpiel 2) wird mit dem Enzym EcoRI für 2 Stunden inkubiert und ebenfallε 15 min mit Klenow-Fragment behandelt. Beide Inkubationsanεätze werden durch Gelelektrophorese aufgetrennt und jeweils der Vektor (pCR2.1-εRNAil) bzw. das Insert (aus pCR2.1-AtCRB-RNAi) aus dem Agarosegel auεgeεchnitten und die DNA-Fragmente wie oben beεchrieben aufgereinigt . Für die Ligation werden 1 μL deε Eluateε vom Vektor und 8 μL deε Eluates vom Insert eingeεetzt und bei 4°C über Nacht inkubiert. Daε reεultierende Konεtrukt wird mit pCR2. l-sRNAi2 bezeichnet. Der resultierende Vektor wird mit dem Enzym Xbal und anschließend mit Klenow-Fragment inkubiert. Der Vektor pCR2.1-4 wird mit den Enzymen EcoRV und Xbal und anschließend mit Klenow-Fragment inkubiert. Nach Gelelektrophoreεe und -reinigung wird das Fragment auε pCR2.1-4 mit dem Vektor pCR2. l-εRNAi2 ligiert, resultierend in dem .Konstrukt pCR2. l-sRNAi3. Der resultierende Vektor wird dann mit dem Enzym Apal für 2 Stunden und dann mit Klenow-Fragment für 15 min inkubiert. Als Insert wird der Vektor pCR2. l-At2S3-RNAi mit dem Enzym EcoRI für 2 Stunden und dann mit Klenow- Fragment für 15 min inkubiert. Nach Gelelektrophorese und -reinigung werden die Eluate ligiert, reεultierend in dem Vektor pCR2. l-sRNAi4. Aus diesem Vektor wird dann daε sRNAi4-Fragment (SEQ ID NO: 144), kodierend für die super- supprimierende dsRNA, durch Inkubation mit'Hindlll und Pvul ausgeschnitten und in den binären Vektor pSUN-USP (SEQ ID NO: 179) ligiert. Das Konstrukt dient der gleichzeitigen Suppression von Arabidopsiε thaliana Speicherproteinen CRB (SEQ ID NO: 4), CRU3 (SEQ ID NO: 112) und At2S3 (SEQ ID NO: 118) .The vectors pCR2. l-AtCRU3-RNAi and pCR2.1-4 (see Example 2) 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 μL elution buffer. 1 μL of the vector and 8 μL 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-εRNAil) or the insert (from pCR2.1-AtCRB-RNAi) is cut out of the agarose gel and the DNA fragments are purified as described above. For the ligation, 1 μL of the eluate from the vector and 8 μL of the eluate from the insert are used and incubated at 4 ° C. overnight. The resultant construct is made with pCR2. designated 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-εRNAi2 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. After gel electrophoresis and purification, the eluates are ligated, resulting in the vector pCR2. 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).
Herεtellung von Super-Suppreεεionεkonstrukt 2Production of Super Suppression Construction 2
Ausgehend von Arabidopsis thaliana cDNA wird ein Fragment aus dem Speicherprotein AtCRU3 (SEQ ID NO: 111, 112) mit dem nachfolgenden Oligonukleotid-Primerpaar unter den in Beispiel 2 angegebenen PCR-Bedingungen amplifiziert:Starting from Arabidopsis thaliana cDNA, 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 11: 5 -AAAAGGCCTGTGTTCCATTTGGCCGGAAACAAC-3 '(SEQ ID NO: 148)
OPN 12 : 5 ' -AAAGATATCACCCTGGAGAACGCCACGAGTG-3 ' (SEQ ID NO: 149) .OPN 12: 5 '-AAAGATATCACCCTGGAGAACGCCACGAGTG-3' (SEQ ID NO: 149).
Das erhaltene Fragment wird in den Vektor pCR2.1-TOPO Vektor (Invitrogen) gemäß Herstellerangaben kloniert, resultierend in den pCR2.1-6 und die Sequenzen überprüft.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.
Auεgehend von Arabidopεiε thaliana cDNA wird ein Fragment auε dem Speicherprotein At2S3 (SEQ ID NO: 3, 4) mit dem nachfolgenden Oligonukleotid-Primerpaar unter den in Beiεpiel 2 angegebenen PCR-Bedingungen amplifiziert :Starting from Arabidopsis thaliana cDNA, a fragment from the storage protein At2S3 (SEQ ID NO: 3, 4) is amplified with the following pair of oligonucleotides-primers under the PCR conditions given in Example 2:
OPN 13 : 5 ' -AAAAGGCCTATGGCTAACAAGCTCTTCCTCGTC-3 ' (SEQ ID NO: 150)OPN 13: 5 '-AAAAGGCCTATGGCTAACAAGCTCTTCCTCGTC-3' (SEQ ID NO: 150)
OPN 14 : 5 -AAAGATATCCTAGTAGTAAGGAGGGAAGAAAG-3 ' (SEQ ID NO: 151) .OPN 14: 5 -AAAGATATCCTAGTAGTAAGGAGGGAAGAAAG-3 '(SEQ ID NO: 151).
Daε erhaltene Fragment wird in den Vektor pCR2.1-T0P0 Vektor (Invitrogen) gemäß Herεtellerangaben kloniert, resultierend in den pCR2.1-7 und die Sequenzen überprüft.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.
Aus den pCR2.1-3, pCR2.1-4 (siehe Beispiel 2) und pCR2.1-6 und pCR2.1-7 werden dann die Konstrukte folgendermaßen zusammen ligiert: Der Vektor pCR2.1-3 wird 2 Stunden mit EcoRV inkubiert und anεchließend 15 min mit alkaliεcher Phoεphataεe dephoεphoryliert . Der Vektor pCR2.1-6 wird mit den Enzymen StuI und EcoRV für 2 Stunden inkubiert und daε PCR-Insert über Gelelektrophorese und -reinigung isoliert. Vektor pCR2.1-3 und Insert aus pCR2.1-6 werden dann über Nacht bei 4°C ligiert, resultierend in dem Konstrukt pCR2. l-sRNAi5. Dieser Vektor wird dann mit EcoRV inkubiert und dephosphoryliert und mit dem StuI/ EcoRV inkubierten und gelaufgereinigten Fragment aus pCR2.1-7 ligiert, resultierend in dem Konstrukt pCR2. l-εRNAi6. Dieser Vektor wird dann mit Xhol inkubiert und dephosphoryliert. Der Vektor pCR2.1-4 wird mit Sall und Xhol inkubiert und daε Inεert auε pCR2.1-4 mit dem vorbereiteten Vektor pCR2. l-εRNAi6 ligiert, resultierend in dem Konstrukt pCR2.l-sRNAi7. Ausgehend von pCR2. l-sRNAi7 wird eine PCR mit den nachfolgenden Primerpaar unter den in Beispiel 2 gegebenen Bedingungen durchgeführt: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 dephoεphorylated 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. l-sRNAi5. 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-εRNAi6. 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-εRNAi6 ligated, resulting in the construct pCR2.l-sRNAi7. Starting from 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 15: 5 CCGCTCGAGCTCAGGGTCTTTTCTTGCCCACT (SEQ ID NO: 152)
OPN 16: 5 λ-CCGGTCGACCTAGTAGTAAGGAGGGAAGAAAG (SEQ ID NO: 153) .OPN 16: 5 λ -CCGGTCGACCTAGTAGTAAGGAGGGAAGAAAG (SEQ ID NO: 153).
Das resultierende PCR-Produkt wird mit den Enzymen Xhol und Sall inkubiert. Das Fragment wird dann in den Vektor pCR2.l-sRNAi7 (inkubiert mit Xhol) ligiert, reεultierend in dem Konεtrukt pCR2.1-sRNAiδ . Aus diesem Vektor wird dann das sRNAi8-Fragment (SEQ ID NO: 146) , kodierend für die super-supprimierende dsRNA, durch Inkubation mit Hindlll und Xbal ausgeεchnitten und in den binären Vektor pSUN-USP (SEQ ID NO: 179) ligiert. Daε Konstrukt dient der gleichzeitigen Suppression von Arabidopsiε thaliana Speicherproteinen CRB (SEQ ID NO: 4), CRU3 (SEQ ID NO: 112) und At2S3 (SEQ ID NO: 118) .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-sRNAiδ. 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).
Vergleichεverεuch 1 :Comparative test 1:
Die Auεwirkung der transgenen Expression der cytosoliεcher, nicht-lichtregulier-ten Arabidopεis thaliana ACCase (GenBank Acc.- No. : D34630) als Fuεionεprotein mit mit Signalεequenz für Trans- port in Chloroplasten aus der Transketolaεe auε Tabak (Henkes S et al. (2001) Plant Cell 13 (3 ): 535-51) auf den Ölgehalt der Pflanzen wurde untersucht. Dazu wurde das Fusionsprotein (vgl. SEQ ID NO: 176, 177) unter Kontrolle des Napin-Promotors in Arabidopsiε thaliana Pflanzen exprimiert. Die für dieεes Ex- pressionεkonstrukt kodierende Nukleinsäuresequenz ist durch SEQ ID NO: 175 beεchrieben. Die Herεtellung und Unterεuchung der Pflanzen erfolgte mit den oben genannten Verfahren. Es konnte keine signifikante Änderung deε Ölgehalteε in Samen verglichen mit Wildtypkontrollpflanzen feεtgeεteilt werden (siehe Fig. 8) . The effect of the transgenic expression of the cytosolic, non-light-regulated Arabidopεis thaliana ACCase (GenBank Acc.- No.: D34630) as a fusion protein with a signal sequence for transport in chloroplasts from the transketolase from tobacco (Henkes S et al. (2001 ) Plant Cell 13 (3): 535-51) for the oil content of the plants was examined. For this purpose, the fusion protein (cf. SEQ ID NO: 176, 177) was expressed in Arabidopsis thaliana plants under the control of the napin promoter. The nucleic acid sequence coding for this expression construct is described by SEQ ID NO: 175. The plants were produced and examined using the above-mentioned methods. No significant change in the oil content in seeds compared to wild-type control plants could be determined (see FIG. 8).

Claims

Patentansprüche claims
1. Verfahren zum Erhöhen des Gesamtölgehalt in einem pflanzlichen Organiεmus, dadurch gekennzeichnet, dasε nachfolgende Arbeitsεchritte umfaεεt εind1. A method for increasing the total oil content in a plant organism, characterized in that the subsequent work steps include
a) Verminderung der Proteinemenge eines oder mehrerer Speicherproteine in einem pflanzlichen Organismus oder einem Gewebe, Organ, Teil oder Zelle des besagten pflanzlichen Organismus unda) reducing the amount of protein of one or more storage proteins in a plant organism or a tissue, organ, part or cell of said plant organism and
b) Auswahl von pflanzlichen Organiεmen, bei denen - im Unterεchied oder Vergleich zur Auεgangεorganismus - der Gesamtölgehalt in dem besagten pflanzlichen Organismuε oder einem Gewebe, Organ, Teil oder Zelle des besagten pflanzlichen Organismuε erhöht iεt.b) Selection of plant organisms in which - in contrast to or compared to the starting organism - the total oil content in said plant organism or in a tissue, organ, part or cell of said plant organism is increased.
2. Verfahren nach Anεpruch 1, dadurch gekennzeichnet, daεε der Gesamtölgehalt im Samen einer Pflanze erhöht wird.2. The method according to claim 1, characterized in that the total oil content in the seed of a plant is increased.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass das Speicherprotein ausgewählt iεt aus den Speicher- protein-Klaεεen der 2S-Albumine, 7S-Globuline, 11S/12S- Globuline oder Zein-Prolamine.3. The method according to claim 1 or 2, characterized in that the storage protein is selected from the storage protein classes of the 2S albumins, 7S globulins, 11S / 12S globulins or zein prolamines.
4. Verfahren nach einem der Anεprüche 1 bis 3, dadurch gekennzeichnet, dass das Speicherprotein ausgewählt ist aus4. The method according to any one of claims 1 to 3, characterized in that the storage protein is selected from
a) der Gruppe der Polypeptid gemäß SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 112, 114, 116, 118, 120, 122, 133, 155, 157, 159, 161, 163, 165, 167, 169, 171, 172 oder 174 odera) the group of polypeptides according to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 , 42, 44, 46, 48, 50, 52, 112, 114, 116, 118, 120, 122, 133, 155, 157, 159, 161, 163, 165, 167, 169, 171, 172 or 174 or
b) einem funktionellen Äquivalent eines der Polypeptide unter a) mit einer Homologie zu dieεem von mindeεtenε 65 %.b) a functional equivalent of one of the polypeptides under a) with a homology to this of at least 65%.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dasε das Speicherprotein beschrieben ist durch ein Polypeptid gemäß SEQ ID NO: 112 oder einem funktionellen Äquivalent deεεelben mit einer Homologie zu dieεem von minde- stens 65 %.5. The method according to any one of claims 1 to 4, characterized in that the storage protein is described by a polypeptide according to SEQ ID NO: 112 or a functional equivalent thereof with a homology to this of at least 65%.
18 Zeichn. 18 drawings
6. Verfahren nach einem der Anεprüche 1 bis 5, dadurch gekennzeichnet, dass die Proteinmenge von mindestens zwei Speicherproteinen vermindert wird, wobei die Homologie von mindestens zwei der in ihrer Proteinmenge verminderten Speicherproteine untereinander geringer als, 90 % iεt.6. The method according to any one of claims 1 to 5, characterized in that the amount of protein of at least two storage proteins is reduced, the homology of at least two of the storage proteins reduced in their amount of protein among each other being less than 90%.
7. Verfahren nach einem der Anεprüche 1 biε 6, dadurch gekennzeichnet, daεs mindestenε zwei der zu vermindernden Speicherproteine zu unterεchiedlichen Klaεεen auεgewählt auε der Gruppe der Klaεεen der 2S-Albumine, 7S-Globuline, 11S/12S- Globuline oder Zein-Prolamine zählen.7. The method according to one of claims 1 to 6, characterized in that at least two of the storage proteins to be reduced count to different classes selected from the group of classes of 2S-albumins, 7S-globulins, 11S / 12S-globulins or zein-prolamines.
8. Verfahren nach einem der Anεprüche 1 biε 7, dadurch kennzeichnet, dass die Verminderung der Proteinemenge mindeεtens eines Speicherproteins gewährleistet wird durch Anwendung eineε Verfahrenε auεgewählt aus der Gruppe bestehend auε8. The method according to one of claims 1 to 7, characterized in that the reduction in the amount of protein of at least one storage protein is ensured by using a method selected from the group consisting of
a) Einbringung einer doppelεträngigen RNA-Nukleinsäure- sequenz oder einer deren Expression gewährleistenden Expresεionεkaεεette oder Expressionεkassetten, wobei die doppelsträngige RNA-Sequenz nachfolgende Elemente umfassta) introduction of a double-stranded RNA nucleic acid sequence or an expression cassette or expression cassettes ensuring its expression, the double-stranded RNA sequence comprising the following elements
i) mindestens eine "sense"-Ribonukleotidεequenz, die im weεentlichen identiεch ist zu mindestens einem Teil deε "sense"-RNA-Tranεkriptes einer Speicherprotein- Nukleinεäureεequenz undi) 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
ii) "antiεenεe"-Ribonukleotidεequenzen, die zu besagtenii) "antiεenεe" ribonucleotide sequences to be said
"sense"-Ribonukleotidsequenz unter i) im wesentlichen komplementären εind"sense" ribonucleotide sequence under i) are essentially complementary
b) Einbringung einer Speicherprotein antisense-RNA-Nuklein- εäureεequenz oder einer deren Expreεεion gewährleiεtenden Expressionskasεette, wobei die Speicherprotein antisenεe- RNA-Nukleinεäureεequenz im weεentlichen komplementär iεt zu indeεtenε einem Teil deε "εense"-RNA-Transkriptes einer Speicherprotein-Nukleinsäuresequenzb) introduction of a storage protein antisense-RNA-nucleic acid sequence or an expression cassette guaranteeing its expression, the storage protein antisense-RNA-nucleic acid sequence being essentially complementary to an independent part of the "εense" -RNA transcript nucleic acid of a storage protein
c) Einbringung einer Speicherprotein antisenεe-RMA-Nuklein- εäureεequenz kombiniert mit einem Ribozym oder einer deren Expreεsion gewährleistenden Expressionskasεette,c) introduction of a storage protein antisenεe-RMA-nucleic acid sequence combined with a ribozyme or an expression cassette ensuring its expression,
d) Einbringung von Speicherprotein sense-RNA-Nukleinsäure- sequenzen zur Induktion einer Kosuppreεsion oder einer deren Expresεion gewährleiεtenden Expressionskaεεette, wobei die Speicherprotein εense-RNA-Nukleinsäureεequenz im weεentlichen identiεch iεt zu mindeεtens einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein- Nukleinεäureεequenzd) Introduction of storage protein sense RNA nucleic acid sequences for inducing a co-suppression or an expression cassette guaranteeing its expression, the storage protein sense RNA nucleic acid sequence being essentially identical to at least part the "sense" RNA transcript of a storage protein nucleic acid sequence
e) Einbringung DNA-bindende Faktoren gegen Speicherprotein -Gene oder -RNAs oder einer deren Expression gewährleistenden Expresεionskassette,e) introduction of DNA-binding factors against storage protein genes or RNAs or an expression cassette ensuring their expression,
f) Einbringung von den Speicherprotein RNA-Abbau bewirkende virale Nukleinεäuresequenzen und Expresεionskonstrukten oder einer deren Expression gewährleistenden Expreεsionskassette,f) introduction of viral nucleic acid sequences and expression constructs causing the degradation of RNA in the protein and expression constructs or an expression cassette ensuring their expression,
g) Einbringung von Konstrukten zur Induktion einer homologen Rekombination an endogenen Speicherprotein-Genen undg) introduction of constructs for the induction of a homologous recombination on endogenous storage protein genes and
h) Einführung von Mutationen in ein endogenes Speicherprotein Gen.h) Introduction of mutations into an endogenous storage protein gene.
9. Verfahren nach einem der Ansprüche 1 bis -8, -umfassend9. The method according to any one of claims 1 to -8, comprising
' I) die stabile Transformation einer pflanzlichen Zelle mit einer rekombinanten Expressionskassette enthaltend in funktioneller Verknüpfung mit einem in Pflanzen aktiven Promotor eine Nukleinsäuresequenz kodierend für 'I) stably transforming a plant cell with a recombinant expression cassette comprising, in functional linkage with a promoter active in plants, a nucleic acid sequence coding for
a) eine doppelsträngigen Speicherprotein RNA-Nuklein- säuresequenz, wobei die doppelsträngige RNA-Sequenz nachfolgende Elemente umfaεsta) a double-stranded storage protein RNA nucleic acid sequence, the double-stranded RNA sequence comprising the following elements
i) mindestenε eine "senεe"-Ribonukleotidsequenz, die im wesentlichen- identisch ist zu mindeεtens einem Teil des "εenεe"-RNA-Tranεkriptes einer Speicherprotein-Nukleinεäuresequenz undi) at least one "senεe" ribonucleotide sequence which is essentially identical to at least part of the "εenεe" RNA transcript of a storage protein nucleic acid sequence and
ii) "antisense"-Ribonukleotidεequenzen, die zu be- εagten "εense"-Ribonukleotidsequenzen unter i) im wesentlichen komplementären sind,ii) “antisense” ribonucleotide sequences which are essentially complementary to the said “εense” ribonucleotide sequences under i),
oderor
b) eine Speicherprotein antisense-RNA-Nukleinεäure- εequenz, wobei die Speicherprotein antisense-RNA- Nukleinεäureεequenz im weεentlichen komplementär ist zu mindestenε einem Teil des "sense"-RNA-Transkriptes einer Speicherprotein-Nukleinsäureεequenz, oder c) eine Speicherprotein antiεenεe-Nukleinsäuresequenz kombiniert mit einem Ribozym oderb) a storage protein antisense-RNA-nucleic acid sequence, 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, or c) a storage protein antiεenεe nucleic acid sequence combined with a ribozyme or
d) eine Speicherprotein sense-Nukleinεäureεequenzen zur Induktion einer Kosuppresεion, wobei die Speicherprotein sense-RNA-Nukleinsäureεequenz im weεentlichen identiεch iεt zu mindeεtenε einem Teil deε "εenεe"-RNA-Tranεkriptes einer Speicherprotein- Nukleinsäuresequenz , oderd) a storage protein sense-nucleic acid sequences for inducing a cosuppression, the storage protein sense-RNA nucleic acid sequence being essentially identical to at least part of the "εenεe" RNA transcript of a storage protein nucleic acid sequence, or
f) DNA-bindende Faktoren gegen Speicherprotein-Gene oder -RNAsf) DNA binding factors against storage protein genes or RNAs
g) den Speicherprotein RNA-Abbau bewirkende virale NukleinsäureSequenzeng) viral nucleic acid sequences causing the storage protein RNA degradation
II) Regeneration der Pflanze aus der pflanzlichen Zelle, undII) regeneration of the plant from the plant cell, and
III) Expresεion beεagter Nukleinsäuresequenz in einer Menge und für eine Zeit hinreichend um den Geεamtölgehalt in beεagter Pflanze zu erhöhen.III) Expression of said nucleic acid sequence in an amount and for a time sufficient to increase the total oil content in said plant.
10. Doppelsträngiges RNA-Molekül, umfassend10. Double-stranded RNA molecule comprising
a) mindestens eine "εenεe"-Ribonukleotidεequenz, die im weεentlichen identisch ist zu mindestenε einem Teil des "senεe"-RNA-Tranεkriptes einer Speicherprotein-Nuklein- εäureεequenz gemäß 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 oder 173 unda) at least one “εenεe” ribonucleotide sequence which is essentially identical to at least part of the “senεe” 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 and
b) "antisense"-Ribonukleotidsequenzen, die zu besagten "senεe"-Ribonukleotidεequenzen unter a) im weεentlichen komplementären εind.b) “antisense” ribonucleotide sequences which are essentially complementary to said “senεe” ribonucleotide sequences under a).
11. Doppelεträngigeε RNA-Molekül nach Anεpruch 10, umfaεsend11. Double-stranded RNA molecule according to Claim 10, comprising
a) mindestens zwei "sense"-Ribonukleotidsequenen, wobeia) at least two "sense" ribonucleotide sequences, wherein
i) jede dieser "senεe"-Ribonukleotidεequenzen im wesentlichen identisch ist zu mindeεtenε einem Teil des "senεe"-RNA-Tranεkripteε einer Speicherprotein-Nu- kleinεäureεequenz gemäß 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 oder 173 und wobeii) each of these “senεe” ribonucleotide sequences is essentially identical to at least part of the “senεe” 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 and where
ii) zumindest zwei der Speicherprotein-Nukleinεäure- εequenzen, zu deren "εenεe"-RNA-Tranεkript die beεag- ten "εense"-Ribonukleotidsequenzen im wesentlichen identisch sind, untereinander eine Homologie von unter 90 % haben, undii) at least two of the storage protein nucleic acid sequences, to whose "εenεe" RNA transcript the said "εense" ribonucleotide sequences are essentially identical, have a homology of less than 90% with one another, and
b) "antisenεe"-Ribonukleotidεequenzen, die zu besagtenb) "antisenεe" ribonucleotide sequences to be said
"senεe"-Ribonukleotidsequenzen unter a) im wesentlichen komplementären sind."senεe" ribonucleotide sequences under a) are essentially complementary.
12. Doppelεträngigeε RNA-Molekül nach Anεpruch 10 oder 11, dadurch gekennzeichnet, dasε zumindest zwei der Speicher- protein-Nukleinsäuresequenzen, zu deren "sense"-RNA- Tranεkript die beεagten "εenεe"-Ribonukleotidsequenzen im wesentlichen identisch εind, zu zwei unterεchiedlichen Klaεεen von Speicherproteinen zählen auεgewählt auε der Gruppe von Speicherproteinklaεεen beεtehend auε 2S-Albuminen, 7S-Globulinen, HS/12S-Globulinen und Zein-Prolaminen.12. Double-stranded RNA molecule according to claim 10 or 11, characterized in that at least two of the storage protein nucleic acid sequences, for whose "sense" RNA transcript the said "εenεe" ribonucleotide sequences are essentially identical, to two different classes storage proteins include selected from the group of storage protein classes consisting of 2S-albumin, 7S-globulin, HS / 12S-globulin and zein-prolamine.
13. Doppelsträngiges RNA-Molekül nach einem der Ansprüche 10 bis 12, dadurch gekennzeichnet, daεs die "εense"-Ribonukleotidεe- quenzen und die "antisense"-Ribonukleotidsequenzen kovalent miteinander verbunden sind.13. Double-stranded RNA molecule according to one of claims 10 to 12, characterized in that the "εense" ribonucleotide sequences and the "antisense" ribonucleotide sequences are covalently linked to one another.
14. Transgene Expressionskasεette enthalten in funktioneller Verknüpfung mit einem Promotor eine Nukleinεäureεequenz kodierend für doppelεträngigeε RNA-Molekül gemäß einem der Ansprüche 10 biε 13.14. In functional linkage with a promoter, transgenic expression cassettes contain a nucleic acid sequence coding for double-stranded RNA molecule according to one of claims 10 to 13.
15. Tranεgeneε Expreεεionεεyεtem enthaltend15. Containing transgenic expression system
a) in funktioneller Verknüpfung mit einem Promotor eine Nukleinεäureεequenz kodierend für "sense"-Ribonukleotid- sequenzen eines doppelsträngigen RNA-Molekül gemäß einem der Ansprüche 10 bis 12 unda) in functional linkage with a promoter encoding a nucleic acid sequence for "sense" ribonucleotide sequences of a double-stranded RNA molecule according to one of claims 10 to 12 and
b) in funktioneller Verknüpfung mit einem Promotor eine Nukleinεäuresequenz kodierend für "antiεenεe"-Ribonukleo- tidεequenzen eines doppelsträngigen RNA-Molekül gemäß einem der Anεprüche 10 bis 12, wobei die Promotoren so gewählt sind, daε in einem bestimmten Organismus die gleichzeitige Expresεion von' "εenεe"-Ribonu- kleotidεequenzen und "antisense"-Ribonukleotidsequenzen gewährleistet ist.b) in functional linkage with a promoter, a nucleic acid sequence coding for "antiεenεe" ribonucleotide sequences of a double-stranded RNA molecule according to one of Claims 10 to 12, the promoters being selected such that the simultaneous expression of ' "εenεe" ribonucleotide sequences and "antisense" ribonucleotide sequences is guaranteed in a particular organism.
16. Tranεgener Vektor enthaltend eine tranεgene Expressionskassette gemäß Anspruch 14 oder ein transgeneε Expreεεions- εyεtem gemäß Anεpruch 15.16. A transgenic vector containing a transgenic expression cassette according to claim 14 or a transgenic expression system according to claim 15.
17. Tranεgener Organismus enthaltend eine transgene Expresεionε- kassette gemäß Anspruch 14 oder ein transgenes Expressionssystem gemäß Anspruch 15 oder einen transgenen Vektor gemäß Anspruch 16.17. A transgenic organism containing a transgenic expression cassette according to claim 14 or a transgenic expression system according to claim 15 or a transgenic vector according to claim 16.
18. Transgener Organismus nach Anspruch 17 ausgewählt auε der Gruppe bestehend aus Bakterien, Hefen, nicht-menschlichen Tieren und Pflanzen.18. Transgenic organism according to claim 17 selected from the group consisting of bacteria, yeast, non-human animals and plants.
19. Transgener Organismus nach einem der Ansprüche 17 oder 18, dadurch gekennzeichnet, dasε die Pflanze auεgewählt ist aus der Gruppe beεtehend aus Arabidopsiε, Rapε, Sonnenblume, Se- εa , Färberdiεtel, Ölbaum, Soja, Maiε, Weizen und Nuεsarten.19. Transgenic organism according to one of claims 17 or 18, characterized in that the plant is selected from the group consisting of Arabidopsiε, rapeseed, sunflower, Se-εa, coloring agent, olive tree, soybean, maye, wheat and nut varieties.
20. Verwendung eines tranεgenen Organiεmuε nach einem der Anεprüche 17 biε 19 zur Herεtellung von Ölen, Fetten, freien Fettεäuren oder Derivaten der vorgenannten. 20. Use of a transgenic organism according to one of claims 17 to 19 for the production of oils, fats, free fatty acids or derivatives of the aforementioned.
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