DE10226413A1 - Increasing the total oil content of plants, useful e.g. in human or animal nutrition, by expressing a yeast transgene encoding glycerol-3-phosphate dehydrogenase - Google Patents

Abstract

Method for increasing the total oil content (TOC) in a plant (or its tissue, organs, parts, cells or replicative material) by: (a) transgenic expression of a yeast glycerol-3-phosphate dehydrogenase (GPDH) gene (A) in a plant (or tissue etc.); and (b) selecting those plants with increased TOC. Independent claims are also included for the following: (1) transgenic expression cassette (EC) that contains, under control of a plant-functional promoter, the nucleic acid encoding a yeast GPHD; (2) transgenic vectors that contain EC; and (3) transgenic plants, or their tissues etc., containing GPHD, EC or the vector of (2).

Description

The invention relates to methods for increasing the oil content in Plants, preferably in plant seeds, by expression of Glycerol-3-phosphate dehydrogenases (G3PDH) from yeast, preferred from Saccharomyces cerevisiae. The invention further relates to Expression constructs for the expression of yeast G3PDH in plants, preferred in plant seeds, expressing transgenic plants Yeast G3PDH, and the use of said transgenic plants for the production of food, animal feed, seeds, Pharmaceuticals or fine chemicals, in particular for the production of Oil.

The increase in the oil content in plants and especially in Plant seeds are for the classic as well as for the modern Plant breeding and especially plant biotechnology of great interest. Due to the increasing consumption of Vegetable oils for food or industrial applications Possibilities for increasing or modifying vegetable oils is increasingly the subject of current research (e.g. Töpfer et al. (1995) Science 268: 681-686). The goal is in particular the Increasing the content of fatty acids in seed oils.

The fatty acids available from vegetable oils are also of special interest. For example, they come as Raw materials for plasticizers, lubricants, surfactants, cosmetics, etc. used or are used in the food and feed industry used as valuable raw materials. For example, the Provision of rapeseed oils with medium chain fatty acids of special interest, since this particular in the Surfactant production are in demand.

Through the targeted modulation of plant metabolic pathways The herbal Metabolism can be altered in a way that is beneficial classic breeding methods only through lengthy steps or could not be reached at all. So be unusual Fatty acids, for example certain polyunsaturated fatty acids, only in certain plants or not at all in plants synthesized and can therefore only by expression of the corresponding enzyme can be produced in transgenic plants (e.g. Millar et al. (2000) Trends Plant Sci 5: 95-101).

Triacylgylceride and other lipids are made from fatty acids synthesized. Fatty acid and triacylglyceride biosynthesis can be separated because of the compartmentalization Biosynthetic pathways, but in terms of the final product, as a View the biosynthetic pathway. Lipid synthesis can be carried out in two Sub-mechanisms are divided, 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 localized in the plastids and involves the biosynthesis of free fatty acids in the cytosol exported where they are used as fatty acid acyl-CoA esters in the enter into eukaryotic mechanism and with glycerol-3-phosphate (G3P) are esterified to phosphatidic acid (PA). PA is the Starting point for the synthesis of neutral and polar lipids. The neutral lipids are in the Kennedy way on Endoplasmic reticulum synthesized (Voelker (1996) Genetic Engineering, Setlow (ed.) 18: 111-113; Shankline & Cahoon (1998) Annu Rev Plant Physiol Plant Mol Biol 49: 611-649; Frentzen (1998) Lipids 100: 161-166). In addition to the biosynthesis of triacylglycerides G3P also serves to synthesize glycerol (e.g. for osmoregulation and against cold stress).

The G3P essential for the synthesis is thereby reduced of dihydroxyacetone phosphate (DHAP) using the Glycerin-3-phosphate dehydrogenase (G3PDH), also called Dihydroxyacetone phosphate reductase called, synthesized. It usually works NADH as a reducing cosubstrate (EC 1.1.1.8). Another Class of glycerol-3-phosphate dehydrogenases (EC 1.1.99.5) uses FAD as cosubstrate. Catalyze the enzymes of this class the reaction of DHAP to G3P. In eukaryotic cells are the two enzyme classes in different compartments distributed, the NAD-dependent cytosolic and the FAD- dependent mitochondrial localization (for Saccharomyces cerevisiae see e.g. B. Larsson et al., 1998, Yeast 14: 347-357). EP-A 0 353 049 describes an NAD-independent G3PDH from Bacillus sp. Saccharomyces cerevisiae was also found to be independent of NAD G3PDH identified (Miyata K, Nagahisa M (1969) Plant Cell Physiol 10 (3): 635-643).

G3PDH is an essential enzyme in prokaryotes and eukaryotes, in addition to the function in lipid biosynthesis also for Maintaining cellular redox status by influencing the NAD + / NADH relationship is jointly responsible. The deletion of the GPD2 gene in Saccharomyces cerevisae (one of two isoforms the G3PDH in this yeast) has decreased growth under anaerobic conditions. In addition, the G3PDH appears a role in the stress response of yeast v. a. against osmotic Playing stress. The deletion of the GPD1 gene results in Saccharomyces cerevisae hypersensitivity to salt.

Sequences for G3PDHs have been described for insects (Drosophila melanogaster, Drosophila virilis), plants (Arabidopsis thaliana, Cuphea lanceolata), mammals (Homo sapiens, Mus musculus, Sus scrofa, Rattus norvegicus), fishing (Salmo salar, Osmerus mordax), birds (Ovis aries), amphibians (Xenopus laevis), Nematodes (Caenorhabditis elegans), algae and bacteria.

Plant cells have at least two isoforms of G3PDH on, a cytoplasmic and a plastid (Gee RW et al. (1988) Plant Physiol 86: 98-103; Gee RW et al. (1988) Plant Physiol 87: 379-383). The enzymatic activity of glycerin-3 Phosphate dehydrogenase was first found in plants Potato tubers found (Santora GT et al. (1979) Arch Biochem Biophys 196: 403-411). Further cytosolic and plastid localized G3PDH activities were found in other plants such as pea, corn or soy detected (Gee RW et al. (1988) PLANT PHYSIOL 86 (1): 98-103). Also described are G3PDHs from algae such. B. two plastid and a cytosolic G3PDH isoform from Dunaliella tertiolecta (Gee R et al. (1993) Plant Physiol 103 (1): 243-249; Gee R et al. (1989) PLANT PHYSIOL 91 (1): 345-351). For the herbal G3PDH from Cuphea lanceolata has been suggested by Overexpression in plants an increase in oil content or to achieve a shift in the fatty acid pattern (WO 95/06733). However, no corresponding effects could be proven.

Bacterial G3PDHs and their function have been described (Hsu and Fox (1970) J Bacteriol 103: 410-416; Bell (1974) J Bacterial 117: 1065-1076).

WO 01/21820 describes the heterologous expression of a mutant E.coli G3PDH for increased stress tolerance and change in Fatty acid composition in storage oils. The mutated E.coli G3PDH (gpsA2FR) has a single amino acid exchange that a reduced inhibition caused by G3P. The heterologous Expression of the gpsA2FR mutant leads to glycerolipids with a increased proportion of C16 fatty acids and a concomitant reduced proportion of C18 fatty acids. The changes in Fatty acid patterns are relatively low: an increase of 2 to 5% 16: 0 fatty acids and 1.5 to 3.5% for 16: 3 fatty acids, as well a reduction of 18: 2 and 18: 3 fatty acids by 2 to 5% was observed. The total content as glycerolipids remained unaffected.

• a) Schizosaccharomyces pombe (Pidoux AL et al. (1990) Nucleic Acids Res 18 (23): 7145; GenBank Acc.-No.: X56162; Ohmiya R et al. (1995) Mol Microbiol 18(5): 963-73; GenBank Acc.-No.: D50796, D50797),
• b) Yarrowia lipolytica (GenBank Acc.-No.: AJ250328)
• c) Zygosaccharomyces rouxii (Iwaki T et al. Yeast (2001) 18(8): 737-44; GenBank Acc.-No: AB047394, AB047395, AB047397) oder
• d) Saccharomyces cerevisiae (Albertyn J et al. (1994) Mol Cell Biol 14(6): 4135-44; Albertyn J et al. (1992) FEBS LETT 308(2): 130-132; Merkel JR et al. (1982) Anal Biochem 122 (1): 180-185; Wang HT et al. (1994) J Bacteriol. 176 (22): 7091-5; Eriksson P et al. (1995) Mol Microbiol. 17(1): 95-107; GenBank Acc.-No.: U04621, X76859, Z35169).
• e) Emericella nidulans (GenBank Acc.-No.: AF228340)
• f) Debaryomyces hansenii (GenBank Acc.-No.: AF210060).

Also described are G3PDH from yeasts (Ascomycetes) such as

• a) Schizosaccharomyces pombe (Pidoux AL et al. (1990) Nucleic Acids Res 18 (23): 7145; GenBank Acc.-No .: X56162; Ohmiya R et al. (1995) Mol Microbiol 18 (5): 963-73 ; GenBank Acc.-No .: D50796, D50797),
• b) Yarrowia lipolytica (GenBank Acc.-No .: AJ250328)
• c) Zygosaccharomyces rouxii (Iwaki T et al. Yeast (2001) 18 (8): 737-44; GenBank Acc.-No: AB047394, AB047395, AB047397) or
• d) Saccharomyces cerevisiae (Albertyn J et al. (1994) Mol Cell Biol 14 (6): 4135-44; Albertyn J et al. (1992) FEBS LETT 308 (2): 130-132; Merkel JR et al. ( 1982) Anal Biochem 122 (1): 180-185; Wang HT et al. (1994) J Bacteriol. 176 (22): 7091-5; Eriksson P et al. (1995) Mol Microbiol. 17 (1): 95 -107; GenBank Acc.-No .: U04621, X76859, Z35169).
• e) Emericella nidulans (GenBank Acc.-No .: AF228340)
• f) Debaryomyces hansenii (GenBank Acc.-No .: AF210060).

It was therefore the task of alternative methods To provide an increase in the oil content in plants. This The object is achieved by the present invention.

• a) transgene Expression einer Glycerol-3-phosphat Dehydrogenase aus einer Hefe in besagtem pflanzlichen Organismus oder einem Gewebe, Organ, Teil oder Zelle desselben und
• 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 desselben erhöht ist.

A first object of the invention comprises a method for increasing the total oil content in a plant organism or a tissue, organ, part or cell thereof, comprising

• a) transgenic expression of a glycerol-3-phosphate dehydrogenase from a yeast in said plant organism or a tissue, organ, part or cell thereof and
• 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 thereof is increased.

It was surprisingly found that the seed-specific heterologous expression of the yeast protein Gpd1p (G3PDH from Saccharomyces cerevisiae; SEQ ID NO: 2) in Arabidopsis seeds leads to a significant increase in the content of triacylglycerides (storage oils). The oil content was increased by about 22%, in a transgenic line even by 41%, compared to wild-type control plants (see FIG. 1). The transgenic expression of the glycerol 3-phosphate dehydrogenase from yeast showed no adverse effects on the growth or other properties of the transformed plants.

Because G3PDH is a key enzyme of biosynthesis in all plant In principle, the method according to the invention can be organisms on all plant types - in addition to the one used as a model plant Art Arabidopsis thaliana - can be applied. Is preferred the method according to the invention applied to oil plants, the already naturally have a high oil content and / or be used for industrial extraction of oils.

"Plant organism or tissue, organ, part, cell or Propagation material of the same "generally means every one or multicellular organism or a cell, tissue, parts or Propagation material (such as seeds or fruits) of the same, which for Is capable of photosynthesis. Included in the Invention of all genera and species of higher and lower plants of the plant kingdom. Annuals, perennials, monocotyledons and dicotyledonous plants are preferred. Included are mature ones Plant, seeds, shoots and seedlings, and those derived from them Parts, propagation material (for example tubers, seeds or fruits) and cultures, for example cell or callus cultures.

"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 from them, Propagation material, plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures, as well as all others Types of groupings from plant cells to functional or structural units. Mature plants mean plants to everyone any stage of development beyond the seedling. seedling means a young, immature plant in an early Development.

"Plant" includes all annual and perennial, monocot and dicot plants and closes, however, by way of example not restricting 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, cichorium, helianthus, lactuca, bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea and Populus.

Plants from the following plant families are preferred: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.

Preferred monocot plants are selected in particular from the monocotyledonous crops, such as the Gramineae such as rice, corn, wheat or other cereals such as Barley, millet, rye, triticale or oats and sugar cane as well as all types of grasses.

• - Asteraceae wie Sonnenblume, Tagetes oder Calendula und andere mehr,
• - Compositae, besonders die Gattung Lactuca, ganz besonders die Art sativa (Salat) und andere mehr,
• - 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 Emperor (Broccoli) und weitere Kohlarten; und der Gattung Arabidopsis, ganz besonders die Art thaliana sowie Kresse oder Canola und andere mehr,
• - Cucurbitaceae wie Melone, Kürbis oder Zucchini und andere mehr,
• - Leguminosae besonders die Gattung Glycine, ganz besonders die Art max (Sojabohne) Soja sowie Alfalfa, Erbse, Bohnengewächsen oder Erdnuss und andere mehr,
• - Rubiaceae, bevorzugt der Unterklasse Lamiidae wie beispielsweise Coffea arabica oder Coffea liberica (Kaffeestrauch) und andere mehr,
• - Solanaceae besonders die Gattung Lycopersicon, ganz besonders die Art esculentum (Tomate), die Gattung Solanum, ganz besonders die Art tuberosum (Kartoffel) und melongena (Aubergine) und die Gattung Capsicum, ganz besonders die Art annum (Pfeffer) sowie Tabak oder Paprika und andere mehr,
• - Sterculiaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Theobroma cacao (Kakaostrauch) und andere mehr,
• - 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 (Selarie)) und andere mehr;

sowie Lein, Baumwolle, Hanf, Flachs, Gurke, Spinat, Möhre, Zuckerrübe und den verschiedenen Baum-, Nuss- und Weinarten, insbesondere Banane und Kiwi. 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,
• - Compositae, especially the genus Lactuca, especially the species sativa (lettuce) and others,
• - 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) and other types of cabbage; and the genus Arabidopsis, especially the species thaliana as well as cress or canola and others,
• - Cucurbitaceae such as melon, pumpkin or zucchini and others,
• - Leguminosae especially the genus Glycine, especially the type max (soybean) soy as well as alfalfa, peas, beans or peanuts 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), the genus Solanum, especially the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, especially the species annum (pepper) as well as tobacco or paprika 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 (selaria)) and others;

as well as flax, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various types of trees, nuts and wines, especially banana and kiwi.

Also includes ornamental plants, useful or ornamental trees, Flowers, cut flowers, shrubs or lawn. Exemplary, however non-restrictive angiosperms, bryophytes such as Hepaticae (liverwort) and Musci (moss); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms like conifers, cycads, ginkgo and gnetals, the families the rosaceae like rose, ericaceae like rhododendrons and azaleas, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae like cloves, Solanaceae like petunias, Gesneriaceae like that African violets, Balsaminaceae such as balsam, Orchidaceae like orchids, 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.

Plant organisms in the sense of the invention are still other photosynthetically active competent organisms, such as Example algae, cyanobacteria and mosses. Preferred algae are Green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. In particular Synechocystis is preferred.

Oil plants are most preferred. Oil plants mean plants that already naturally have a high oil content and / or are used for industrial extraction of oils. These plants can have a high oil content and / or a special, industrially interesting fatty acid composition. Plants which have a lipid content of at least 1% by weight are preferred. Examples of oil plants include:
Borago officinalis (borage); Brassica species such as B. campestris, B. napus, B. rapa (mustard, rape or turnip); Cannabis sativa (hemp); Curthamus tinctorius (dye diesel); Cocos nucifera (coconut); Crambe abyssinica (Krambe); Cuphea species (Cuphea species provide medium chain fatty acids especially for industrial applications); Elaeis guinensis (African oil palm); Ekeis oleiferu (American oil palm); Glycine max (soybean); Gossypium hirsitum (American cotton); Gossypium barbadense (Egyptian cotton); Gossypium herbaceum (Asian cotton); Helianthus annus (sunflower); Linum usitatissimum (flax or flax); Oenethern biennis (evening primrose); Ozea europea (olive); Oryza sativa (Rice); Ricinus communis (Castor); Sesamum indicum (Sesame); Triticum species (wheat); Zea maize (maize) and various types of nuts such as walnut or almond.

"Total oil content" means the sum of all oils, preferably the sum the triacylglycerides.

"Oils" includes neutral and / or polar lipids and mixtures thereof. Examples listed, but not restrictive, are those listed in Table 1. Table 1 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. Table 2 Overview of various fatty acids (selection)

¹ chain length: number of double bonds * not naturally occurring in plants

Oils preferably means seed oils.

"Increasing" the total oil content means increasing the content on oils in a plant or part, tissue or organ the same, preferably in the seed organs of the plant. It is the oil content compared to that not according to the invention Procedures subject to, but otherwise unchanged Initial plant by at least otherwise under the same general conditions 5%, preferably at least 10%, particularly preferably at least 15%, most preferably at least 20%, most preferably increased at least 25%. Framework means here all for the germination, cultivation or growth of the plant relevant conditions such as soil, climate or light conditions, Fertilization, irrigation, crop protection measures, etc.

"Glycerol 3-phosphate dehydrogenase from yeast" (as a result of "yeast G3PDH ") generally means all those enzymes that are able to Dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) - preferably using a cosubstrate such as NADH and naturally expressed in a yeast.

Yeast means a group of unicellular fungi with pronounced Cell wall and formation of pseudomycelium (in the opposite to molds). They are propagated vegetatively by sprouting and / or Cleavage (Schizosaccharomyces or Saccharomycodes).

So-called fake yeasts are included, preferably those Families Cryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon, Rhodotorula or Sporobolomyces and Bullera, as well as real yeasts (yeasts with sexual reproduction; Ascus), preferring families Endou. Saccharomycetaceae, with the genera Saccharo-, Debaro-, Lipomyces, Hansenula, Endomycopsis, Pichia, Hanseniaspora. Most preferred are the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans, Debaryomyces hansenii and Torulaspora hansenii.

• a) die Umsetzung von Dihydroxyacetonphosphat zu Glycerin-3-phosphat unter Verwendung von NADH als Cosubstrat (EC 1.1.1.8), und
• b) eine Peptidsequenz umfassend mindestens ein Sequenzmotiv ausgewählt aus der Gruppe von Sequenzmotiven bestehend aus
  • a) GSGNWGT(A/T)IAK (SEQ ID NO: 22)
  • b) CG(V/A)LSGAN(L/I/V)AXE(V/I)A (SEQ ID NO: 26)
  • c) (L/V)FXRPYFXV (SEQ ID NO: 27)
  bevorzugt ist das Sequenzmotiv ausgewählt aus der Gruppe bestehend aus
  • a) GSGNWGTTIAKV(V/I)AEN (SEQ ID NO: 29)
  • b) NT(K/R)HQNVKYLP (SEQ ID NO: 30)
  • c) D(I/V)LVFN(I/V)PHQFL (SEQ ID NO: 31)
  • d) RA(I/V)SCLKGFE (SEQ ID NO: 32)
  • e) CGALSGANLA(P/T)EVA (SEQ ID NO: 33)
  • f) LFHRPYFHV (SEQ ID NO: 34)
  • g) GLGEII(K/R)FG (SEQ ID NO: 35)

besonders bevorzugt enthält die Peptidsequenz mindestens 2 oder 3, ganz besonders bevorzugt mindestens 4 oder 5, am meisten bevorzugt alle der Sequenzmotive ausgewählt aus der Gruppe der Sequenzmotive i), ii) und iii) oder ausgewählt aus der Gruppe der Sequenzmotive iv), v), vi), vii), viii), ix) und xiv). (Angaben in Klammern meinen alternativ mögliche Aminosäuren an dieser Position; z. B. mein (V/I), dass an dieser Position Valin oder Isoleucin möglich ist). Yeast G3PDH means in particular polypeptides which have the following properties as "essential properties":

• a) the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate using NADH as cosubstrate (EC 1.1.1.8), and
• b) a peptide sequence comprising at least one sequence motif selected from the group of sequence motifs consisting of
  • a) GSGNWGT (A / T) IAK (SEQ ID NO: 22)
  • b) CG (V / A) LSGAN (L / I / V) AX (V / I) A (SEQ ID NO: 26)
  • c) (L / V) FXRPYFXV (SEQ ID NO: 27)
  the sequence motif is preferably selected from the group consisting of
  • a) GSGNWGTTIAKV (V / I) AEN (SEQ ID NO: 29)
  • b) NT (K / R) HQNVKYLP (SEQ ID NO: 30)
  • c) D (I / V) LVFN (I / V) PHQFL (SEQ ID NO: 31)
  • d) RA (I / V) SCLKGFE (SEQ ID NO: 32)
  • e) CGALSGANLA (P / T) EVA (SEQ ID NO: 33)
  • f) LFHRPYFHV (SEQ ID NO: 34)
  • g) GLGEII (K / R) FG (SEQ ID NO: 35)

the peptide sequence particularly preferably contains at least 2 or 3, very particularly preferably at least 4 or 5, most preferably all of the sequence motifs selected from the group of sequence motifs i), ii) and iii) or selected from the group of sequence motifs iv), v) , vi), vii), viii), ix) and xiv). (Information in parentheses means alternatively possible amino acids at this position; e.g. my (V / I) that valine or isoleucine is possible at this position).

• a) H(E/Q)NVKYL (SEQ ID NO: 23)
• b) (D/N)(I/V)(L/I)V(F/W)(V/N)(L/I/V)PHQF(V/L/I) (SEQ ID NO: 24)
• c) (A/G)(I/V)SC(L/I)KG (SEQ ID NO: 25)
• d) G(L/M)(L/G)E(M/I)(I/Q)(R/K/N)F(G/S/A) (SEQ ID NO: 28)

Furthermore, a yeast G3PDH can optionally - in addition to at least one of the sequence motifs i) to x) - comprise further sequence motifs selected from the group consisting of

• a) H (E / Q) NVKYL (SEQ ID NO: 23)
• b) (D / N) (I / V) (L / I) V (F / W) (V / N) (L / I / V) PHQF (V / L / I) (SEQ ID NO: 24)
• c) (A / G) (I / V) SC (L / I) KG (SEQ ID NO: 25)
• d) G (L / M) (L / G) E (M / I) (I / Q) (R / K / N) F (G / S / A) (SEQ ID NO: 28)

Most preferably, yeast G3PDH means the yeast protein Gpd1p according to SEQ ID NO: 2, as well as functional equivalents as well functionally equivalent parts of the aforementioned.

Functional equivalents mean in particular natural or Artificial mutations of the yeast protein Gpd1p according to SEQ ID NO: 2 as well as homologous polypeptides from other yeasts that are the same essential properties of a yeast G3PDH, accordingly above definition. Include mutations Substitutions, additions, deletions, inversions or Insertions of one or more amino acid residues. In particular The polypeptides are preferably described by SEQ ID NO: 4, 5, 7, 9, 11, 12, 14, 16, 38 or 40.

Those to be used advantageously in the context of this invention Yeast G3PDH can be done by searching the database or by browsing Gene or cDNA libraries - using the exemplary listed yeast G3PDH sequence according to SEQ ID NO: 2 or for this coding nucleic acid sequence according to SEQ ID NO: 1 as Search sequence or probe - can be easily found.

Said functional equivalents preferably have one Homology of at least 60%, particularly preferably at least 70%, particularly preferred at least 80%, most preferred at least 90% of the protein with SEQ ID NO: 2.

Homology between two polypeptides means the identity of the amino acid sequence over the entire sequence length, which is calculated 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 becomes:
Gap Weight: 8 Length Weight: 2
Average Match: 2,912 Average Mismatch: -2,003

An example is a sequence that has a homology of at least 80% protein based with the sequence SEQ ID NO: 2 has understood a sequence that when compared with the sequence SEQ ID NO: 2 according to the above program algorithm The above parameter set has a homology of at least 80%. Functional equivalents also include those proteins that encoded by nucleic acid sequences that have homology of at least 60%, particularly preferably at least 70%, particularly preferably at least 80%, most preferably at least Have 90% of the nucleic acid sequence with SEQ ID NO: 1.

Homology between two nucleic acid sequences is understood to mean the identity of the two nucleic acid sequences over the 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
Average Match: 10 Average Mismatch: 0

An example is a sequence that has a homology of at least 80% nucleic acid based on the sequence SEQ ID NO: 1, understood a sequence that occurs in a Comparison with the sequence SEQ ID NO: 1 according to the above Program algorithm with the above parameter set a homology of has at least 80%.

Functional equivalents also include those proteins that can be encoded by nucleic acid sequences which under Standard conditions with one of those described by SEQ ID NO: 1 Nucleic acid sequence that is complementary to this Hybridize nucleic acid sequence or parts of the aforementioned and the have essential properties of a yeast G3PDH.

"Standard hybridization conditions" is to be understood broadly but preferably means stringent hybridization conditions. Such Hybridization conditions are among others at 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 & Sons, N.Y. (1989), 6.3.1-6.3.6. described. For example, the conditions during the washing step be selected from the range of high stringency conditions (with approximately 0.2 × SSC at 50 ° C, preferably at 65 ° C) (20 × SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). During the Hybridization can also use denaturing agents such as Formamide or SDS can be used. In the presence of 50% Formamide hybridization is preferably carried out at 42 ° C.

Another object of the invention relates to transgenic Expression constructs that transgene expression of a Yeast G3PDH, in a plant organism or in a Tissue, organ, part of cells or propagation material of the can guarantee said plant organism.

The above definition applies to yeast G3PDH, transgenic expression of a yeast is particularly preferred G3PDH described by the sequence with SEQ ID NO: 2.

In said transgenic expression constructs is a Nucleic acid molecule coding for a yeast G3PDH preferred in functional association with at least one genetic Control element (for example a promoter), the one Expression in a plant organism or a tissue, Organ, part, cell or propagation material of the same guaranteed.

• a) eine Sequenz mit der SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 oder 39 oder
• b) eine Sequenz, die sich entsprechend dem degenerierten genetischen Code von einer Sequenz mit der SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 oder 39 ableitet, oder
• c) eine Sequenz, die eine Identität von mindestens 60% zu der Sequenz mit der SEQ ID NO: 1 aufweist.

Transgenic expression cassettes are particularly preferred, the nucleic acid sequence coding for a glycerol-3-phosphate dehydrogenase being described by

• a) a sequence with SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 or 39 or
• b) a sequence which is derived from a sequence with SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 or 39 in accordance with the degenerate genetic code, or
• c) a sequence which is at least 60% identical to the sequence with SEQ ID NO: 1.

A functional link is understood to mean Example the sequential arrangement of a promoter with the expressing nucleic acid sequence coding for a yeast G3PDH (for example the sequence according to SEQ ID NO: 1) and possibly others regulatory elements such as a terminator such that each of the regulatory elements has its function in the can meet transgenic expression of the nucleic acid sequence. To is not necessarily a direct link in the chemical sense required. Genetic control sequences, such as Enhancer sequences can also continue their function distant positions or even from other DNA molecules exercise the target sequence. Arrangements are preferred in which the nucleic acid sequence to be expressed transgenically behind the as Promoter acting sequence is positioned so that both Sequences are covalently linked. Is preferred the distance between the promoter sequence and the transgene nucleic acid sequence to be expressed less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

Establishing a functional link as well Production of a transgenic expression cassette can be carried out using common recombination and cloning techniques realized as 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 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 and at Gelvin et al. (1990) In: Plant Molecular Biology Manual are. However, further sequences can also be between the two sequences positioned, for example, the function of a linker with certain restriction enzyme interfaces or one Have signal peptides. The insertion of sequences lead to the expression of fusion proteins. Preferably, the Expression cassette, consisting of a linkage of promoter and nucleic acid sequence to be expressed, integrated in one Vector present and through for example transformation into a plant genome can be inserted.

But are also under a transgenic expression cassette to understand such constructions in which the Nucleic acid sequence coding for a yeast G3PDH so behind an endogenous, plant promoter is placed that this expression the yeast causes G3PDH.

Preferred in the transgenic expression cassettes Promoters used in a plant organism or a tissue, organ, part, cell or Propagation material of the same are functional. In plant organisms functional promoters basically means any promoter that the expression of genes, especially foreign genes, in plants or control plant parts, cells, tissues, cultures. Expression can be constitutive, inducible, for example or be development dependent.

• a) Konstitutive Promotoren
  "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 (Benfey et al. (1989) EMBO J 8: 2195-2202). Vorzugsweise verwendet man insbesondere einen pflanzlichen Promotor oder einen Promotor, der einem Pflanzenvirus entstammt. Insbesondere bevorzugt ist der Promotor des 35S-Transkriptes des CaMV Blumenkohlmosaikvirus (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 195 CaMV Promotor (US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8: 2195-2202). Ein weiterer geeigneter konstitutiver Promotor ist der "Rubisco small subunit (SSU)"-Promotor (US 4,962,028), der LeguminB-Promotor (GenBank Acc.-Nr. X03677), der Promotor der Nopalinsynthase aus 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 (Christensen et al. (1992) Plant Mol Biol 18: 675-689; Bruce et al. (1989) Proc Natl Acad Sci USA 86: 9692-9696), den Smas Promotor, den Cinnamylalkoholdehydrogenase-Promotor (US 5,683,439), die Promotoren der vakuolärer ATPase Untereinheiten, den Promotor des Nitrilase-1 Gens aus Arabidopsis thaliana (GenBank Acc.-No.: U38846, Nukleotide 3862 bis 5325 oder alternativ 5342) oder der Promotor eines prolinreichen Proteins aus Weizen (WO 91/13991), sowie weitere Promotoren von Genen, deren konstitutive Expression in Pflanzen dem Fachmann bekannt ist. Besonders bevorzugt sind der CaMV 35S-Promotor und der Nitrilase-1 Promotor aus Arabidopsis thaliana.
• b) Gewebespezifische Promotoren
  Bevorzugt sind ferner Promotoren mit Spezifitäten für Samen, wie zum Beispiel der Promotor des Phaseolins (US 5,504,200; Bustos mm et al. (1989) Plant Cell 1(9): 839-53), des 25 Albumingens (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), des Legumins (Shirsat A et al. (1989) Mol Gen Genet 215(2): 326-331), des USP (unknown seed protein; Bäumlein H et al. (1991) Mol Gen Genet 225(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 U 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 Pyrophosphatase (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 lpt1-Gen (WO 95/15389, WO 95/23230) oder die Promotoren beschrieben in WO 99/16890 (Promotoren des Hordein-Gens, des Glutelin-Gens, des Oryzin-Gens, des Prolamin-Gens, des Gliadin-Gens, des Glutelin-Gens, des Zein-Gens, des Kasirin-Gens oder des Secalin-Gens).
• c) Chemisch induzierbare Promotoren
  Die Expressionskassetten können auch einen chemisch induzierbaren Promotor enthalten (Übersichtsartikel: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), durch den die Expression des exogenen Gens in der Pflanze zu einem bestimmten Zeitpunkt gesteuert 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-induzierbarer Promotor (EP 0 388 186), ein durch Tetrazyklin-induzierbarer Promotor (Gatz et al. (1992) Plant J 2: 397-404), ein durch Abscisinsäure induzierbarer Promotor (EP 0 335 528) bzw. ein durch Ethanol- oder Cyclohexanon-induzierbarer Promotor (WO 93/21334) können ebenfalls verwendet werden. Ferner geeignet ist der Promotor des Glutathione-S-transferase Isoform II Gens (GST-II-27), der durch exogen applizierte Safener wie z. B. N,N-Diallyl-2,2-dichloroacetamid aktiviert werden kann (WO 93/01294) und in zahlreichen Geweben von sowohl Monokotyledonen als auch Dikotyledonen funktionell ist.

Preferred are:

• a) Constitutive promoters
  “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). In particular, a plant promoter or a plant virus-derived promoter is preferably used. Particularly preferred is the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (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 195 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 nopaline synthase 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 (Christensen et al. (1992) Plant Mol Biol 18: 675-689 ; Bruce et al. (1989) Proc Natl Acad Sci USA 86: 9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (US 5,683,439), the promoters of the vacuolar ATPase subunits, the promoter of the nitrilase-1 gene from Arabidopsis thaliana (GenBank Acc.-No .: U38846, nucleotides 3862 to 5325 or alternatively 5342) or the promoter of a proline-rich protein from wheat (WO 91/13991), as well as further promoters of genes whose constitutive expression in plants is known to the person skilled in the art. The CaMV 35S promoter and the nitrilase-1 promoter from Arabidopsis thaliana are particularly preferred.
• b) Tissue-specific promoters
  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 25 albumingen (Joseffson LG et al. ( 1987) J Biol Chem 262: 12196-12201), the legumin (Shirsat A et al. (1989) Mol Gen Genet 215 (2): 326-331), the USP (unknown seed protein; Bäumlein H et al. (1991 ) Mol Gen Genet 225 (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 U et al. (1995) Biotechnology ( NY) 13 (10): 1090f), the oleosin promoter from Arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980).
  Further suitable seed-specific promoters are those of the 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 lpt1 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 be used advantageously Prolamin gene, gliadin gene, glutelin gene, zein gene, kasirin gene or secalin gene).
• c) Chemically inducible promoters
  The expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu 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, such as. B. the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracycline inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), a promoter inducible by abscisic acid (EP 0 335 528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334) can also be used be used. Also suitable is the promoter of the glutathione S-transferase isoform II gene (GST-II-27), which is administered by exogenously administered safeners such as. B. N, N-diallyl-2,2-dichloroacetamide can be activated (WO 93/01294) and is functional in numerous tissues of both monocotyledons and dicotyledons.

Constitutive and very special are particularly preferred preferably seed-specific promoters, especially the napin and the USP promoter.

Further promoters can also be functional with the to be linked to express nucleic acid sequence, the expression in other plant tissues or in others Enable organisms such as E.coli bacteria. As Plant promoters come in principle all described above Promoters in question.

The in the transgenic expression cassettes according to the invention or nucleic acid sequences containing transgenic vectors with additional genetic control sequences in addition to a promoter be functionally linked. The concept of genetic Control sequences is to be understood broadly and means all such sequences, that have an impact on the formation or function of the have expression cassette according to the invention. genetic Control sequences modify, for example, the transcription and Translation in prokaryotic or eukaryotic organisms. The transgenic agents according to the invention preferably comprise Expression cassettes 5 'upstream from the respective transgene expressing a plant-specific nucleic acid sequence Promoter and 3'-downstream a terminator sequence as additional genetic control sequence, as well as further, if necessary usual regulatory elements, each functional linked to the nucleic acid sequence to be expressed transgenically.

Genetic control sequences also include other promoters Promoter elements or minimal promoters that the can modify expression-controlling properties. So through genetic control sequences, for example the tissue-specific ones Expression also depends on certain stress factors respectively. Corresponding elements are for example for Water stress, abscisic 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).

Further advantageous control sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast or Fungus promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters can with their Regulatory sequences like the above for the inventive methods are used. You can also synthetic promoters can be used advantageously.

Genetic control sequences also include the 5 'untranslated regions, introns or 3' non-coding region of Genes such as the Actin-1 intron, or the Adh1-S Introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It is been shown to have significant functions in can play a role in regulating gene expression. So it was demonstrated that 5'-untranslated sequences made the transient Can enhance expression of heterologous genes. Exemplary for Translation enhancer is the 5 'leader sequence from the tobacco To name the mosaic virus (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. You can also use the Promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).

The transgenic expression cassette can advantageously be a or several so-called "enhancer sequences" functionally linked to the promoter containing an increased transgenic Enable expression of the nucleic acid sequence. Also at the 3 'end of the transgenic nucleic acid sequences to be expressed additional advantageous sequences are inserted, like others regulatory elements or terminators. The transgenic too expressing nucleic acid sequences can be in one or multiple copies may be included in the gene construct.

Suitable polyadenylation signals as control sequences vegetable polyadenylation signals, preferably those which essentially T-DNA polyadenylation signals from Agrobacterium tumefaciens, especially gene 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 for particularly suitable terminator sequences are the OCS (octopine Synthase) terminator and the NOS (nopaline synthase) terminator.

Control sequences are also to be understood as those which a homologous recombination or insertion into the genome of a Allow host organism or removal from the genome allow. For example, with homologous recombination the coding sequence of a particular endogenous gene against specifically exchanged the coding sequence for a dsRNA become. Methods like cre / lox technology allow one 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 are certain flanking Sequences added to the target gene (lox sequences), which later become a Allow removal using the cre recombinase.

• a) Selektionsmarker, die eine Resistenz gegen einen Metabolismusinhibitor wie 2-Desoxyglucose-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 Resistenz gegen Herbizide verleihen. Beispielhaft seien genannt: DNA Sequenzen, die für Phosphinothricinacetyltransferasen (PAT) kodieren und Glutaminsynthaseinhibitoren inaktivieren (bar und pat Gen), 5-Enolpyruvylshikimat-3-phosphatsynthasegene (EPSP Synthasegene), die eine Resistenz gegen Glyphosat® (N-(phosphonomethyl)glycin) verleihen, das für das Glyphosat® degradierende Enzyme kodierende gox Gen (Glyphosatoxidoreduktase), das deh Gen (kodierend für eine Dehalogenase, die Dalapon inaktiviert), Sulfonylurea- und Imidazolinon inaktivierende Acetolactatsynthasen sowie bxn Gene, die für Bromoxynil degradierende Nitrilaseenzyme kodieren, das aasa-Gen, das eine Resistenz gegen das Antibiotikum Apectinomycin verleih, das Streptomycinphosphotransferase (SPT) Gen, das eine Resistenz gegen Streptomycin gewährt, das Neomycinphosphotransferas (NPTII) Gen, das eine Resistenz gegen Kanamycin oder Geneticidin verleiht, das Hygromycinphosphotransferase (HPT) Gen, das eine Resistenz gegen Hygromycin vermittelt, das Acetolactatsynthas Gen (ALS), das eine Resistenz gegen Sulfonylharnstoff-Herbizide verleiht (z. B. mutierte ALS-Varianten mit z. B. der 54 und/oder Hra Mutation).
• b) Reportergene, die für leicht quantifizierbare Proteine kodieren und über Eigenfarbe oder Enzymaktivität eine Bewertung der Transformationseffizienz oder des Expressionsortes oder -zeitpunktes gewährleisten. Ganz besonders bevorzugt sind dabei Reporter-Proteine (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) wie das "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784), die Chloramphenicoltransferase, eine Luziferase (Ow et al. (1986) Science 234: 856-859), das Aequorin- Gen (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268), die β-Galactosidase, ganz besonders bevorzugt ist die β-Glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).
• c) Replikationsursprünge, die eine Vermehrung der erfindungsgemäßen Expressionskassetten oder Vektoren in zum Beispiel E.coli gewährleisten. Beispielhaft seien genannt ORI (origin of DNA replication), der pBR322 ori oder der P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2^nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• d) Elemente, die für eine Agrobakterium vermittelte Pflanzentransformation erforderlich sind, wie zum Beispiel die rechte oder linke Begrenzung der T-DNA oder die vir-Region.

A transgenic expression cassette and the transgenic vectors derived from it can contain further functional elements. The term functional element is to be understood broadly and means all those 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) Selection markers which confer resistance to a 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: DNA sequences which code for phosphinothricin acetyltransferases (PAT) and inactivate glutamine synthase inhibitors (bar and pat gene), 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which have resistance to Glyphosat® (Ncin) phosphonomethyl confer the gox gene (glyphosate oxidoreductase) coding for the glyphosate® degrading enzyme, the deh gene (coding for a dehalogenase which inactivates dalapon), sulfonylurea and imidazolinone inactivating acetolactate synthases, and bxn genes which degenerate nitrodenase-degrading enzyme, bromoxynilase Gene conferring resistance to the antibiotic apectinomycin, the streptomycin phosphotransferase (SPT) gene conferring resistance to streptomycin, the neomycin phosphotransferas (NPTII) gene conferring resistance to kanamycin or geneticidin, the hygromycin phosphotransferase (HPT) gene, the Resistance to hygromycin mediates the Acetolacta tsynthas gene (ALS), which confers resistance to sulfonylurea herbicides (e.g. B. mutated ALS variants with z. B. the 54 and / or Hra mutation).
• b) reporter genes which code for easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an assessment of the transformation efficiency or the place or time of expression. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as the "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784), chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234: 856-859), the aequorin gene (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3) : 1259-1268), the β-galactosidase, the β-glucuronidase is very particularly preferred (Jefferson et al. (1987) EMBO J 6: 3901-3907).
• c) origins of replication, which ensure an increase in the expression cassettes or vectors according to the invention in, for example, E. coli. Examples are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al .: Molecular Cloning. A Laboratory Manual, ^2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• d) Elements which are required for an agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.

For the selection of successfully homologously recombined or transformed cells, it is usually necessary to use one selectable marker to introduce the successfully recombined cells resistant to a biocide (for Example a herbicide), a metabolism inhibitor such as 2-Deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic. The selection marker allows the selection of the transformed ones Untransformed cells (McCormick et al. (1986) Plant Cell Reports 5: 81-84).

In addition, said transgenic expression cassette or Vectors contain further nucleic acid sequences that are not for encode a yeast G3PDH, and its transgenic expression to a leads to an additional increase in fatty acid biosynthesis (as a result proOIL). This additionally expressed transgene proOIL Nucleic acid sequence can be exemplary but not restrictive be selected from nucleic acids coding for acetyl-CoA carboxylase (ACCase), glycerol-3-phosphate acyltransferase (GPAT), Lysophosphatidate acyltransferase (LPAT), diacylglycerol acyltransferase (DAGAT) and phospholipid: diacylglycerol acyltransferase (PDAT). Corresponding sequences are known to the person skilled in the art and are known from Databases or corresponding cDNA banks of the respective plants easily accessible.

The introduction of an expression cassette according to the invention in an organism or cells, tissues, organs, parts or seeds the same (preferably in plants or plant cells, tissues, Organs, parts or seeds), can be beneficial using Vectors are realized in which the transgenic Expression cassettes are included. Another object of the invention therefore relates to said transgenic vectors which are a transgenic Include expression cassette for a yeast G3PDH.

Vectors can include plasmids, cosmids, phages, viruses or be agrobacteria. The expression cassette can be in the vector (preferably a plasmid vector) via a suitable one Restriction interface will be introduced. The resulting one Vector is first introduced in E. coli. Correctly transformed E. coli are selected, grown and the recombinant vector with methods familiar to the person skilled in the art won. Restriction analysis and sequencing can do this serve to check the cloning step. Those are preferred Vectors showing a stable integration of the expression cassette into the host genome.

Another object of the invention relates to transgenic vegetable organisms or tissue, organ, part, cells or Propagate the same, corresponding to a yeast G3PDH definition given above, a transgenic expression cassette for a yeast G3PDH or a transgenic vector comprising one contain such an expression cassette.

The preparation of a corresponding transgenic herbal Organism takes place z. B. by means of transformation or transfection by means of the corresponding proteins or nucleic acids. The Production of a transformed organism (or a transformed cell or tissue) requires the corresponding DNA (e.g. the expression vector), RNA or protein in the appropriate host cell is introduced. For this operation, as a transformation (or transduction or transfection) a variety of methods are available (Keown et al. (1990) Methods in Enzymology 185: 527-537). So can the DNA or RNA, for example, directly by microinjection or by bombardment with DNA-coated microparticles be introduced. The cell can also be chemical, for example with Polyethylene glycol, permeabilized so that the DNA through Diffusion can get into the cell. The DNA can also pass through Protoplast fusion with other DNA-containing units such as Mini cells, cells, lysosomes or liposomes are made. Electroporation is another suitable method of introducing DNA in which the cells are reversible by an electrical Impulse to be permeabilized. It is also possible to swell Plant parts in DNA solutions as well as pollen or Pollen tube transformation. Appropriate procedures 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; Horsch et al. (1985) Science 227: 1229-1231; DeBlock et al. (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).

In plants, the methods described are used Transformation and regeneration from plants Plant tissues or plant cells for transient or stable Transformation used. Suitable methods are, above all Protoplast transformation by polyethylene glycol-induced DNA Recording, the biolistic method with the gene gun, the so-called "particle bombardment" method, electroporation, the incubation of dry embryos in DNA-containing solution and the microinjection.

In addition to these "direct" transformation techniques, a Transformation also by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes and transmission of corresponding recombinant Ti plasmids or Ri plasmids through or through infection with transgenic plant viruses be performed. The Agrobacterium-mediated transformation is best suited for dicotyledonous plant cells. The Methods are described for example in Horsch RB et al. (1985) Science 225: 1229f).

If Agrobacteria are used, the expression cassette is integrate into special plasmids, either into one Shuttle or intermediate vector or one binary vector. Becomes a Ti or Ri plasmid for transformation is to be used is at least the right limit, but mostly the right and left boundaries of the Ti or Ri plasmid T-DNA as a flanking region with the one to be introduced Expression cassette connected.

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 restriction sequence. You can go straight to Agrobacterium can be transformed (Holsters et al. (1978) Mol Gen Genet 163: 181-187). The selection marker gene allows selection transformed agrobacteria and is for example the nptII gene, which confers resistance to kanamycin. In this case Agrobacterium acting as the host organism should already be a Contain plasmid with the vir region. This is for the Transfer of T-DNA to the plant cell required. On Agrobacterium thus transformed can be used for transformation plant cells can be used. The use of T-DNA for Transformation of plant cells has been studied intensively and (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4: 277-287). Different binary vectors are known and some are commercially available such as Example pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).

Further promoters suitable for expression in plants are (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 Sci USA 86: 8402-8406).

Direct transformation techniques are suitable for everyone Organism and cell type. In the case of injection or Electroporation of DNA or RNA into plant cells are not made special demands on the plasmid used. Simple plasmids such as the pUC series can be used. Should complete plants from the transformed cells be regenerated, so it is necessary that on the Plasmid is an additional selectable marker gene.

Stably transformed cells d. H. those that the introduced DNA integrated into the DNA of the host cell can contain from untransformed can be selected if a selectable marker is part of the introduced DNA. As a marker can act as an example of any gene that is resistant to Antibiotics or herbicides (such as kanamycin, G 418, bleomycin, Hygromycin or phosphinotricin etc.) is able to confer (see above). Transformed cells that express such a marker gene are able to in the presence of concentrations of one corresponding antibiotic or herbicide to survive the one kill untransformed wild type. Example are mentioned above and preferably include the bar gene that resistance to the herbicide Phosphinotricin confers (Rathore KS et al. (1993) Plant Mol Biol 21 (5): 871-884), the nptII gene that resistance to kanamycin conferred the hpt gene conferring resistance to hygromycin, or the EPSP gene, which is resistant to the herbicide glyphosate gives. The selection marker allows the selection of transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84). The plants obtained can in are bred and crossed in the usual way. Two or more Generations should be cultivated to ensure that genomic integration is stable and inheritable.

The above methods are described in, for example That B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by SD Kung and R Wu, Academic Press, pp. 128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225). The construct to be expressed is preferably converted into a vector cloned, which is suitable for Agrobacterium tumefaciens transform, for example pBin19 (Bevan et al. (1984) Nucl Acids Res 12: 8711f).

Once a transformed plant cell has been made, a whole plant using the skilled person known methods can be obtained. Here you go as an example from callus cultures. From these still undifferentiated Cell masses can cause sprout and root formation in a known manner be induced. The sprouts obtained can be planted out and be bred.

The person skilled in the art is also aware of processes for extracting from plant cells Regenerate parts of plants and whole plants. For example For this purpose, methods 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 used.

• a) die Nukleinsäuresequenz kodierend für eine Hefe G3PDH, oder
• b) eine mit besagter Nukleinsäuresequenz unter a) funktionell verknüpfte genetische Kontrollsequenz, zum Beispiel ein in pflanzlichen Organismen funktioneller Promotor, oder
• c) (a) und (b).

sich nicht in ihrer natürlichen, genetischen Umgebung befinden oder durch gentechnische Methoden modifiziert wurden, wobei die Modifikation beispielhaft eine Substitutionen, Additionen, Deletionen, Inversion oder Insertionen eines oder mehrerer Nukleotidreste sein kann. Natürliche genetische Umgebung meint den 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 mindestens 500 bp, besonders bevorzugt mindestens 1000 bp, ganz besonders bevorzugt mindestens 5000 bp. Eine natürlich vorkommende Expressionskassette - beispielsweise die natürlich vorkommende Kombination des Promotors eines Gens kodierend für eine Hefe G3PDH mit dem entsprechenden Hefe G3PDH- Gen wird zu einer transgenen Expressionskassette, wenn diese durch nicht-natürliche, synthetische ("künstliche") Verfahren wie beispielsweise einer Mutagenisierung geändert wird. Entsprechende Verfahren sind beschrieben (US 5,565,350; WO 00/15815; siehe auch oben). With regard to, for example, a yeast G3PDH nucleic acid sequence, an expression cassette or a vector containing said G3PDH nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector, “transgene” means all such constructions which have been obtained by genetic engineering methods and in which either

• a) the nucleic acid sequence coding for a yeast G3PDH, or
• b) a genetic control sequence functionally linked to said nucleic acid sequence under a), for example a promoter functional in plant organisms, or
• c) (a) and (b).

are not in their natural, genetic environment or have been modified by genetic engineering methods, the modification being for example a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment means the 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 promoter of a gene coding for a yeast G3PDH with the corresponding yeast G3PDH gene becomes a transgenic expression cassette if this is carried out by non-natural, synthetic ("artificial") methods such as mutagenization will be changed. Corresponding methods are described (US 5,565,350; WO 00/15815; see also above).

Preferred host or as transgenic organisms Starting organisms are mainly plants according to the above Definition. All are included within the scope of the invention Genera and species of higher and lower plants of the Plant-rich, especially plants that are for extraction of oils such as rapeseed, sunflower, Sesame, safflower, olive tree, soy, corn, wheat and types of nuts. The mature plants, seeds and sprouts are also included and seedlings, as well as parts derived therefrom, propagation material and cultures, for example cell cultures. Ripe plants means Plants at any stage of development beyond Seedling. Seedling means a young, immature plant in one early development stage.

The production of the transgenic organisms can be done with the above described methods for transformation or transfection be realized by organisms.

Another object of the invention relates to the use the transgenic organisms according to the invention and that of them derived cells, cell cultures, parts - such as in transgenic plant organisms roots, leaves etc. -, and transgenic propagation material such as seeds or fruits, for Manufacture of food or feed, pharmaceuticals or Fine chemicals, especially oils, fats, fatty acids or Derivatives of the aforesaid.

In addition to influencing the oil content, the transgenic Expression of a yeast G3PDH in plants still more convey beneficial effects, such as increased Stress resistance e.g. B. against osmotic stress. The yeast G3PDH provides protection against high glycerol levels such stress by using glycerol as an osmoprotective. Such osmotic stress occurs, for example, in saline Soils and water on and is an increasing problem in the Agriculture. An increased stress tolerance offers here for example the possibility of agricultural use of Areas in which common arable plants cannot thrive.

Furthermore, the transgenic expression of the yeast G3PDH can the NADH Mirror and so the REDOX balance in the vegetable Affect the organism. Stress, such as drought, Heat, cold, UV light etc. can increase and increase NADH levels lead to an increased formation of reactive oxygen (ROS). The transgenic expression of the yeast G3PDH can be one of said Reduce NADH excess stress conditions, so that REDOX balance stabilize and the effects of stress mitigate.

sequences

• 1. SEQ ID NO: 1
  Nucleic acid sequence coding for Saccharomyces cerevisiae G3PDH (Gpd1p)
• 2. SEQ ID NO: 2
  Protein sequence coding for Saccharomyces cerevisiae G3PDH (Gpd1p)
• 3. SEQ ID NO: 3
  Nucleic acid sequence coding for Saccharomyces cerevisiae G3PDH (Gpd2p)
• 4. SEQ ID NO: 4
  Protein sequence coding for Saccharomyces cerevisiae G3PDH (Gpd2p)
• 5. SEQ ID NO: 5
  Protein sequence coding for Saccharomyces cerevisiae G3PDH (Gpd2p) with a second, alternative start codon
• 6. SEQ ID NO: 6
  Nucleic acid sequence coding for Schizosaccharomyces pombe G3PDH
• 7. SEQ ID NO: 7
  Protein sequence coding for Schizosaccharomyces pombe G3PDHD
• 8. SEQ ID NO: 8
  Nucleic acid sequence coding for Schizosaccharomyces pombe G3PDH
• 9. SEQ ID NO: 9
  Protein sequence coding for Schizosaccharomyces pombe G3PDH
• 10. SEQ ID NO: 10
  Nucleic acid sequence coding for Yarrowinia lipolytica G3PDH
• 11. SEQ ID NO: 11
  Protein sequence coding for Yarrowinia lipolytica G3PDH
• 12. SEQ ID NO: 12
  Protein sequence coding for Yarrowinia lipolytica G3PDH, with a second, alternative start codon
• 13. SEQ ID NO: 13
  Nucleic acid sequence coding for Zygosaccharomyces rouxii G3PDH
• 14. SEQ ID NO: 14
  Protein sequence coding for Zygosaccharomyces rouxii G3PDH
• 15. SEQ ID NO: 15
  Nucleic acid sequence coding for Zygosaccharomyces rouxii G3PDH
• 16. SEQ ID NO: 16
  Protein sequence coding for Zygosaccharomyces rouxii G3PDH
• 17. SEQ ID NO: 16
  Expression vector based on pSUN-USP for S.cerevisiae G3PDH (Gpd1p; insert from bp 1017-2190)
• 18. SEQ ID NO: 18
  Oligonucleotide primer ONP1
  5'-ACTAGTATGTCTGCTGCTGCTGATAG-3 '
• 19. SEQ ID NO: 19
  Oligonucleotide prime ONP2
  5'-CTCGAGATCTTCATGTAGATCTAATT-3 '
• 20. SEQ ID NO: 20
  Oligonucleotide prime ONP3
  5'-GCGGCCGCCATGTCTGCTGCTGCTGATAG-3 '
• 21. SEQ ID NO: 21
  Oligonucleotide prime ONP4
  5'-GCGGCCGCATCTTCATGTAGATCTAATT-3 '
• 22. SEQ ID NP 22 to 35:
  Sequence motifs for yeast G3PDHs with possible sequence variations. The variations of a single motif can occur individually but also in the different combinations with each other.
• 23. SEQ ID NO: 36
  Expression vector pGPTV-gpd1 based on pGPTV-Napin for S.cerevisiae G3PDH (Gpd1p; Insert gdp1 from bp 11962-13137; nos terminator: 13154-13408; Napin promoter: 10807-11951).
• 24. SEQ ID NO: 37
  Nucleic acid sequence coding for Emericella nidulans G3PDH.
• 25. SEQ ID NO: 38
  Amino acid sequence coding for Emericella nidulans G3PDH.
• 26. SEQ ID NO: 39
  Nucleic acid sequence coding for Debaryomyces hansenii G3PDH (partial).
• 27. SEQ ID NO: 40
  Amino acid sequence coding for Debaryomyces hansenii G3PDH (partial).

pictures

Fig. 1 oil content in transgenic GPD1p lines
Measurement of the TAG content in T2 seeds from transgenic Arabidopsis lines with the Gpd1p gene from Saccharomyces cerevisiae (G2 to G30). For comparison, the content in corresponding untransformed (wilt-type plants; W1 to W10) was determined. Eight Arabidopsis lines with a significantly increased oil content were identified. The specified error deviation results from 3 independent measurements.

Examples General methods

All chemicals, unless otherwise stated, are from the Companies Fluka (Buchs), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Restriction enzymes, DNA modifying enzymes and molecular biology kits have been developed by the Companies Amersham-Pharmacia (Freiburg), Biometra (Göttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden), Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom). The used Reagents were made according to the manufacturer's instructions used.

The chemical synthesis of oligonucleotides can, for example, in a known manner, according to the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The performed within the scope of the present invention Cloning steps such as B. restriction splits, Agarose gel electrophoresis, purification of DNA fragments, transfer of Nucleic acids on nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E.coli cells, cultivation of Bacteria, phage propagation and recombinant sequence analysis DNA is, as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules are sequenced using a Laser fluorescence DNA sequencer from ABI using the method by Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74: 5463-5467).

example 1 General procedures

The Arabidopsis thaliana plant represents a member of the higher plants (seed plants). This plant is closely related with other plant species from the cruciferous family such as z. B. Brassica napus, but also with other plant families of dicotyledons. Due to the high degree of homology of their Arabidopsis thaliana can use DNA sequences or polypeptide sequences can be used as a model plant for other plant species.

a) Cultivation of Arabidopsis plants

The plants are grown on either Murashige-Skoog Medium with 0.5% sucrose (Ogas et al. (1997) Science 277: 91-94) or pulled to earth (Focks & Benning (1998) Plant Physiol 118: 91-101). To ensure uniform germination and flowering times reach, the seeds after plating or Spread on earth stratified for two days at 4 ° C. To the pods are marked during flowering. According to the Markings will then be pods ages 6 to Harvested 20 days after flowering.

Example 2 Cloning of the Gpd1 gene from yeast

Genomic DNA from Saccharomyces cerevisiae strain S288C (Mat alpha SUC2 times mel gal2 CUP1 flo1 flo8-1; Invitrogen, Karlsruhe, Germany) was isolated according to the following protocol:
A 100 ml culture was grown to an optical density of 1.0 at 30 ° C. 60 ml were centrifuged off at 3000 xg for 3 min. The pellet was resuspended in 6 ml of double-distilled H ₂ O and distributed into 1.5 ml tubes, centrifuged and the supernatant discarded. The pellets were resuspended with 200 μl solution A, 200 μl phenol / chloroform (1: 1) and 0.3 g glass beads by vortexing and lysed. After adding 200 .mu.l TE buffer pH 8.0 was centrifuged for 5 min. The supernatant was subjected to ethanol precipitation with 1 ml of ethanol. The pellet obtained after precipitation was dissolved in 400 μl TE buffer pH 8.0 + 30 μg / ml RNaseA. After incubation for 5 min at 37 ° C., 18 μl of 3 M sodium acetate solution pH 4.8 and 1 ml of ethanol were added and the precipitated DNA was pelleted by centrifugation. The DNA pellet was dissolved in 25 ul double distilled H ₂ O. The concentration of the genomic DNA was determined by its absorption at 260 nm. Solution A 2% Trition-X100
1% SDS
0.1 M NaCl
0.01 M Tris-HCl pH 8.0
0.001 M EDTA

For the cloning of the Gpd1 gene, the isolated yeast DNA was used in a PCR reaction with the oligonucleotide primers ONP1 and ONP2.
ONP1: 5'-ACTAGTATGTCTGCTGCTGCTGATAG-3 '(SEQ ID NO: 18)
ONP2: 5'-CTCGAGATCTTCATGTAGATCTAATT-3 '(SEQ ID NO: 19) Composition of the PCR approach (50 µl) 5.00 µl 5 µg genomic yeast DNA
5.00 µl 10x buffer (Advantage polymerase) + 25 mM MgCl ₂
5.00 µl 2 mM dNTP
1.25 µl per primer (10 µmol / ul)
0.50 µl Advantage polymerase

Clontech's Advantage polymerase was used. 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.

The PCR product was in the pCR2.1-TOPO vector (Invitrogen) cloned according to manufacturer's instructions, resulting in the vector pCR2.1-gpd1 and the sequence was checked by sequencing.

For cloning in the agrotransformation vector pGPTV were 0.5 µg of the vector pCR2.1-gpd1 with the restriction enzyme XhoI (New England Biolabs) incubated for 2 hours and then Incubated for 15 min with Klenow fragment (New England Biolabs). To Incubation for 2 hours with SpeI, the DNA fragments were by Gel electrophoresis separated. The next to the vector (3.9 kb) 1185 bp fragment of the gpd1 sequence was removed from the gel cut out and with the "Gelpurification" kit from Qiagen Manufacturer's information purified and with 50 µl elution buffer eluted. 0.1 µg of the vector pGPTV was first used for 1 hour digested the restriction enzyme SacI, then for 15 min with Klenow Incubated fragment (New England Biolabs). 10 µl of the eluate of the gpd1 fragment and 10 ng of the treated vector pGPTV were ligated overnight at 16 ° C (T4 ligase from New England Biolabs). The ligation products are then in TOP10 cells (Stratagene) transformed according to the manufacturer's instructions and accordingly selected, resulting in the vector pGPTV-gpd1. positive Clones are primed with ONP1 and ONP2 by PCR and Sequencing verified.

For the production of the vector pSUN-USP-gpd1, a PCR of the vector pCR2.1-gpd1 was carried out with the primers ONP3 and ONP4.
ONP3: 5'-GCGGCCGCCATGTCTGCTGCTGCTGATAG-3 '(SEQ ID NO: 20)
ONP4: 5'-GCGGCCGCATCTTCATGTAGATCTAATT-3 '(SEQ ID NO: 21) Composition of the PCR mixture (50 μl) 5 ng DNA plasmid pCR2.1-gpd1
5.00 µl 10x buffer (Advantage polymerase) + 25 mM MgCl ₂
5.00 µl 2 mM dNTP
1.25 µl per primer (10 µmol / ul)
0.50 µl Advantage polymerase

Clontech's Advantage polymerase was used. 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.

The 1190 bp PCR product was treated with the restriction enzyme NotI digested for 24 hours. The vector pSUN-USP was used for Digested for 2 hours with NotI, then 15 minutes with alkaline Phosphatase (New England Biolabs) incubated. 100 ng of pretreated gpd1 fragment and 10 ng of the treated vector pGPTV were ligated overnight at 16 ° C (T4 ligase from New England Biolabs). The ligation products will then be in TOP10 Cells (Stratagene) transformed according to the manufacturer's instructions and selected accordingly, resulting in the vector pSUN-USP- gpd1. Positive clones are primed with ONP3 and ONP4 PCR and sequencing verified.

Example 3 Plasmids for plant transformation

Binary vectors such as pBinAR can be used for plant transformation can be used (Höfgen and willmitzer (1990) Plant Science 66: 221-230). The construction of the binary vectors can be done by Ligation of the cDNA in sense or antisense orientation in T-DNA done. 5 'of the cDNA activates a plant promoter Transcription of the cDNA. There is a polyadenylation sequence 3 'away from the cDNA.

Tissue-specific expression can be used using of a tissue-specific promoter. For example seed-specific expression can be achieved by or the LeB4 or USP promoter 5 'of the cDNA is cloned becomes. Any other seed-specific promoter element can also be used. For constitutive expression in the whole The CaMV-35S promoter can be used in plants.

Another example of binary vectors is the vector pSUN-USP and pGPTV-Napin in which the fragment from Example 2 is cloned has been. The vector pSUN-USP contains the USP promoter and the OCS Terminator. The vector pGPTV-Napin contains a shortened one Version of the Napin promoter and the nos terminator.

The fragments from Example 2 were multiple Cloning site of the vector pSUN-USP or pGPTV-Napin cloned to the seed-specific expression of the suppression constructs enable. The corresponding construct pSUN-USP-gpd1 is with the SEQ ID NO: 17, the construct of G3PDH in pGPTV-Napin (pGPTV-gpd1) is described by SEQ ID NO: 36.

Example 4 Agrobacterium transformation

The Agrobacterium -mediated plant transformation can Example using the Agrobacterium tumefaciens strains GV3101 (pMP90) (Koncz and Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech). The Transformation can be done through standard transformation techniques (Deblaere et al. (1984) Nucl Acids Res 13: 4777-4788).

Example 5 plant transformation

The Agrobacterium-mediated plant transformation can be found at Use of standard transformation and Regeneration techniques are carried out (Gelvin SB, Schilperoort R, Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Central signature: BT11-P ISBN 0-7923-2731-4; Glick BR, Thompson JE, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 p., ISBN 0-8493-5164-2).

The transformation using Agrobacterium from Arabidopsis thaliana was by the method according to Bechthold et al., 1993 (C.R. Acad. Sci. Ser. III Sci. Vie., 316, 1194-1199).

For example, rapeseed can be made using cotyledons or Hypocotyl transformation can be transformed (Moloney et al. (1989) Plant Cell Report 8: 238-242; De Block et al. (1989) Plant Physiol 91: 694-701). The use of antibiotics for the Agrobacterium and plant selection depends on that for transformation binary vector and Agrobacterium strain used. The Rapeseed selection is usually done using Kanamycin as selectable plant marker performed.

Agrobacterium-mediated gene transfer in linseed (Linum usitatissimum) can be used using, for example one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 perform the technique described. The transformation of soy can, for example, use one 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) Technique.

Plant transformation using Particle bombardment, polyethylene glycol-mediated DNA recording or is about silicon carbon fiber technology, for example described by Freeling and Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York).

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 based on the transcription and / or the Translation plane measured.

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 a Northern blot as outlined below (For reference, see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the ones mentioned above Example part), where a primer that is designed to match binds the gene of interest with a detectable label (usually radioactive or chemiluminescent) is marked so that when the total RNA is extracted from a culture of the organism, separated on a gel, transferred to a stable matrix and incubated with this probe, binding and extent the presence of the probe and the amount of mRNA for this gene. This information shows the degree of Transcription of the transformed gene. Total cellular RNA can be made from cells, tissues or organs using several methods, who are all known in the art, such as that by Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326 described, prepared.

Northern hybridization

For the RNA hybridization, 20 ug total RNA or 1 ug poly (A) ⁺ -RNA were gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in Amasino (1986, Anal. Biochem. 152 , 304) separated, transferred by capillary attraction using 10 × SSC to positively charged nylon membranes (Hybond N +, Amersham, Braunschweig), immobilized by means of UV light and 3 hours at 68 ° C. using hybridization buffer (10% dextran sulfate w / w Vol., 1 M NaCl, 1% SDS, 100 mg herring sperm DNA) prehybridized. The DNA probe was labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the pre-hybridization using alpha- ³² P-dCTP (Amersham Pharmacia, Braunschweig, Germany). 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 exposure of the closed filter was carried out at -70 ° C for a period of 1 to 14 T.

To examine the presence or relative amount of this mRNA translated protein can use standard techniques such as a Western blot can be used (see for example Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, the total cellular Proteins extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose and with a Probe, such as an antibody, specific to the desired one Protein binds, incubated. This probe is usually with one chemiluminescent or colorimetric marking, that is easy to prove. The presence and the amount the observed mark shows the presence and the amount of the desired mutant protein present in the cell.

Example 7 Analysis of the impact of the recombinant proteins on the production of the desired product

The impact of genetic modification in plants, fungi, Algae, ciliates or on the production of a desired one Compound (such as a fatty acid) can be determined by the modified microorganisms or the modified plant under suitable conditions (such as those described above) be grown and the medium and / or the cellular Components to the increased production of the desired product (i.e. of lipids or a fatty acid) is examined. This Analysis techniques are known to those skilled in the art and include spectroscopy, Thin layer chromatography, staining methods of various types, enzymatic and microbiological processes as well as analytical Chromatography, such as high performance liquid chromatography (see for example Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp. 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) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy JF and Cabral JMS (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz JA and Henry JD (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to the methods mentioned above, plant lipids are made from Plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145. The qualitative and quantitative lipid or fatty acid analysis is described with 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 Press Lipid Library; 1); "Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977) u. d. T .: Progress in the Chemistry of Fats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation it is also possible to add other components of the metabolic pathways analyze that to produce the desired connection used as intermediates and by-products to the To determine overall efficiency of 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 production more common Metabolites of biosynthetic pathways and measurements of gases during fermentation. Standard procedures for this Measurements are in Applied Microbial Physiology; A practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, Pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and therein described literature references.

An example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid chromatography mass spectrometry; TAG, triacylglycerol; TLC, TLC).

The unambiguous proof of the existence of Fatty acid products can be analyzed using recombinant organisms Standard analysis methods are obtained: GC, GC-MS or TLC, as variously described by Christie and the References therein (1997, in: Advances on Lipid Methodology, Fourth Ed .: Christie, Oily Press, Dundee, 119-169; 1998 Gas Chromatography Mass Spectrometry, Lipids 33: 343-353).

The material to be analyzed can be Grinding in a glass mill, liquid nitrogen and grinding or be broken up through other applicable procedures. The Material must be centrifuged after breaking open. The Sediment is in aqua dest. resuspended, 10 min at 100 ° C heated, cooled on ice and centrifuged again, followed by Extraction in 0.5 M sulfuric acid in methanol with 2% Dimethoxypropane for 1 h at 90 ° C, resulting in hydrolyzed oil and Leads to lipid compounds that result in transmethylated lipids. This Fatty acid methyl esters are extracted into 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 received Fatty acid methyl esters must be made using standards that are out commercial sources are available (i.e. sigma) become.

The following protocol was used for the quantitative oil analysis of the Arabidopsis plants transformed with the Gpd1 gene:
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 Arabidopsis seeds are weighed in 1.2 ml Qiagen microtubes (Qiagen, Hilden) on a Sartorius (Göttingen) microbalance. The seed material is homogenized with 500 μl chloroform / methanol (2: 1; contains mono-C17-glycerol from Sigma as internal standard) in the MM300 mill from Retsch (Haan) and incubated at RT for 20 min. After adding 500 μl of 50 mM potassium phosphate buffer pH 7.5, the phases are separated. 50 μl are removed from the organic phase, diluted with 1500 μl chloroform and 5 μl applied to the capillaries Chromarods SIII from Iatroscan (SKS, Bechenheim). After the samples have been applied, they are separated for 15 min in a thin-layer chamber which is saturated with 6: 2: 2 chloroform: methanol: toluene in a first step. After the time has elapsed, the capillaries are dried for 4 min at room temperature and then placed in a thin-layer chamber which is saturated with 7: 3 n-hexane: dieethyl ether for 22 min. After a further drying step for 4 min at room temperature, the samples are in an Iatroscan MK-5 (SKS, Bechenheim) according to Fraser & Taggart, 1988 J. Chromatogr. 439: 404 analyzed. The following parameters were set for the measurements: Slice width 50 msec, Threshold 20 mv, Noise 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).

T2 seeds were used for the quantitative determination of the oil contents of several independent transgenic lines with the constructs pSUN-USP-gpd1 or pGPTV-gpd1 analyzed. From the seed pools Three independent extractions were performed on each line and the extracts measured independently. From the three independent Measurements were the mean as well as the standard deviation calculated.

The result of the measurements for the lines with the construct pGPTV-gpd1 showed a significantly higher oil content in several (10) transgenic lines ( FIG. 1), compared with the measurements of 10 wild-type plants. Similar oil contents are measured for the construct pSUN-USP-gpd1 (not shown).

The average oil content of the lines listed above is 34.86 ± 1.56%, while the average of wild type plants is 27.75 ± 2.64%. This corresponds to an increase in the oil content of absolutely 7.1% (realistically 25.5%). SEQUENCE LISTING

Claims (16)

1. A method for increasing the total oil content in a plant organism or a tissue, organ, part, cell or propagation material thereof, comprising a) transgenic expression of a glycerol-3-phosphate dehydrogenase from a yeast in said plant organism or a tissue, organ, part, cell or propagation material of the same and 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, cell or propagation material of the same is increased. 2. The method of claim 1, wherein the glycerol-3-phosphate Dehydrogenase from a yeast is selected from the Genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon, Rhodotorul, Sporobolomyces, Bullera, Saccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia and Hanseniaspora. 3. The method according to any one of claims 1 or 2, wherein the Glycerol-3-phosphate dehydrogenase comes from a yeast selected from the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans, Debaryomyces hansenii and Torulaspora hansenii. 4. The method according to any one of claims 1 to 3, wherein the glycerol-3-phosphate dehydrogenase causes the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate using NADH as cosubstrate and has a peptide sequence which comprises at least one sequence motif selected from the group of sequence motifs consisting of a) GSGNWGT (A / T) IAK b) CG (V / A) LSGAN (L / I / V) AX (V / I) A c) (L / V) FXRPYFXV 5. The method according to any one of claims 1 to 4, wherein the glycerol-3-phosphate dehydrogenase causes the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate using NADH as cosubstrate and has a peptide sequence which comprises at least one sequence motif selected from the group of sequence motifs consisting of a) GSGNWGTTIAKV (V / I) AEN b) NT (K / R) HQNVKYLP c) D (I / V) LVFN (I / V) PHQFL d) RA (I / V) SCLKGFE e) CGALSGANLA (P / T) EVA f) LFHRPYFHV g) GLGEII (K / R) FG 6. The method according to any one of claims 4 or 5, wherein the glycerol-3-phosphate dehydrogenase additionally comprises at least one sequence motif selected from the group of sequence motifs consisting of a) H (E / Q) NVKYL b) (D / N) (I / V) (L / I) V (F / W) (V / N) (L / I / V) PHQF (V / L / I) c) (A / G) (I / V) SC (L / I) KG d) G (L / M) (L / G) E (M / I) (I / Q) (R / K / N) F (G / S / A) 7. The method according to any one of claims 1 to 6, wherein the glycerol-3-phosphate dehydrogenase from a yeast is described by a) a sequence with SEQ ID NO: 2, 4, 5, 7, 9, 11, 12, 14, 16, 38 or 40, or b) a functional equivalent of a) with an identity of at least 60%, a sequence with SEQ ID NO: 2. 8. The method according to any one of claims 1 to 7, wherein the plant is an oil plant. 9. The method according to any one of claims 1 to 8, wherein the Total oil content in the seed of a plant is increased. 10. Transgenic expression cassette fully under control one in a plant organism or tissue, Organ, part or cell of the same functional promoter a nucleic acid sequence coding for a glycerol-3 phosphate dehydrogenase from a yeast corresponding to Definitions in one of claims 2 to 7. 11. The transgenic expression cassette according to claim 10, wherein the nucleic acid sequence coding for a glycerol-3-phosphate dehydrogenase is described by a) a sequence with SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 or 39 or b) a sequence which is derived from a sequence with SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 or 39 in accordance with the degenerate genetic code, or c) a sequence which is at least 60% identical to the sequence with SEQ ID NO: 1. 12. Transgenic expression cassette according to one of claims 10 or 11, wherein the promoter is a seed-specific promoter is. 13. Transgenic vector containing an expression cassette one of claims 10 to 12. 14. Transgenic plant organism or tissue, organ, Part, cell or propagation material of the same, containing one Glycerol-3-phosphate dehydrogenase from a yeast accordingly the definitions in one of claims 2 to 7 or one Expression cassette according to one of claims 10 to 12 or a vector according to claim 13. 15. A transgenic plant organism according to claim 14, wherein the plant organism is selected from the group of oil plants consisting of Borago officinalis, Brassica campestris, brassica napus, brassica rapa, cannabis sativa, Curthamus tinctorius, Cocos nucifera, Crambe abyssinica, Cuphea species, Elaeis guinensis, Ekeis oleiferu, Glycine max, Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum, Helianthus annus, Linum usitatissimum, Oenethern biennis, Ozea europea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticum species, Zea maize, and walnut Almond. 16. Use of a transgenic plant organism or Tissue, organ, part, cell or propagation material of the same according to one of claims 14 or 15 for the production of oils, Fats, free fatty acids or derivatives of the aforementioned.