EP1948805A1 - Method for increasing the total oil content in oil plants - Google Patents

Abstract

The invention relates to methods for increasing the total oil content and/or the content of glycerol-3-phosphate in transgenic oil plants, which contain at least 20 wt.-% oleic acid in relation to the total fatty acid content, preferably in plant seeds, by the expression of glycerol-3-phosphate dehydrogenases (G3PDH) from yeasts, preferably from Saccharomyces cerevisiae. Advantageously, the oil obtained by this method and/or the free fatty acids are added to polymers, foodstuffs and feedstuffs, cosmetics, pharmaceutical products or products with industrial applications.

Description

Process for increasing the total oil content in oil plants

description

The invention relates to processes for increasing the total oil content and / or the content of glycerol-3-phosphate in transgenic oil plants containing at least 20% by weight of oleic acid based on the total fatty acid content, preferably in plant seeds, by expression of glycerol-3-phosphate dehydrogenases ( G3PDH) from yeasts, preferably from Saccharomyces cerevisiae. Advantageously, the oil obtained in the process and / or the free fatty acids are added to polymers, foods, animal feeds, cosmetics, pharmaceuticals or industrial applications.

The increase in the total oil content and / or the glycerol-3-phosphate content in transgenic oil plants and in particular in plant seeds is of great interest for classical as well as for modern plant breeding and especially plant biotechnology. Due to the increasing consumption of vegetable oils for nutrition or industrial applications, possibilities for increasing or modifying vegetable oils are increasingly the subject of current research [e.g. Potter et al. (1995) Science 268: 681-686]. The aim is, in particular, to increase the content of fatty acids in seed oils.

The fatty acids obtainable from vegetable oils are also of particular interest. They are used, for example, as basic substances for plasticizers, lubricants, surfactants, cosmetics, etc., or are used as valuable raw materials in the food and animal feed industry. For example, the provision of rapeseed oils with medium chain fatty acids is of particular interest, as they are particularly sought after in surfactant production.

Through the targeted modulation of plant metabolic pathways by means of genetic engineering methods, the plant metabolism can be advantageously changed in a way that would only be possible by means of classical breeding methods through tedious steps or not at all. Thus, unusual fatty acids, for example certain polyunsaturated fatty acids, are only synthesized in certain plants or not at all in plants and can therefore only be produced by expression of the corresponding enzyme in transgenic plants [eg Miliar et al. (2000) Trends Plant 5: 95-101]. Triacylglycerides and other lipids are synthesized from fatty acids. Fatty acid and triacylglycerol biosynthesis may be considered as a biosynthetic pathway due to compartmentalization as separate biosynthetic pathways, but with respect to the final product. The lipid synthesis can be subdivided into two partial mechanisms, a quasi "prokaryotic" and a quasi "eukaryotic" [Browse et al. (1986) Biochemical J 235: 25-31; Ohlrogge & Browse (1995) Plant Cell 7: 957-970]. The prokaryotic mechanism of the synthesis is localized in the plastids and comprises the biosynthesis of the free fatty acids, which are exported to the cytosol, where they enter the eukaryotic mechanism as fatty acid acyl-CoA esters and are treated with glycerol-3-phosphate (G3P, Glycerol-3-phosphate) to phosphatidic acid (PA). PA is the starting point for the synthesis of neutral and polar lipids. The neutral lipids are synthesized via, inter alia, the Kennedy pathway on the endoplasmic reticulum [Voelker (1996) Genetic Engineering, Setlow (ed.) 18: 1 11-1 13; 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 (eg for osmoregulation and against cold stress).

The G3P essential for the synthesis is synthesized by reduction of dihydroxyacetone phosphate (DHAP) by means of glycerol-3-phosphate dehydrogenase (G3PDH), also known as dihydroxyacetone phosphate reductase. As a rule, NADH acts 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. The enzymes of this class catalyze the reaction of DHAP to G3PDH. In eukaryotic cells, the two classes of enzymes are distributed in different compartments, with the NAD-dependent cytosolic and the FAD-dependent mitochondrially localized (for Saccharomyces cerevisiae see, for example, Larsson et al., 1998, Yeast 14: 347-357).

EP-A 0 353 049 describes an NAD-independent G3PDH from Bacillus sp. Also in Saccharomyces cerevisiae an NAD-independent G3PDH was identified [Miyata K, Nagahisa M (1969) Plant Cell Physiol 10 (3): 635-643].

G3PDH is an essential enzyme in prokaryotes and eukaryotes, which, in addition to its function in lipid biosynthesis, is also responsible for maintaining the cellular redox status by influencing the NAD + / NADH ratio. The Deletion of the GPD2 gene in Saccharomyces cerevisae (one of two isoforms of G3PDH in this yeast) results in decreased growth under anaerobic conditions. In addition, G3PDH seems to play a role in the yeast's stress response, especially against osmotic stress. The deletion of the GPD1 gene causes a hypersensitivity to salt in Saccharomyces cerevisae.

Furthermore, sequences for G3PDHs for insects (Drosophila melanogaster, Drosophila virilis), plants (Arabidopsis thaliana, Cuphea lanceolata), mammals (Homo sapiens, Mus musculus, Sus scrofa, Rattus norvegicus), fish (Salmo salar, Osmerus mordax), birds ( Ovis aries), amphibians (Xenopus laevis), namatods (Caenorhabditis elegans), algae and bacteria.

Plant cells have at least two isoforms of G3PDH, one cytoplasmic and one 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 glycerol-3-phosphate dehydrogenase was first detected in potato tubers in plants [Santora GT et al. (1979) Arch Biochem Biophys 196: 403-411]. Other cytosolic and plastid localized G3PDH activities have been detected in plants such as pea, corn or soy [Gee RW et al. (1988) PLANT PHYSIOL 86 (1): 98-103]. Also described are g3PDHs from algae such as e.g. two plastid and a cytosolic G3PDH isoform of 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 plant G3PDH from Cuphea lanceolata, it has been proposed to achieve an increase in the oil content or a shift in the fatty acid pattern by overexpression in plants (WO 95/06733). However, corresponding effects could not be proven.

Bacterial G3PDHs and their function are described in Hsu and Fox [(1970) J Bacteriol 103: 410-416] and Bell [1974] J Bacteriol 117: 1065-1076].

WO 01/21820 describes the heterologous expression of a mutant E. coli G3PDH for increased stress tolerance and alteration of the fatty acid composition in storage oils. The mutant E. coli G3PDH (gpsA2FR) has a single amino acid change that causes decreased inhibition by G3P. The heterologous expression of the gpsA2FR mutant leads to glycerolipids with an increased proportion of C16 fatty acids and a concomitant reduced proportion of C18 fatty acids. The changes in the fatty acid pattern are relatively small: an increase of 2 to 5% in 16: 0 fatty acids and 1, 5 to 3.5% in 16: 3 fatty acids, as well as a decrease in 18: 2 and 18: 3 fatty acids by 2 to 5% were observed. The total content as glycerolipids was unaffected.

WO 03/095655 describes the expression of the yeast protein Gpd i p in Arabidopsis. The oil content of the analyzed Arabidopsis plants could be increased by about 22%. Single seeds of a single transgenic line showed an increase of 41% compared to wild-type control plants. A disadvantage of this method is that Arabidopsis is a model plant, which is unsuitable for the commercial production of oils due to their agronomic properties. In addition, Arabidopsis accumulates significant amounts of eicosenoic acid (20: 1), which prohibits use of the oil in foods or pharmaceuticals.

Described are also 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; Eiksson 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)

None of the processes described so far for increasing the oil content in transgenic plants leads to an increase of the oil content in culturable plants which is sufficient for a technical process. Therefore, there is still a great need to increase the total oil content in transgenic culturable plants, preferably in the seeds of these plants. Such a procedure should meet the following criteria: • In order to increase the total oil content in the transgenic plants, as few genes as possible should be introduced into the plant.

• The procedure should be as simple and inexpensive as possible.

• In order to have the highest possible oil yield plants should be used, which have a high oil content.

• With the plants used, a high harvest yield of oil should be achieved.

• Saturated C ₁₄ - C ₈ fatty acids should be present in as low as possible quantities in the produced oil.

• The fatty acid profile should as far as possible not be changed or only slightly changed between the wild type and the transgenic plant.

• Furthermore, any bottlenecks in the precursors of the oil or fatty acid synthesis should be eliminated in the process.

It was therefore the task of developing a method for increasing the total oil content in crops that has as many of the aforementioned properties.

This object has been achieved by a process for increasing the total oil content in transgenic oil plants, characterized in that the transgenic oil plants contain at least 20% by weight of oleic acid based on the total fatty acid content and comprising the following process steps

a) introducing a nucleic acid sequence coding for a glycerol-3-phosphate dehydrogenase a yeast, in the oil plant, and

b) expression of the encoded by the nucleic acid glycerol-3-phosphate dehydrogenase in the oil plant, and

c) Selection of oil plants in which the total oil content is at least 25% by weight increased in the plant compared to the non-transgenic plant.

Advantageously, the transgenic oil plants contain at least 21, 22, 23, 24 or 25% by weight of oleic acid, advantageously at least 26, 27, 28, 29 or 30% by weight of oleic acid based on the total fatty acid content, more preferably at least 35, 40, 45, 50, 55 or 60% by weight of oleic acid based on the total fatty acid content, most preferably at least 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70% by weight of oleic acid based on the total fatty acid content or more. Plants which are advantageous for the method according to the invention also have a preferred palmitic acid content of at most 30, 29, 28, 27 or 26% by weight, advantageously 25, 24, 23, 22, 21 or 20% by weight, particularly advantageously of 15, 14, 13, 12, 1, 1, 10 or 9% by weight, based on the total fatty acid content. Further advantageous plants have a linoleic acid content of at least 20, 25, 30, 35, 40, 45 or 50% by weight, preferably 55, 60, 65 or 70% by weight, based on the total fatty acid content. Advantageous plants may also have a combination of the aforementioned fatty acids, the total fatty acid content being 100% by weight.

By the method, the total oil content in the transgenic Ölplfanzen by at least 26, 27, 28, 29 or 30% by weight, preferably by at least 31, 32, 33, 34 or 35% by weight, more preferably by at least 36, 37, 38 , 39 or 40% by weight, most advantageously increased by at least 41, 42, 43, 44 or 45% by weight.

Preferred oil plants used in the process have a high oil content in the seed. Advantageous plants have an oil content of at least 20, 25, 30, 35 or 40 wt .-%, advantageously of at least 41, 42, 43, 44 or 45 wt .-%, particularly advantageously of at least 46, 47, 48, 49 or 50% by weight or more.

Preferred oil plants in the process produce oils, lipids and / or free fatty acids containing less than 4, 3, 2 or 1% by weight, advantageously less than 0.9; 0.8; 0.7; 0.6 or 0.5% by weight, more preferably less than 0.4; 0.3; 0.2; Contain 0.1 or 0.09 wt% or less myristic acid. Further advantageous oil plants contain less than 5, 4 or 3 wt .-% palmitic acid and / or less than 2; 1, 5 or 1 wt .-% stearic acid.

Advantageous oil plants should also have a low protein content in the seed in addition to a high oil content in the seed. This proportion of protein should be as low as possible to be 30, 25 or 20 wt .-%, advantageously less than 19, 18, 17, 16 or 15 wt .-%.

The oil plants preferred in the process advantageously have no significant change in the fatty acid profile of the fatty acids C16: 0, C16: 3, C18: 0, C18: 1, C18: 2, C18: 3 and C20: 0 after introduction of the nucleic acid sequences coding for the G3PDH That is, the relative percentages of each of the stated fatty acids in total fatty acid content in weight percent remains substantially the same. Substantially equal means that the variations in the fatty acid values of the fatty acids differ by less than 5 percentage points.

Advantageous plants used in the process have a high yield of oil per hectare. This oil crop yield is at least 100, 110, 120, 130, 140 or 150 kg oil / ha, advantageously at least 250, 300, 350, 400, 450 or 500 kg oil / ha, preferably at least 550, 600, 650, 700 , 750, 800, 850, 900 or 950 kg oil / ha, more preferably at least 1000 kg oil / ha or more.

In principle, all culturable oil plants are suitable for the process according to the invention. Preferably used for the inventive method oil plants selected from the group of plants consisting of the families Anacardiaceae, Arecaceae, Asteraceae, Brassicaceae, Cannabaceae, Euphorbiaceae, Fabaceae, Juglandaceae, Linaceae, Lythraceae, Oleaceae, Poaceae and Rosaceae, which already naturally high oil content and / or used for industrial recovery of oils.

Particularly advantageously, the plants used in the process are selected from the group of oil plants selected from the group consisting of the genera and species Anacardium occidentale, Arachis hypogaea, Borago officinalis, Brassica campestris, Brassica napus, Brassica rapa, Brassica juncea, Camelina sativa, Cannabis. to sativa, Carthamus tinctorius, Cocos nucifera, Crambe abyssinica, Cuphea ciliata, Elaeis guineensis, Glycine max, Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum, Helianthus annus, Linum usitatissimum, Oenothera biennis, Olea europaea, Ricinus communis, Zea mays, Juglans regia and Prunus dulcis, more preferably from the genera and species Brassica campestris, Brassica napus, Brassica rapa, Brassica juncea, Camelina sativa, Helianthus annus, Linum usitatissimum and Carthamus tinctorius, most preferably Brassica campestris, Brassica napus, Brassica rapa, Brassica juncea and Camelina sativa.

In the present method, the seed-specific, heterologous expression of the yeast gpdi p gene in the preferred plant family of Brassicaceae eg in Brassica napus and especially in the seed leads to a significant increase in the oil content as described above. The oil content increase advantageously leads to an increase in the triacylglycerides (storage oils). The oil content was thereby in 3 independent lines about 35% increased compared to wild-type control plants (FIG. 4). The transgenic expression of yeast glycerol 3-phosphate dehydrogenase advantageously showed no adverse effects on the growth or other properties of the transformed oil plants such as the rape plants.

It has been shown that the increase in the content of triacylglycerides (storage oils) is advantageously achieved by an increase in G3PDH activity. Advantageously, in addition to the oil content, the glycerol-3-phosphate content in the inventive process is advantageously increased in the maturing seed of the G3PDH-expressing oil plants, preferably the transgenic Brassicaceae. Glycerol-3-phosphate is an important precursor in the biosynthesis of triacylglycerides and is thus an essential precursor for increasing oil content in oil plants, especially in seeds.

For the purposes of the invention, plants or oil plants include plant cells and certain tissues, organs and parts of plants, propagation material (such as seeds, tubers and fruits) or seeds of plants, and plants in all their forms, such as anthers, fibers, Root hair, stems, leaves, embryos, ciliates, kotelydons, petioles, shoots, germs, crops, plant tissue, reproductive tissue and cell cultures derived from the actual transgenic plant and / or used to produce the transgenic plant. Also included are mature plants. Mature plants are to be understood as meaning plants at any developmental stage on the side of the seedling. Keimling means a young, immature plant at an early stage of development.

"Plant" includes all annuals and perennials, monocotyledonous and dicotyledonous plants and includes the above-mentioned beneficial oil plants.

Preferred monocotyledonous plants are in particular selected from the monocotyledonous crop plants, for example the family Poaceae such as corn.

In the process according to the invention dicotyledonous oil plants are advantageously used. Preferred dicotyledonous plants are especially selected from the dicotyledonous crops, such as for example

Asteraceae like Sunflower, Tagetes or Calendula and others more,

- Brassicaceae, especially the genus Brassica, especially the species napus (rape), napus var. Napus or rapa ssp. oleifera (canola), juncea (sarepta mustard) Camelina sative (Leindotter) and others more,

Leguminosae especially the genus Glycine, especially the species max (soybean) soy or peanut and others more

as well as flax, soy, cotton or hemp.

Advantageously, transgenic plants containing increased oil content can be directly marketed without having to isolate the synthesized oil. Plants in the process according to the invention include whole plants and all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, callosis, kotelydons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that can be derived from the transgenic plant and / or used to produce the transgenic plant. The seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue. However, the oils produced in the process according to the invention can also be isolated from the plants in the form of their oils, fat, lipids and / or free fatty acids. Oils produced in the process can be obtained by harvesting the plants either from the culture in which they grow or from the field. This can be done by pressing or extraction of the plant parts, preferably the plant seeds. The oils can be extracted by pressing them "cold or cold" without adding heat.To make it easier for the plant parts to digest the seeds in particular, they are first crushed, steamed or roasted.The seeds that have been pretreated can then be pressed or solvent, such as warm hexane, and then the solvent is removed, allowing more than 96% of the oils produced in the process to be isolated, and then further processing, ie, refining, the resulting products The so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid such as phosphoric acid, and then the free fatty acids can be removed by treatment with a base, for example sodium hydroxide solution It is thoroughly washed with water and dried to remove the lye remaining in the product. In order to remove the dyes still contained in the product, the products are bleached with, for example, bleaching earth or activated carbon. At the end, the product is still deodorized, for example, with steam.

An embodiment of the invention is the use of the oils prepared by the process of the invention or obtained by mixing these oils with animal, microbial or vegetable oils, lipids or fatty acids in feed, food, cosmetics or pharmaceuticals. The oils produced in the process according to the invention may be blended with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, e.g. Fish oils are used. Also contained in the oils produced according to the invention, the fatty acids

Base treatment can be released from the oils, feed, food, cosmetics and / or pharmaceuticals in conventional amounts directly or after mixing with other oils, lipids, fatty acids or fatty acid mixtures of animal origin such. Fish oils are added.

The oils produced in the process contain compounds such as sphingolipids,

Phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerols, diacylglycerides, triacylglycerides or other fatty acid esters, preferably triacylglycerides (see Table 1).

From the oils thus prepared in the process according to the invention, the saturated and unsaturated fatty acids can be released, for example via an alkali treatment, for example with aqueous KOH or NaOH or acid hydrolysis, advantageously in the presence of an alcohol, such as methanol or ethanol, or via enzymatic cleavage and isolated via, for example, phase separation and subsequent acidification over eg H ₂ SO ₄ . The release of the fatty acids can also be carried out directly without the workup described above.

The term "oil" is also understood as meaning "lipids" or "fats" or "fatty acid mixtures" which contain unsaturated, saturated, preferably esterified fatty acid (s), advantageously bound in triglycerides. It is preferred that the oil. The oil may contain various other saturated or unsaturated fatty acids, e.g. Palmitin, palmitoleic, stearic, oleic, linoleic or α-linolenic acid, etc. included.

In particular, depending on the starting plant, the proportion of the various fatty acids in the oil may vary. By "total oil content" is meant the sum of all oils, lipids, fats or fatty acid mixtures, preferably the sum of all triacylglycerides.

"Oils" includes neutral and / or polar lipids and mixtures thereof. By way of example but not by way of limitation, those listed in Table 1 should be mentioned.

Tab. 1: Vegetable lipid classes

Neutral lipids favored triacylglycerides. Both the neutral and the polar lipids may contain a wide range of different fatty acids. By way of example, but not by way of limitation, the fatty acids listed in Table 2 should be mentioned. Table 2: Overview of various fatty acids (selection) ¹ Chain length: number of double bonds ⁺ occurring only in very few plant genera ^* not naturally occurring in higher plants

Oils prefers seed oils.

"Increasing" the total oil content means increasing the content of oils in a plant or part, tissue or organ thereof, preferably in the seed organs of the plant. In this case, the oil content compared to a not subject to the novel process, but otherwise unchanged starting plant under otherwise identical conditions by at least 25%, preferably at least 30%, more preferably at least 35%, very particularly preferred increased at least 40%, most preferably at least 45% or more. Basic conditions means all conditions relevant to the germination, cultivation or growth of the plant, such as soil, climate or light conditions, fertilization, irrigation, crop protection measures, etc.

By increasing the content of glycerol-3-phosphate in an oil plant is meant increasing the level in a plant or part of the plant, in tissues or organs of the same, preferably in the seed of the plant. In this case, the content of glycerol-3-phosphate is at least 25, 30, 35, 40, 45 or 50 wt .-%, preferably at least 60 compared to a not subject to the inventive process, but otherwise unchanged starting plant under otherwise identical conditions 70, 80, 90 or 100%, more preferably at least 1 10, 120, 130, 140 or 150%, most preferably at least 200, 250 or 300%, most preferably at least 350 or 400% or more increased. Basic conditions mean all conditions relevant to the germination, cultivation or growth of the plant, such as soil, climate or light conditions, fertilization, irrigation, crop protection measures, etc.

"Glycerol 3-phosphate dehydrogenase from yeast" (due to "yeast G3PDH") generally means all such enzymes capable of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) optionally using a cosubstrate such as NADH or NADPH - and naturally expressed in a yeast.

Hefen means one of the group of unicellular fungi with pronounced cell wall and formation of pseudomyzel (in the Ggs. To molds). Their propagation is vegetative by budding and / or cleavage (Schizosaccharomyces or Saccharomyces).

It includes so-called spurious yeasts, preferably the families Cryptococceae, Sporobolomycetaceae with the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon, Rhodotorula and Sporobolomyces and Bullera, and also real yeasts ( Yeasts with also sexual reproduction, Ascus), and prefers the families Endo u. Saccharomycetaceae, with the genera Saccharomyces, 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, Deparymyces hansenii and Torulaspora hansenii.

In particular, yeast G3PDH means polypeptides which have the following properties as "essential properties":

a) the reaction of Dihydroxyacetonph osphat 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

c) GSGNWGT (A / T) IAK (SEQ ID NO: 22)

d) CG (V / A) LSGAN (L / I / V) AX (V / I) A (SEQ ID NO: 26)

e) (UV) FXRPYFXV (SEQ ID NO: 27)

Preferably, the sequence motif is selected from the group consisting of

f) GSGN WGTTI AKV (V / I) AEN (SEQ ID NO: 29)

g) NT (K / R) HQNVKYLP (SEQ ID NO: 30)

h) D (I / V) LVFN (I / V) PHQFL (SEQ ID NO: 31)

i) RA (1 / V) SCLKGF E (SEQ ID NO: 32)

j) CGALSGANLA (P / T) EVA (SEQ ID NO: 33)

k) LFHRPYFHV (SEQ ID NO: 34)

I) GLGEII (K / R) FG (SEQ ID NO: 35)

most preferably, the peptide sequence contains at least 2 or 3, most 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 x. (In parentheses indicate alternative possible amino acids at this position, eg means (V / l) that at this position VaNn or isoleucine is possible.) In the sequence listing only one of the possible variants is reproduced.). Furthermore, a yeast G3PDH may optionally - in addition to at least one of the above sequence motifs i) to x) - further sequence motifs selected from the group consisting of

m) H (E / Q) NVKYL (SEQ ID NO: 23)

n) (0 / N) (IZV) (LZI) V (FZW) (VZN) (LZIZV) PHQF) (VZL /!) (SEQ ID NO: 24)

o) (A / G) (IZV) SC (LZI) KG (SEQ ID NO: 25)

p) G (LZM) (LZG) E (MZI) (IZQ) (RZKZN) F (GZSZA) (SEQ ID NO: 28)

Most preferably, yeast G3PDH means the yeast protein Gpdi p according to SEQ ID NO: 2, as well as functional equivalents also functionally equivalent parts of the aforementioned. Functionally equivalent parts are to be understood as sequences which are at least 51, 60, 90 or 120 bp, advantageously at least 210, 300, 330, 420 or 450 bp, particularly advantageously at least 525, 540, 570 or 600 bp, very particularly preferably at least 660 , 720, 810, 900 or 1101 bp or more.

Functional equivalents means, in particular, natural or artificial mutations of the yeast protein Gpdi p according to SEQ ID NO: 2 and homologous polypeptides from other yeasts which have the same essential properties of a yeast G3PDH, as defined above. Mutations include substitutions, additions, deletions, inversions or insertions of one or more amino acid residues. More preferably, the polypeptides are described by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 38 or SEQ ID NO: 40.

The yeast G3PDH useful for the purposes of this invention can be obtained by database search or screening of gene or cDNA libraries - using the yeast exemplified G3PDH sequence according to SEQ ID NO: 2 or the nucleic acid sequence according to SEQ ID NO: 1 as a search sequence or probe - easily found.

Preferably, functional equivalents have a homology of at least 50 or 60%, more preferably at least 70 or 80%, more preferably at least 85 or 90%, most preferably at least 91, 92, 93, 94, 95 or 96% or more to the protein with SEQ ID NO: 2. Homology between two polypeptides is understood to mean the identity of the amino acid sequence over the entire length of the sequence, by comparison with the aid of the GAP program algorithm (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following parameters is calculated:

Gap Weight: 8 Length Weight: 2

Average Match: 2,912 Average Mismatch: -2,003

By way of example, a sequence having a homology of at least 80% protein-based with the sequence SEQ ID NO: 2, a sequence understood that in a comparison with the sequence SEQ ID NO: 2 according to the above program algorithm with the above parameter set a homology of at least 80%. Functional equivalents also include those proteins which are encoded by nucleic acid sequences having a homology of at least 60, 70 or 80%, more preferably at least 85, 87, 88, 89 or 90%, more preferably at least 91, 92, 93, 94 or 95%, most preferably at least 96, 97, 98 or 99% to the nucleic acid sequence having SEQ ID NO: 1.

Homology between two nucleic acid sequences is understood to mean the identity of the two nucleic acid sequences over the respective sequence length, which are compared by means of the GAP program algorithm (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al (1997) Nucleic Acids Res. 25: 3389ff) is calculated with the following parameters:

Gap Weight: 50 Length Weight: 3

Average Match: 10 Average MismatdrO

By way of example, a sequence having a homology of at least 80% based on nucleic acid with the sequence SEQ ID NO: 1, understood a Seq uenz, which in a comparison with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set a homology of at least 80%.

Functional equivalents also include those proteins which are expressed by nucleic acid Sequences are coded, which hybridize under standard conditions with one of the nucleic acid sequence described by SEQ ID NO: 1, which hybridize to this complementary nucleic acid sequence or parts of the aforementioned and have the essential properties of a yeast G3PDH.

"Standard hybridization conditions" is to be understood broadly and means stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, in Sambrook J, Fritsch EF, Maniatis T et al., In Molecular Cloning (A Laboratory Manual), 2nd edition, CoId Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1 -e.S.e.

By way of example, the conditions during the wash step can be selected from the range of conditions limited by those with low stringency (with approximately 2 × SSC at 50 ⁰ C.) and preferably at high stringency (approximately 0.2X SSC at 50 ⁰ C at 65 ° C) (2OX SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). During hybridization, denaturing agents such as formamide or SDS may also be used. In the presence of 50% formamide, hybridization is preferably carried out at 42 ° C.

In the method according to the invention, the nucleic acid sequences used are advantageously introduced into a transgenic expression construct which can ensure a transgenic expression of a yeast G3PDH, in a plant or a tissue, organ, part, cell or propagation material of the plant.

In the expression constructs, a nucleic acid molecule coding for a yeast G3PDH is preferably in functional linkage with at least one genetic control element (for example a promoter and / or terminator) which expresses in a plant organism or a tissue, organ, part, cell or Propagating material of the same guaranteed.

Particular preference is given to using transgenic expression cassettes which contain a nucleic acid sequence coding for a glycerol-3-phosphate dehydrogenase which is selected from the group consisting of the sequences a) a sequence having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 37 or SEQ ID NO: 39 or

b) a sequence corresponding to the degenerate genetic code of a sequence with the in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID

NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 37 or SEQ ID NO: 39, or

a sequence having an identity of at least 60% to the sequence of SEQ ID NO: 1.

A functional linkage is understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence coding for a yeast G3PDH (for example the sequence according to SEQ ID NO: 1) and optionally further regulatory elements such as a terminator such that each the regulatory elements can fulfill its function in the transgenic expression of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as enhancer sequences, may also exert their function on the target sequence from more distant locations or even from other DNA molecules. Preference is given to arrangements in which the nucleic acid sequence to be transgenically expressed is positioned behind the sequence acting as a promoter, so that both sequences are covalently linked to one another. In this case, the distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is preferably less than 200 base pairs, more preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

The production of a functional link as well as the production of a

Expression cassette can be made using standard recombinant and cloning techniques as described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, ColD Spring Harbor Laboratory, ColD Spring Harbor (NY), Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusion, CoId 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 Gelvin et al. (1990) In: Plant Molecular Biology Manual are described. Between both sequences but also more Sequences are positioned, for example, have the function of a linker with certain restriction enzyme cleavage or a signal peptide. Also, insertion of sequences may result in the expression of fusion proteins. Preferably, the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed, integrated in a vector and be inserted by, for example, transformation into a plant genome.

However, an expression cassette is also to be understood as those constructions in which the nucleic acid sequence coding for a yeast G3PDH is placed behind an endogenous promoter, which causes it to express the yeast G3PDH.

In the transgenic expression cassettes, promoters which are functional in a plant organism or a tissue, organ, part, cell or propagation material are preferably used. In principle, functional promoters in plant organisms means any promoter which can control the expression of genes, in particular foreign genes, in plants or parts of plants, cells, tissues or cultures. The expression may be, for example, constitutive, inducible or developmentally dependent.

Preferred are:

a) Constitutive promoters

"Constitutive" promoters means those promoters which ensure expression in numerous, preferably all, tissues over a longer period of plant development, preferably at all times in plant development (Benfey et al. (1989) EMBO J 8: 2195-2202). In particular, it is preferable to use a plant promoter or a promoter derived from a plant virus. Particularly preferred is the promoter of the 35S transcript of 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 19S CaMV promoter (U.S. 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 legumin B promoter (GenBank Acc No. X03677), the promoter of nopaline synthase. The Agrobacterium thase, the TR double promoter, the Agrobacterium OCS (Octopin synthase) promoter, 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 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. Preferred are the CaMV 35S promoter or the nitrilase-1 promoter us Arabidopsis thaliana.

b) Tissue specific promoters

Also preferred are promoters having specificities for seeds, such as the promoter of phaseolin (US 5,504,200, Bustos MM et al., (1989) Plant Cell 1 (9): 839-53), 2S albumining (Joseffson LG et al. 1987) J Biol Chem 262: 12196-12201), the legume (Shirsat A et al., (1989) Mol Gen Genet 215 (2): 326-331), the USP (unknown seed protein, Baumlein H et al ) 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 Biolein 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 Arabidopsis oleosin promoter (WO 98/45461), the Brassica Bce4 promoter (WO 91/13980).

Other 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 which allow seed-specific expression in monocotyledons such as corn, barley, wheat, rye, rice, etc. The promoters 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, of the Prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the kasirin gene or the secalin gene). c) Chemically Inducible Promoters

The expression cassettes may also contain a chemically inducible promoter (reviewed in: Gatz et al., (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), which controls the expression of the exogenous gene in the plant at a particular time , Such promoters as e.g. 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 tetracyclic inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), a scisinic acid-inducible promoter (EP 0 335 528) or an ethanol- or cyclohexanone-inducible promoter (WO

93/21334) can also be used. Also suitable is the promoter of the glutathione-S-transferase isoform II gene (GST-II-27), which is produced by exogenously applied safeners, such as e.g. N, N-diallyl-2,2-dichloroacetamide can be activated (WO 93/01294) and is functional in numerous tissues of both monocotyledons and dicotyledons.

Particular preference is given to constitutive and very particularly preferably seed-specific promoters, in particular the napin and the USP promoters.

Furthermore, further promoters can be functionally linked to the nucleic acid sequence to be expressed, which can be expressed in further plant tissues or in other organisms, such as E. coli bacteria allow. In principle, all promoters described above are suitable as plant promoters.

The nucleic acid sequences contained in the expression cassettes or vectors may be functionally linked to other genetic control sequences in addition to a promoter. The term "genetic control sequences" is to be understood broadly and means all those sequences which have an influence on the production or the function of the expression cassette according to the invention. For example, genetic control sequences modify transcription and translation in prokaryotic or eukaryotic organisms. The expression cassettes according to the invention preferably comprise a plant-specific promoter 5'-upstream of the respective transgene-to-be-expressed promoter and 3'-downstream a terminator sequence as additional genetic control sequence, and optionally further customary regulatory elements, in each case functionally linked to the nucleic acid sequence to be transgeneically expressed. Genetic control sequences also include other promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. For example, by means of genetic control sequences, the tissue-specific expression can additionally take place as a function of specific stress factors. Corresponding elements are exemplified by 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 fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used to advantage.

Genetic control sequences also include the 5 'untranslated regions n, introns or non-coding 3' region of genes such as actin-1

Intron, or the Adh1-S introns 1, 2 and 6 (commonly: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). These have been shown to play a significant role in the regulation of gene expression. It has been shown that 5'-untranslated sequences can enhance the transient expression of heterologous genes. Exemplary of translation enhancers is the 5 'leader sequence from the tobacco mosaic virus (GaINe et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. They may also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).

The expression cassette may advantageously contain one or more so-called enhancer sequences functionally linked to the promoter, which allow increased transgenic expression of the nucleic acid sequence. Also at the 3 'end of the transgenic nucleic acid sequences to be expressed additional advantageous sequences can be inserted, such as other regulatory elements or terminators. The transgenic nucleic acid sequences to be expressed may be contained in one or more copies in the gene construct.

Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which are essentially T-DNA polyadenylation signals. Signals from Agrobacterium tumefaciens, in particular gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACHS correspond (Gielen et al. (1984) EMBO J 3: 835 ff) or functional equivalents thereof. Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopaline synthase) terminator.

Control sequences are furthermore to be understood as meaning those which permit homologous recombination or insertion into the genome of a host organism or permit removal from the genome. In homologous recombination, for example, the coding sequence of a particular endogenous gene can be selectively exchanged for the sequence coding for a dsRNA. Methods such as the cre / lox technology allow for tissue-specific, possibly inducible removal of the expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14 (4): 381-92). Here, certain flanking sequences are added to the target gene (lox sequences), which later enable removal by means of the cre recombinase.

An expression cassette and the vectors derived from it can contain further functional elements. The term functional element is to be understood broadly and means all those elements which have an influence on the production, multiplication or function of the expression cassettes, vectors or transgenic organisms according to the invention. By way of example but not by way of limitation:

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 kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc. Particularly preferred selection markers are those which confer resistance to herbicides. Examples include:

DNA sequences which encode phosphinothricin (PAT) and inactivate glutamin synthase inhibitors (bar and pat genes), 5-enolpyruvylshikimate-3-phosphate (EPSP synthase genes), which confer resistance to glyphosate ^® (N- (phosphonomethyl) glycine), which for the glyphosate ^® degrading enzymes encoding gox gene (Glyphosatoxidoreduk- tase), the deh gene (encoding a dehalogenase which inactivates dalapon), sulfonylurea- and imidazolinone-inactivating acetolactate synthases, and bxn genes encoding the bromoxynil-degrading nitrilase enzymes, the aasa- Gene conferring resistance to the antibiotic apectinomycin, streptomycin phosphotransferase (SPT) gene conferring resistance to streptomycin, neomycin phosphotransferase (NPTII) gene conferring resistance to kanamycin or geneticin, hygromycin phosphotransferase (HPT) A gene conferring resistance to hygromycin, the acetolactate synthase gene (ALS), which confers resistance to sulfonylurea herbicides (eg mutant ALS variants with, for example, the S4 and / or Hra mutation).

b) Reporter genes which code for easily quantifiable proteins and ensure an evaluation of the transformation efficiency or of the expression site or time point via intrinsic color or enzyme activity. Very particular preference is given to 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 galactosidase, most preferably the β-glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).

c) Replication origins that ensure an increase of the expression cassettes or vectors according to the invention in, for example, E. coli. Examples include ORI (origin of DNA replication), the pBR322 ori or the P15A oh

(Sambrook et al.: Molecular Cloning, A Laboratory Manual, 2 ^nd Ed., CoId Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989).

d) Elements required for Agrobacterium-mediated plant transformation, such as 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 generally necessary to additionally introduce a selection marker which confers on the successfully recombined cells a resistance 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 selection of the transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5:81 -84). In addition, the transgenic expression cassette or the expression vectors may contain further nucleic acid sequences which do not code for a yeast G3PDH and whose transgenic expression leads to an additional increase in fatty acid biosynthesis (as a result of pro-OL). This additional transgenically expressed proOIL nucleic acid sequence may be selected by way of example but not limitation from nucleic acids encoding 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 readily available from databases or corresponding cDNA libraries of the respective plants.

The introduction of an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably in plants or plant cells, tissues, organs, parts or seeds) can advantageously be realized using vectors in which the Expression cassettes are included. A further subject of the invention therefore relates to said transgenic vectors comprising a transgenic expression cassette for a yeast G3PDH.

Vectors may be, for example, plasmids, cosmids, phages, viruses or else agrobacteria. The expression cassette can be introduced into the vector (preferably a plasmid vector) via a suitable restriction site. The resulting vector is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombinant vector obtained by methods familiar to those skilled in the art. Restriction analysis and sequencing can serve to test the cloning step. Preference is given to those vectors which enable a stable integration of the expression cassette into the host genome.

The production of a corresponding transgenic plant organism takes place, for example, by means of transformation or transfection using the corresponding proteins or nucleic acids. The production of a transformed organism (or a transformed cell or tissue) requires that the appropriate DNA (eg the expression vector), RNA or protein be introduced into the corresponding host cell. For this process, referred to as transformation (or transfection), a variety of methods are available (Keown et al., (1990) Methods in Enzymology 185: 527-537). For example, DNA or RNA can be directly injected by microinjection or by bombardment with DNA-coated microtubes. Particles are introduced. Also, the cell can be permeabilized chemically, for example with polyethylene glycol, so that the DNA can enter the cell by diffusion. The DNA can also be made by protoplast fusion with other DNA-containing moieties such as minicells, cells, lysosomes or liposomes. Electroporation is another suitable method for introducing DNA, in which the cells are reversibly permeabilized by an electrical pulse. Also possible are the swelling of plant parts in DNA solutions as well as pollen or pollen tube transformation. Corresponding methods have been described (for example, Bilang et al., (1991) Gene 100: 247-250, Scheid et al (1991) Mol Gen Gen 228: 104-112, Guerche et al (1987) Plant Science 52: 11 Neuhaus 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 for the transformation and regeneration of plants from plant tissues or plant cells are used for transient or stable transformation. Suitable methods are in particular the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called particle bombardment method, the electroporation, the incubation of dry embryos in DNA-containing solution and the microinjection.

Besides these "direct" transformation techniques, transformation can also be performed by bacterial infection by Agrobacterium tumefaciens or Agrobacterium rhizogenes and transfer of corresponding recombinant Ti plasmids or Ri plasmids by or through infection with transgenic plant viruses. 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 to be integrated into special plasmids, either into an intermediate vector (shuttle or intermediate vector) or a binary vector. If a Ti or Ri plasmid is to be used for transformation, at least the right boundary, but most often the right and left borders of the Ti or Ri plasmid T-DNA as flanking region connected to the expression cassette to be introduced.

Preferably, binary vectors are used. Binary vectors can replicate in both E. coli and Agrobacterium. They usually contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA restriction sequence. They can be transformed directly into Agrobacterium (Holsters et al., (1978) Mol Gen Genet 163: 181-187). The selection marker gene allows selection of transformed Agrobacteria and is, for example, the nptll gene conferring resistance to kanamycin. The Agrobacterium acting as a host organism in this case should already contain a plasmid with the vir region. This is required for the transfer of T-DNA to the plant cell. Such transformed Agrobacterium can be used to transform plant cells. The use of T-DNA for the transformation of plant cells has been extensively studied and described (EP 120 516, Hoekema, In: The Binary Planet Vector System, Offsetdrukkerij Kanters BV, Alblasserdam, Chapter V, An et al., (1985) EMBO J 4 : 277-287). Various binary vectors are known and partially commercially available such as pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).

Other promoters suitable for expression in plants are described (Rogers et al., (1987) Meth in Enzymol 153: 253-277; Schardl et al. (1987) Gene 61: 1-11; Berger et al. (1989) Proc Natl Acad See USA 86: 8402-8406).

Direct transformation techniques are suitable for every organism and cell type. In the case of injection or electroporation of DNA or RNA into plant cells, no special requirements are placed on the plasmid used. Simple plasmids such as the pUC series can be used. Should be complete

Plants are regenerated from the transformed cells, it is necessary that there is an additional selectable marker gene on the plasmid.

Stably transformed cells, ie those containing the introduced DNA integrated into the DNA of the host cell, can be selected from untransformed if a selectable marker is part of the introduced DNA. By way of example, any gene which can confer resistance to antibiotics or herbicides (such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin, etc.) can act as a marker (see above). Transformed cells expressing such a marker gene are in able to survive in the presence of concentrations of a corresponding antibiotic or herbicide that kill an untransformed wild type. Examples are mentioned above and preferably include the bar gene that confers resistance to the herbicide phosphinotricin (Rathore KS et al. (1993) Plant Mol Biol 21 (5): 871-884), the nptll gene that confers resistance to kanamycin, the hpt gene, that

Conferring resistance to hygromycin, or the EPSP gene conferring resistance to the herbicide glyphosate. The selection marker allows the selection of transformed cells from transformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84). The resulting plants can be grown and crossed in the usual way. Two or more generations should be cultured to ensure that genomic integration is stable and hereditary.

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

Once a transformed plant cell has been produced, a complete plant can be obtained using methods known to those skilled in the art. This is an example of callus cultures. From these still undifferentiated cell masses, the formation of shoot and root can be induced in a known manner. The obtained sprouts can be planted out and bred.

The person skilled in such methods are known to regenerate from plant cells, plant parts and whole plants. For example, methods for this are described by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al. (1995) Plant Cell Rep. 14: 273-278; Jahne et al. (1994) Theor Appl Genet 89: 525-533.

By "transgene" or "recombinant" is meant, for example, a nucleic acid sequence, an expression cassette or a vector containing said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector any such constructions resulting from genetic engineering in which either a) the nucleic acid sequence encoding 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 organism, or

c) (a) and (b)

are not in their natural genetic environment or have been modified by genetic engineering methods, which modification may exemplarily be substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. Natural genetic environment means the natural chromosome 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 conserved. 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, more preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of the promoter of a gene encoding a yeast G3PDH, becomes a transgenic expression cassette when modified by non-natural, synthetic ("artificial") methods such as mutagenization. Corresponding methods are described (US 5,565,350, WO 00/15815, see also above).

Preferred host or starting organisms as transgenic organisms are, in particular, plants as defined above. Included within the scope of the invention are all genera and species of monocotyledonous and dicotyledonous plants of the plant kingdom, in particular plants used for the production of oils, such as, for example, oilseed rape, sunflower, sesame, safflower, olive tree, soya, maize and nut species. Also included are the mature plants, seeds, sprouts and seedlings, as well as derived parts, propagation material and cultures, for example cell cultures. Mature plants means plants to any developmental stage beyond the seedling. Keimling means a young, immature plant at an early stage of development.

The production of transgenic plants can be carried out by the methods described above to transform or transfect organisms.

Another object of the invention relates to the direct use of the transgenic plants according to the invention and the cells derived from them, cell cultures, parts - such as in transgenic plants roots, leaves, etc.-, and transgenic propagation material such as seeds or fruits, for the production of food or feed, cosmetics or pharmaceuticals, in particular of oils, fats, fatty acids or derivatives of the abovementioned. For this purpose, the plants or plant parts are added to food, feed, cosmetics, pharmaceuticals or products with industrial applications in conventional amounts. Also, from the plants, preferably from the seeds, as described above, the oils and / or optionally free fatty acids can be obtained and added to the foodstuffs, feedstuffs, cosmetics, pharmaceuticals or products with industrial applications in conventional amounts.

In addition to affecting the oil content, transgenic expression of a yeast G3PDH in plants can mediate further beneficial effects, such as increased stress resistance, e.g. against osmotic stress. The yeast G3PDH provides protection against such stress via elevated glycerol levels by acting as an osmoprotective agent. Such osmotic stress occurs, for example, in saline soils and water and is an increasing problem in agriculture. An increased stress tolerance offers, for example, the possibility of agricultural use of land in which conventional crops can not thrive.

Furthermore, the transgenic expression of the yeast G3PDH can influence the NADH level and thus the REDOX balance in the plant organism. Stress, such as dryness, heat, cold, UV light, etc. can increase NADH

Mirroring and leading to increased formation of reactive oxygen (ROS). The transgenic expression of the yeast G3PDH can degrade an excess of NADH under said stress conditions, thus stabilizing the REDOX balance and mitigating the effects of stress.

sequences

1. SEQ ID NO: 1

Nucleic acid sequence encoding Saccharomyces cerevisiae G3PDH (Gpd1p) 2. SEQ ID NO: 2

Protein sequence of Saccharomyces cerevisiae G3PDH (Gpdi p)

3. SEQ ID NO: 3

Nucleic acid sequence encoding Saccharomyces cerevisiae G3PDH (Gpd2p)

4. SEQ ID NO: 4

Protein sequence of Saccharomyces cerevisiae G3PDH (Gpd2p)

5. SEQ ID NO: 5

Protein sequence of Saccharomyces cerevisiae G3PDH (Gpd2p) with second, alternative start codon

6. SEQ ID NO: 6

Nucleic acid sequence encoding Schizosaccharomyces pombe G3PDH

7. SEQ ID NO: 7

Protein sequence of Schizosaccharomyces pombe G3PDH

8. SEQ ID NO: 8 Nucleic acid sequence encoding Schizosaccharomyces pombe G3PDH

9. SEQ ID NO: 9

Protein sequence of Schizosaccharomyces pombe G3PDH

10. SEQ ID NOM O.

Nucleic acid sequence encoding Yarrowinia lipolytica G3PDH

11. SEQ ID NO: 11

Protein sequence of Yarrowinia lipolytica G3PDH

12. SEQ ID NO: 12

Protein sequence of Yarrowinia lipolytica G3PDH, with second, alternative start codon

13. SEQ ID NO: 13

Nucleic acid sequence encoding Zygosaccharomyces rouxii G3PDH

14. SEQ ID NO: 14

Protein sequence of Zygosaccharomyces rouxii G3PDH 15. SEQ ID NO: 15

Nucleic acid sequence encoding Zygosaccharomyces rouxii G3PDH

16. SEQ ID NO: 16

Protein sequence of Zygosaccharomyces rouxii G3PDH

17. SEQ ID NO: 17

Expression vector based on pSUN-USP for S. cerevisiae G3PDH (Gpdip, insert from bp 1017 - 2190)

18. SEQ ID NO: 18 oligonucleotide primer OPNI ö'-ACTAGTATGTCTGCTGCTGCTGATAG-S '

19. SEQ ID NO: 19 oligonucleotide primer OPN2 δ'-CTCGAGATCTTCATGTAGATCT AATT-3 ¹

20. SEQ ID NO: 20 oligonucleotide primer OPN3 5'-GCGGCCGCCATGTCTGCTGCTGCTGATAG-S '

21. SEQ ID NO: 21 oligonucleotide primer OPN4 5'-GCGGCCGCATCTTCATGTAGATCTAATT-S '

22-35: SEQ ID NOs: 22-35: Sequence motifs for yeast G3PDHs indicating possible sequence variations (see above). The variations of a single motif can occur individually but also in different combinations with each other.

36. SEQ ID NO: 36

Expression vector pGPTV-gpd1 based on pGPTV-Napin for S. cerevisiae G3PDH (Gpdip, insert gdp1 of bp 11962-13137, nos terminator: 13154-13408, napin promoter: 10807-11951).

37. SEQ ID NO: 37 Nucleic acid sequence encoding Emericella nidulans G3PDH

38. SEQ ID NO: 38

Protein sequence of the Emericella nidulans G3PDH 39. SEQ ID NO: 39

Nucleic acid sequence encoding Debaryomyces hansenii G3PDH (partial)

40. SEQ ID NO: 40

Protein sequence of Debaryomyces hansenii G3PDH (partial)

pictures

FIG. 1: Northern blot. Proof of transcription of the yeast GPD1 gene in ripening seeds of transgenic rapeseed lines (8, 6, 9 and 3). As a comparison, the same detection has been carried out with wild-type (WT) plants. In lines 8, 6 and 9, the GPD1 transcript could be detected. In line 3 there was no expression of the GPD1 gene.

This line was used as additional control for further analysis.

Figure 2: Glycerol-3-phosphate amount in maturing seeds (40 days after flowering DAF) of transgenic GPD1 rape lines 8, 6 and 9 (black bars). As a comparison, the content has been determined in corresponding untransformed Wilt type plants (WT) and the non-expressing transgenic line 3 (lighter bars). The specified error deviations result from 6 independent measurements of all received seeds.

FIG. 3: Glycerol-3-phosphate dehydrogenase activity in maturing seeds (40

DAF) transgenic GPD1 rapeseed lines 8, 6 and 9 (black bars). As a comparison, the content has been determined in corresponding untransformed Wilt type plants (WT) and the non-expressing transgenic line 3 (lighter bars). The specified error deviations result from 6 independent measurements of all obtained

Seeds.

Figure 4: Total amount of lipid in the seeds of transgenic GPD1 p-rapeseed lines

(black bars) related to the seed mass. For comparison, the content has been determined in corresponding untransformed Wilt type plants (WT) and the non-expressing transgenic line 3 (lighter bars). All 3 transgenic and expressing plants show a significant increase in total lipid levels in mature seeds. The indicated error deviations result from in each case 5 independent measurements of all received seeds.

FIG. 5 shows a sequence comparison of homologs of G3PDH from other yeasts.

tables

Table 1 :

Fatty acid profile of seed oils in the GPD1 p-rape lines 8, 6 and 9 (in mol%). For comparison, the fatty acid profile in the corresponding untransformed wild-type plants (WT) and the non-expressing transgenic line 3 is shown.

General methods:

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

The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The carried out in the context of the present invention cloning steps such. B. restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial growth, propagation of phages and sequence analysis of recombinant DNA are as in Sambrook et al. (1989) CoId Sprig ng Harbor

Laboratory Press; ISBN 0-87969-309-6 described. The sequencing of recombinant DNA molecules is carried out with a laser fluorescence DNA sequencer from the company ABI according to the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74: 5463-5467).

Example 1: Cloning of the Gpd1 gene from yeast

Genomic DNA from Saccharomyces cerevisiae S288C (Mat alpha SUC2 times mel gal2 CUP1 flo1 Flo8-1, Invitrogen, Karlsuhe, Germany) was isolated according to the following protocol:

A 100 ml culture was grown to an optical density of 1.0 at 30 ° C. Of these, 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 to 1.5 ml vials, spun down and the supernatant discarded. The pellets were resuspended by vortexing with 200 μl solution A, 200 μl phenol / chloroform (1: 1) and 0.3 g glass beads and lysed. After addition of 200 μl of TE buffer pH 8.0, centrifugation was carried out for 5 minutes. The supernatant was subjected to ethanol precipitation with 1 ml of ethanol. The resulting pellet after precipitation was dissolved in 400 μL TE buffer pH 8.0 + 30 μg / mL RnaseA. After incubation for 5 min at 37 ⁰ C 18 μl_ 3 M sodium acetate solution pH 4.8 and 1 mL ethanol were added and the precipitated DNA was pelleted by centrifugation. The DNA pellet was dissolved in 25 μl of double distilled H ₂ O. The concentration of genomic DNA was determined by its absorbance 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.

Sequence primer pair 1: δ'-ACTAGTATGTCTGCTGCTGCTGATAG

Sequence primer pair 2: δ'-CTCGAGATCTTCATGTAGATCTAATT

Composition of the PCR approach (50 μL):

5.00 μl_ 5 μg yeast genomic DNA 5.00 μL 10x buffer (Advantage polymerase) + 25 mM MgCl ₂ 5.00 μL ₂ mM dNTP 1.25 μL per primer (10 pmol / μL) 0.50 μL Advantage polymerase

The Advantage polymerase from Clontech was used.

PCR program:

Initial denaturation for 2 min at 95 ° C, then 35 cycles at 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 cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, resulting in the vector pCR2.1-gpd1 and the sequence was checked by sequencing. For cloning into the agrotransformation vector pGPTV, 0.5 μg of the

Vector pCR2.1-gpd1 with the restriction enzyme Xhol (New England Biolabs) for 2 hours and then incubated for 15 min with Klenow fragment (New England Biolabs). After incubation with Spei for 2 hours, the DNA fragments were separated by gel selector phoresis. The 1185 bp fragment of the gpd1 sequence next to the vector (3.9 kb) was excised from the gel and mixed with the

Purified gel of Qiagen according to manufacturer's instructions and mixed with 50 μl_

Elution buffer eluted. 0.1 μg of the vector pGPTV was first incubated with the

Restriction enzyme Sacl digested, then incubated for 15 min with Klenow 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 transformed into TOP10 cells (Stratagene) according to the manufacturer's instructions and selected accordingly, resulting in the vector pGPTV-gpd1. Positive clones are verified with primers 1 and 2 by PCR and sequencing.

For the production of the vector pSUN-USP-gpd1, a PCR of the vector pCR2.1-gpd1 was performed with primers 3 and 4.

Sequence Primer 3: 5'-GCGGCCGCCATGTCTGCTGCTGCTGATAG Sequence Primer 4: 5'-GCGGCCGCATCTTCATGTAGATCTAATT

Composition of the PCR approach (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 pmol / μL) 0.50 μl_ Advantage polymerase

The Advantage polymerase from Clontech was used.

PCR program:

Initial denaturation for 2 min at 95 ° C, then 35 cycles with 45 sec 95 ° C, 45 sec 55 ⁰ C and 2 min 72 ° C. Final extension of 5 min at 72 ° C. The 1190 bp PCR product was digested with the restriction enzyme Notl for 24 hours. The vector pSUN-USP was digested with NotI for 2 hours, then incubated for 15 minutes with alkaline phosphatase (New England Biolabs). 100 ng of the 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 are then transformed into TOP 10 cells (Stratagene) according to the manufacturer's instructions and classified according to selection, resulting in the vector pSUN-USP-gpd1. Positive clones are verified with primers 3 and 4 by PCR and sequencing.

Example 2: Plasmids for plant transformation

For plant transformation, binary vectors such as pBinAR can be used (Hofgen 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 into T-DNA. 5 'of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is 3 'to the cDNA.

The tissue-specific expression can be achieved using a tissue-specific promoter. For example, seed-specific expression can be achieved by cloning in the napin or LeB4 or the USP promoter 5 'of the cDNA. Any other seed-specific promoter element can also be used. For constitutive expression throughout the plant, the CaMV 35S promoter can be used.

Another example of a binary vector is the vector pSUN-USP and pGPTV-Napin in which the fragments of the fragment from Example 2 were cloned. The vector pSUN-USP contains the USP promoter and the OCS terminator. The vector pGPTV-napin contains a truncated version of the napin promoter and the nos terminator.

The fragments of Example 2 were cloned into the multiple cloning site of the vector pSUN-USP and pGPTV-napin, respectively, to allow for seed-specific expression of the GPD1 gene. The corresponding construct pSUN-USP-gpd1 is described by SEQ ID NO: 16, the construct of G3PDH in pGPTV-Napin is described by SEQ ID NO: 36.

Example 3: Transformation of Agrobacterium The Agrobacterium -mediated plant transformation can be carried out, for example, using the Agrobacterium tumefaciens strains GV3101 (pM P90) (Koncz and Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech). The transformation can be performed by standard transformation techniques (Deblaere et al., (1984) Nucl Acids Res 13: 4777-4788).

Example 4: Plant transformation

Agrobacterium-mediated plant transformation was performed using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Central Signature: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, Joh n E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pp ., ISBN 0-8493-5164-2).

Rapeseed was transformed by cotyledon or hypocotyl transformation (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use of antibiotics for Agrobacterium and plant selection depends on the binary vector and Agrobacterium strain used for the transformation. Rapeseed selection was performed using kanamycin as a selectable plant marker.

The Agrobacterium-mediated gene transfer in flax (Linum usitatissimum) can be determined using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285.

Transformation of soy may be accomplished using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 039 7 687, US 5,376,543, US 5,169,770 (University Toledo).

Plant transformation using particle bombardment, polyethylene glycol-mediated DNA uptake or via the silicon carbonate fiber technique is described, for example, by Freeling and Walbot "The Maize Handbook" (1993) ISBN 3-540-97826-7, Springer, New York ).

Example 5: Examination of the Expression of a Recombinant Gene Product in a transformed organism

A suitable method for determining the amount of transcription of the gene (an indication of the amount of RNA available for translation of the gene product) is the performance of a Northern blot as set forth below (for reference, see Ausubel et al. 1988) Current Protocols in Molecular Biology, Wiley: New York, or the Example part mentioned above), wherein a primer designed to bind to the gene of interest is labeled with a detectable label (usually radioactive or chemiluminescent) such that when the total RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, the binding and extent of binding of the probe determines the presence as well as the amount of mRNA for indicating this gene. This information indicates the degree of transcription of the transformed gene. Total cellular RNA may be prepared from cells, tissues or organs by a variety of methods, all known in the art, such as that described by Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326.

Northern hybridization:

For RNA hybridization, 20 μg of total RNA or 1 μg of poly (A) ⁺ RNA were purified by gel electrophoresis in 1.25% strength agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem 152, 304), transferred by capillary attraction using 10 × SSC to positively charged nylon membranes (Hybond N +, Amersham, Braunschweig), immobilized by UV light and incubated for 3 hours at 68 ° C. using hybridization buffer (10%). Dextran sulfate, w / v, 1 M NaCl, 1% SDS, 100 mg herring sperm DNA). The labeling of the DNA probe with the High Prime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization stage using alpha- ³² P-dCTP (Amersham Pharmacia, Braunschweig, Germany). Hybridization was performed after addition of the labeled DNA probe in the same buffer at 68 ° C overnight. The washes were performed twice for 15 minutes using 2X SSC and twice for 30 minutes using 1X SSC, 1% SDS, at 68 ° C. The sealed filters were exposed at -70 ⁰ C for a period of 1 to 14 days. Standard techniques, such as western blotting, can be used to study the presence or relative amount of protein translated from this mRNA (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York). In this method, the total cellular proteins are extracted, separated by gel electrophoresis, on a matrix, such as

Nitrocellulose and incubated with a probe, such as an antibody that binds specifically to the desired protein. This probe is usually provided with a chemiluminescent or colorimetric label that is easily detected. The presence and amount of label observed indicates the presence and amount of the desired mutant protein present in the cell.

FIG. 1 shows the results of the Northern blot of 4 independent transgenic rapeseed lines and of the wild-type. The plants of lines 6, 8 and 9 show a strong detection signal on the Northern blot. The plants consequently express the GPD1 gene in ripening seeds. In contrast, the transcription of the GPD1 gene was not detected in the semen sample of line 3 and served as an additional control in addition to the wild type. In addition, line 3 shows that the expression of the transferred gene, depending on the place of integration into the genome of Brassica napus not always succeed.

Example 6: Analysis of the effect of the recombinant proteins on the production of the desired product

The effect of genetic modification in plants or on the production of a desired compound (such as a fatty acid) may be determined by cultivating the modified plant under appropriate conditions (such as those described above) and the medium and / or cellular components the increased production of the desired product (ie lipids or a fatty acid) is examined. These analytical techniques are known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods as well as analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Volume A2, pages 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 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, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, FJ. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to the above-mentioned methods, plant lipids derived from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Be. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145. Qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W., Advances in Lipid Methodology, Ayr / Scotland: OiIy Press (Oli Press Lipid Library, 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: OiIy Press, 1989, Repr. 1992, IX, 307 p. (OiIy Press Lipid Library, 1); Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977) et al .: Progress in the Chemistry of Fats and Other Lipids CODES.

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

The unambiguous evidence for the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods: GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition. Christie, Oliver Press, Dundee, 119-169; 1998, Gas Chromatography Mass Spectrometry Method, Lipids 33: 343-353).

The material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods. The material must be centrifuged after rupture. The sediment is distilled in aqua. resuspended, heated at 100 ^° C. for 10 minutes, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90 ^° C., resulting in hydrolyzed oil and lipid compounds that produce transmethylated lipids. These fatty acid methyl esters are extracted in petroleum ether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ⁰ C and 240 ° C for 20 min and 5 min at 240 ⁰ C subjected. The identity of the resulting fatty acid methyl esters must be defined using standards available from commercial sources (ie Sigma).

Plant material is first mechanically homogenized by mortars to make it more accessible to extraction.

For the quantitative oil analysis of the Brassica plants transformed with the construct Gpd1 gene, the following was applied to the protocol:

The extraction of the lipids from seeds is carried out according to the method of Bligh & Dyer (1959) Can J Biochem Physiol 37:91 1. For this purpose, 5 mg Arabidopsis Brassica seeds are weighed in 1.2 ml Qiagen microtubes (Qiagen, Hilden) on a Sartorius (Göttingen) microbalance. The seed material is homogenized with 1 ml of chloroform / methanol (1: 1, Sigma's mono-C17-glycerol as internal standard) in the Rätschmühle MM300 from Retsch (Haan) and incubated for 20 min at RT. After centrifugation, the supernatant was transferred to a new vessel and the sediment extracted again with 1 ml of chloroform / methanol (1: 1). The supernatants were combined and concentrated to dryness. The derivatization of the fatty acid was carried out by acid methanolysis. For this purpose, the extracted lipids were treated with 0.5 M sulfuric acid in methanol and 2% (v / v) dimethoxypropane and for 60 min. incubated at 80 ⁰ C. This was followed by extracting twice with petroleum ether, followed by washing steps with 100 mM sodium bicarbonate and water. The fatty acid methyl esters thus prepared were concentrated to dryness and in a defined

Volumes of petroleum ether recorded. 2 μl of the fatty acid methyl ester solution were finally separated by gas chromatography (HP 6890, Agilent Technologies) on a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and analyzed by means of a flame ionization detector. The quantification of the oil was carried out by comparing the signal strengths of the derivatized fatty acids with those of the internal standard mono-C17-glycerol (Sigma). FIG. 4 shows, by way of example, the results for the quantitative determination of the oil contents in T3 seeds of 3 independent transgenic rape lines as well as a non-expressing control line and the untransformed wild-type plants. From the seed pools of each line, five independent extractions were performed and the extracts measured independently. Out The mean and standard deviation were calculated for the three independent measurements.

In the semen samples of the transgenic lines 6, 8, and 9, a significant increase in the total lipid content compared to the wild type and the non-expressing control line could be detected. This was between about 20 and 22% of the

Seed weight. The lipid content in the wild type and in the control line 3 was in contrast only about 15%. This corresponds to a total increase in oil content of 33% or approx. 47%. By contrast, the fatty acid composition was not altered by GPD1 expression (see Table 1).

Table 1 shows the fatty acid composition in the T3 seeds of the transgenic

GDH-expressing lines 8, 6 and 9, as well as a non-expressing control line 3 and the untransformed wild-type plants. From the seed pools of each line, five independent extractions were performed and the extracts measured independently. From the three independent measurements, the mean and the standard deviation were calculated.

Both in the transgenic and expressing GDH lines 8, 6 and 9 and in the non-expressing Kontrollli never 3 and the untransformed wild-type plants makes oleic acid (18: 1) with more than 55%, the largest share in the oil.

Example 7: Extraction of glycerol-3-phosphate

For the extraction of glycerol-3-phosphate from ripening rapeseed they are homogenized in a vibrating mill (Retsch) and mixed with 500 .mu.l of cold 16% (w / v) TCA / diethyl ether and incubated on ice for 20 minutes. Subsequently, 800 μl of cold 16% TCA / H2O, 5 mM EGTA are added and incubated on ice for 3 hours. By centrifugation, the sedimentation of non-soluble components takes place. The liquid upper phases are transferred to a new vessel and washed with 500 .mu.l of cold, water-saturated diethyl ether at 4 ⁰ C and centrifuged again. The hydrophilic lower phase is subjected to 3 more washes and the pH is adjusted to 6-7 with 5 M KOH / 1 M TEA. The hydrophilic phase is flash frozen in liquid nitrogen, dried in a lyophilizer (lyophilizer, Christ) and then dissolved in 800 μl H2O.

Example 8: Determination of glycerol-3-phosphate (G3P) amount The determination of the amount of G3P is carried out by the enzymic cycling assay (Gibon et al., 2002). To this end, 10 .mu.l of the hydrophilic phase (s. Above) or G3PDH replication te (s. Below) with 46 .mu.l Tricine / KOH (pH 7.8 200 mM) / 10 mM MgCl 2 was added and 20 minutes at 95 ⁰ C to to destroy the dihydroxyacetone phosphate. Subsequently, the samples are briefly centrifuged and the supernatant with 45 ul reaction mixture (2 u glycerol-3-phosphate oxidase, 0.4 u glycerol-3-phosphate dehydrogenase, 13 O u catalase, 0.12 .mu.mol NADH) was added. The reaction results in a net consumption of NADH, which can be monitored directly on the photometer by decreasing the absorbance at 340 nm. The calculation of the G3P quantity takes place via a calibration line of different G3P concentrations.

Interestingly, seed-specific overexpression of Saccharomyces GDP resulted in a significant increase in G3P content in maturing seeds of transgenic oilseed rape (40 DAF). The G3P levels in the seeds (40 DAF) of lines 6, 8 and 9 ranged between about 350 and 420 nmol G3P / gm of fresh mass. By contrast, the G3P content in the wild-type seeds and the seeds of the non-expressing line ranged between about 50 and 100 nmol G3P / gm of fresh mass (see FIG. 2).

Example 9: Determination of G3PDH activity

To determine the G3PDH activity in the ripening rape seeds, the ripening seeds are isolated from frozen pods, weighed with a fine balance and homogenized by means of a vibrating mill (Retsch). The samples are then frozen again in liquid nitrogen.

Five hundred microliters of cold extraction buffer (50 mM HEPES pH 7.4, 5 mM MgCl 2, 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, 10% (v / v) glycerol, 2 mM benzamidine, 2 mM caproic acid, 0.5 mM Phenylmethylsulphonylfluorid, 1 g / l Polyvi- nylpolypyrrolidon) are added to the homogenized samples, mixed well and incubated for 30 minutes at 4 ° C in the dark with continuous shaking. Subsequently, the samples are centrifuged at 14,000 rpm and 4 ° C. for 15 minutes (Eppendorf centrifuge). The supernatant containing the soluble proteins is transferred to a new Eppendorf tube and can be used directly for the determination of G3PDH activity or used at -8O ⁰ C.

10 μl of the protein extracts are pipetted with 90 μl of reaction mixture (4 mM dihydroxyacetone phosphate, 0.2 mM NADH in 50 mM HEPES pH 7.4) and for 30 minutes incubated at 24 ⁰ C. Thereafter, the termination of the reaction by heating to 95 ° C for 20 minutes.

From each sample, 3 replicates are used for the determination of G3PDH activity whereby a sample is heated directly and serves as a blank sample. The amount of glycerol-3-phosphate (G3P) formed is then measured by the method of Gibon et al. 2002 (see above).

The transgenic lines 6, 8 and 9, which were positive for transcription, showed a significant increase in glycerol-3-phosphate dehydrogenase activity in the maturing seeds (40 DAF) compared to the wild-type or non-expressing control line 3. In lines 6, 8 and 9 an activity of up to about 400 nmol G3P / g fresh mass per minute could be detected. By contrast, in the wild type and in the non-expressing control line 3 the activity was only about 250 nmol G3P / g fresh mass and minute. This shows that GPD1 is seed-specific at both RNA level and enzyme level in lines 6, 8 and 9.

equivalents:

One skilled in the art will recognize or recognize many equivalents of the specific embodiments of the invention described herein, using only routine experimentation. These equivalents are intended to be encompassed by the claims.

Claims

claims 1. A method for increasing the total oil content in transgenic oil plants, characterized in that the transgenic oil plants contain at least 20% by weight of oleic acid based on the total fatty acid content and comprising the following process steps a) introducing a nucleic acid sequence coding for a glycerol-3-phosphate dehydrogenase from a yeast, in the oil plant, and b) expression of the encoded by the nucleic acid glycerol-3-phosphate dehydrogenase in the oil plant, and c) selection of oil plants where the total oil content is at least 25% by weight in the plant compared to the non-transgenic plant is increased. 2. A method for increasing the total oil content in transgenic oil plants, characterized in that the transgenic oil plants contain at least 20% by weight of oleic acid based on the total fatty acid content and in which the total oil content by at least 45% by weight in the plant compared to the non-transgenic plant is increased. 3. The method according to claim 1 or 2, characterized in that the nucleic acid sequence coding for the glycerol-3-phosphate dehydrogenase, is derived from a yeast which is selected from the group of the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Thchosporon, Rhodotorul, Sporobolomyces, Bullera, Saccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia and Hanseniaspora. 4. The method according to any one of claims 1 to 3, characterized in that the nucleic acid sequence coding for the glycerol-3-phosphate dehydrogenase, is derived from a yeast which is selected from the group of the genera and species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorphe, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Y-arrowia lipolitica, Emericella nidulans, Debaryomyces hansenii and Torulaspora hansenii. 5. The method according to any one of claims 1 to 4, characterized in that the encoded by the nucleic acid sequence glycerol-3-phosphate dehydrogenase, the reaction of dihydroxyacetone phosphate to glycerol-3-phosphate using NADH or NA DPH as cosubstrate causes and a Peptidse- having at least one sequence motif selected from the Group of sequence motifs consisting of i) GSGNWGT (A / T) IAK ii) CG (V / A) LSGAN (L / I / V) AX (V / I) A iii) (L / V) FXRPYFXV 6. The method according to any one of claims 1 to 5, characterized in that the encoded by the nucleic acid sequence glycerol-3-phosphate dehydrogenase, the reaction of dihydroxyacetone phosphate to glycerol-3-phosphate using NADH or NA DPH as Cosubstrat causes and a Peptidse- comprising at least one sequence motif selected from the group of sequence motifs consisting of iv) GSGNWGTTIAKV (V / I) AEN v) NT (K / R) HQNVKYLP vi) D (I / V) LVFN (I / V) PHQFL vii) RA (I / V) SCLKGFE viii) CGALSGAN LA (P / T) EVA ix) LFHRPYFHV x) GLGEII (K / R) FG 7. The method according to any one of claims 5 or 6, characterized in that the encoded by the nucleic acid sequence glycerol-3-phosphate dehydrogenase se additionally comprises at least one sequence motif selected from the group of sequence motifs consisting of xi) H (E / Q) NVKYL xii) (0 / N) (IZV) (LZI) V (FZW) (VZN) (LZIZV) PHQF (VZL /!) xiii) (AZG) (IZV) SC (LZI) KG xiv) G (LZM) (LZG) E (MZI) (IZQ) (RZKZN) F (GZSZA) 8. The method according to any one of claims 1 to 4, characterized in that the encoded by the nucleic acid sequence glycerol-3-phosphate dehydrogenase is selected from the group: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 2, 4, 5, 7, 9, 11, 12, 14, 16, 38 or 40, or b) a functional equivalent of a) which codes for proteins with an identity of at least 60% with the sequence shown in SEQ ID NO: 2. 9. The method according to any one of claims 1 to 8, characterized in that for the expression of the nucleic acid sequence according to claim 1 (a) and (b) this is functionally connected to a promoter or terminator. 10. The method according to any one of claims 1 to 9, characterized in that the total oil content in the seed of the oil plant is increased. 11. The method according to claim 10, characterized in that the seed of the oil plant is harvested after cultivation and optionally the oil contained in the seed is isolated. 12. The method according to any one of claims 1 to 10, characterized in that the oil plant is selected from the group of oil plants consisting of Anacardium occidentale, Arachis hypogaea, Borago officinalis, Brassica campestris, Brassica napus, Brassica rapa, Brassica juncea, Camelina sativa, Cannabis sativa, Curthamus tinctorius, Cocos nucifera, Crambe abyssinica, Cuphea ciliata, Elaeis guineensis, Glycine max, Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum, Helianthus a nnus, Linum usitatissimum, Oenothera biennis, Olea europaea, Ricinus communis, Zea mays, Juglans regia and Prunus dulcis. 13. The method according to any one of claims 1 to 12, characterized in that in addition to increasing the total oil content and the content of glyceol-3-phosphate is increased by at least 20% by weight in the transgenic oil plant. 14. The method according to claim 11, characterized in that the fatty acids contained in the oil are liberated. 15. The method according to claim 10 or 11, characterized in that the oil or the released fatty acids polymers, food, feed, cosmetics, pharmaceuticals or products with industrial applications are added or used as lubricants.