WO2003104463A1 - Transgenic expression constructs and method for increasing the vitamin e content in plant organisms - Google Patents

Abstract

The invention relates to transgenic expression constructs and method for increasing the vitamin E content in plant organisms, preferably in algae, by means of transgenic expression of ORF s110832 from Synechocystis sp. PCC6803. The invention further relates to transgenic expression constructs for the expression of ORF s110832 in plant organisms, preferably in algae, transgenic plant organisms expressing ORF s110832 and the use of said transgenic plant organisms for the production of foodstuffs, animal foodstuffs, seed stock, pharmaceuticals or fine chemicals, in particular for the production of vitamin E.

Description

Transgenic expression constructs and methods for increasing the vitamin E content in plant organisms

description

The invention relates to transgenic expression constructs and methods for increasing the vitamin E content in plant organisms, preferably in algae, by transgenic expression of ORF S110832 from Synechocystis sp. PCC6803. The invention further relates to transgenic expression constructs for the expression of ORF sll0832 in plant organisms, preferably in algae, transgenic plant organisms expressing ORF sll0832, and the use of said transgenic plant organisms for the production of foodstuffs, animal feed, seeds, pharmaceuticals or fine chemicals, especially for the production of vitamin E.

The eight naturally occurring compounds with vitamin E activity are derivatives of 6-chromanol (Ullmann's Encyclopedia of Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft, Chapter 4., 478-488, vitamin E). The group of tocopherols (la-d) has a saturated side chain, the group of tocotrienols (2a-d) has an unsaturated side chain:

la, α-tocopherol: R ¹ = R ² = R ³ = CH ₃ lb, ß-tocopherol [148-03-8]: R ¹ = R ³ = CH _{3 /} R ² = H lc, γ-tocopherol [54 -28-4]: R ¹ = H, R ² = R ³ = CH ₃ id, δ-tocopherol [119-13-1]: R ¹ = R ² = H, R ³ = CH ₃

2a, α-tocotrienol [1721-51-3]: R ¹ = R ² = R ³ = CH ₃ 2b, β-tocotrienol [490-23-3]: R ¹ = R ³ = CH ₃ , R ² = H 2c, γ-tocotrienol [14101-61-2]: R ¹ = H, R ² = R ³ = CH ₃ 2d, δ-tocotrienol [25612-59-3]: Ri = R ² = H, R ³ = CH ₃

In the present invention, vitamin E is understood to mean all eight tocopherols and tocotrienols with vitamin E activity mentioned above.

Vitamin E compounds have a high economic value as additives in the food and feed sector, in pharmaceutical formulations and in cosmetic applications.

There are attempts to increase the metabolic flow to increase the tocopherol or tocotrienol content in transgenic plants by overexpressing individual tocopherol bio-synthesis genes (WO 97/27285; WO 99/04622; WO 99/23231; WO 00/10380;

Shintani and Dellapenna, Science 282 (5396): 2098-2100, 1998;

Tsegaye et al. Annual meeting of the American Society of Plant Physiologists (July 24-28, 1999, Baltimore, USA) Abstract No. 413).

Synechocystis sp. PCC 6803 is a unicellular, non-nitrogen fixing cyanobacterium that has been genetically well studied (Churin et al. (1995) J Bacteriol 177: 3337-3343) and can be easily transformed (Williams (1988) Methods Enzymol 167: 766-778) and has a very active homologous recombination ability. The PCC 6803 strain was isolated from fresh water by R. Kunisawa as Aphanocapsa N-1 in California, USA in 1968 and is now available from the Pasteur Culture Collection of Axenic Cyanobacterial Strains (PCC), Unite de Physiologie Microbienne, Paris, France. The overall genomic sequence of Synechocystis sp. PCC 6803 has been published since 1995 (Kaneko et al. (1995) DNA Research 2: 153-166; Kaneko et al. (1995) DNA Research 2: 191-198; Kaneko et al. (1996) DNA Research 3: 109- 136; Kaneko et al. (1996) DNA Research 3: 185-209; Kaneko and Tabata (1997) Plant Cell Physiol 38: 1171-1176; Kotani and Tabata (1998) Annu Rev Plant Physiol 49: 151-171) published on the Internet (http://www.kazusa.or.jp/cyano/cyano.html) under the name "CyanoBase". Effective expression systems for Synechocystis 6803 have been described in the literature (Mermet-Bouvier et al. (1993) Curr Microbiol 27: 323-327; Mermet-Bouvier and Chauvat (1993) Curr Microbiol 28: 145-148; Murphy and Stevens

(1992) Appl Environ Microbiol 58 1650-1655; Takeshima et al. (1994) Proc Natl Acad Sei USA 91 9685-9689; Xiaoqiang et al. (1997) Appl Environ Microbiol 63 4971-4975; Ren et al. (1998) FEMS Microbiol Lett 158: 127-132). Despite some success, there is still a need to optimize vitamin E biosynthesis. The object of the invention was to provide further methods which increase vitamin E biosynthesis and thus lead to advantageous transgenic plant organisms.

This object is achieved by the present invention. In the context of the invention, the protein was encoded by ORF sll0832 from Synechocystis sp. PCC 6803 (GenBank Acc.-No .: NP_442444; gi: 16331716; SEQ ID NO: 2) was surprisingly identified as a key factor in vitamin E biosynthesis (as a result of Tocen-1 for "tocopherol synthesis enhancing protein" -l). Tocen-1 is classified in the GenBank as an unknown protein without any functional annotation. The protein has no significant homology to other proteins of known function. Surprisingly, it was found that the transgenic expression of this protein can increase the vitamin E content in plant organisms.

A first object of the invention comprises methods for increasing the vitamin E content in plant organisms

a) Transgenic expression of the Tocen-1 protein encoded by

SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the aforesaid in said plant organism or a tissue, organ, part or cell of said plant organism, and

b) Selection of plant organisms in which - in contrast to or compared to the starting organism - the vitamin E content in the said plant organism or in a tissue, organ, part or cell of the said plant organism is increased.

In principle, the method according to the invention can be applied to all plant organisms, preferably to those which naturally produce vitamin E, very particularly preferably to those which are used for industrial production of natural vitamin E.

Another object of the invention relates to transgenic expression constructs for expressing a nucleic acid encoding the Tocen-1 protein encoded by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the aforementioned. The sequence according to SEQ ID N0: 1 and that based on the degeneration of the genetic code, sequences derived therefrom and functionally equivalent parts of the aforementioned.

"Plant organism or cells derived therefrom" generally means any cell, tissue, part or reproductive material (such as seeds or fruits) of an organism which is capable of photosynthesis. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Annual, perennial, monocot and dicot plants are preferred. Included are mature plants, seeds, shoots and seedlings, as well as parts derived from them, propagation material (for example tubers, seeds or fruits) and cultures, for example row or callus cultures.

"Plant" in the context of the invention means all genera and species of higher and lower plants in the plant kingdom. Included under the term are the mature plants, seeds, sprouts and seedlings, as well as parts derived therefrom, worship material, plant organs, tissues, protoplasts, callus and other cultures, for example cell cultures, and all other types of groupings of plant cells to form functional or structural units. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.

"Plant" includes all annual and perennial, monocotyledonous and dicotyledonous plants and includes, by way of example but not by way of limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linu, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscya us, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrallhinum, Nesisoc , Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisu, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Seeale, Triticum, Sorghum, Picea and Populus.

Plants of the following plant families are preferred: Amaranth aceae, Asteraceae, Brassicaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceaeaeae, Rosaceaeaeae, Rosaceaeaeae Tetragoniacea, Theaceae, Umbelliferae. Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants, such as, for example, the family of the Gramineae such as rice, corn, wheat or other types of cereals such as barley, millet, rye, triticale or oats as well as sugar cane and all types of grass.

The invention is very particularly preferably applied from dicotyledonous plant organisms. Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example

- Asteraceae such as sunflower, tagetes or calendula and others,

- Compositae, especially the genus Lactuca, especially the species sativa (salad) and others,

- Cruciferae, especially the genus Brassica, especially the species napus (rape), campestris (turnip), oleracea (e.g. cabbage, cauliflower or broccoli and other types of cabbage); and the genus Arabidopsis, especially the species thaliana and cress or. Canola and others,

- Cucurbitaceae such as melon, pumpkin or zucchini and others,

- Leguminosae especially the genus Glycine, especially the type max (soybean) soy, as well as alfalfa, peas, beans or peanuts and others

Rubiaceae, preferably of the subclass La iidae such as Coffea arabica or Coffea liberica (coffee bush) and others,

- Solanaceae especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant) and the genus Capsicum, especially the species annum (pepper), as well as tobacco or Peppers and others,

Sterculiaceae, preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) and others,

Theaceae, preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others, - Umbelliferae, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (celery)) and others;

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

Also included are ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawn. Examples include, but are not limited to, angiosperms, bryophytes such as hepaticae (liverwort) and musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms such as conifers, cycads, ginkgo and gnetals, the families of rosaceae such as rose, ericaceae such as rhododendrons and azaleas, euphorbiaceae such as poinsettias and croton, caryophyllaceae such as cloves, solanaceae such as petunias, Gesneriaceae such as the Usamalsamineaeaid as the Usambaramineae , Iridaceae like gladiolus, iris, freesia and crocus, Compositae like marigold, Geraniaceae like geranium, Liliaceae like the dragon tree, Moraceae like Ficus, Araceae like Philodendron and others.

Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as

Example algae, cyanobacteria and mosses. Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.

In particular, the plant organism is selected from the group of oil plants consisting of Borago officinalis, Brassica campestris, Brassica napus, Brassica rapa, Cannabis sativa, Carthamus tinctorius, Cocos nucifera, Crambe abyssinica, Cuphea species, Elaeis guineensis, Ekeis oleiferu, Glycine max, Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum, Helianthus annus, Linum usitatissimum, Oenothera biennis, Ozea europea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticum species, Zea maize, walnut and almond.

Most preferred are plant organisms that are suitable for vitamin E production, such as, for example, rapeseed, sunflower, sesame, safflower, olive tree, soybeans, corn, wheat or various types of nuts such as, for example, walnut or almond. "Vitamin E content" means the sum of the tocopherols and tocotrienols in a plant organism or a tissue, organ, part or cell of the same. Tocopherols and tocotrienols preferably include the 8 compounds α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrieno, γ-tocotrienol and δ-tocotrienol described above. The vitamin E content in the seed of a plant organism is particularly preferably increased.

"Increasing" the vitamin E content means increasing the content of vitamin E in a plant organism or a part, tissue or organ thereof, preferably in the seminal organs thereof. The vitamin E content is most at least 5%, preferably at least 10%, particularly preferably at least 15%, very particularly preferably at least 20%, compared to a starting plant which is not subject to the process according to the invention but is otherwise unchanged and is otherwise unchanged preferably increased at least 25%. Framework conditions means all conditions relevant to the germination, cultivation or growth of the plant such as soil, climate or light conditions, fertilization, irrigation, plant protection measures etc.

Functional equivalents means in particular natural or artificial mutations of the Tocen-1 protein according to SEQ ID NO: 2 and homologous polypeptides from other organisms, preferably from plant organisms, which have the same essential properties.

"Essential properties" of the Tocen-1 protein described by SEQ ID NO: 2 means in particular the property of increasing the vitamin E content in the case of transgenic expression in a plant organism in comparison to an identical but not transgenic plant organism according to the definition given above. In a preferred embodiment, at least one property selected from the following group also applies as essential properties:

a) a molecular weight in a range from approximately 10 to approximately 25 kDa, preferably approximately 15 to approximately 20 kDa, particularly preferably approximately 17 kDa

b) a theoretical isoelectric point in a range from approximately 8 to approximately 9.5, preferably approximately 8.4 to approximately 9.1, particularly preferably approximately pH 8.7 c) an acidic character, preferably with a total net charge at neutral pH in a range from approximately 2 to approximately 3, preferably approximately 2.2 to approximately 2.8, particularly preferably approximately 2.6,

d) a helical secondary structure according to computer-aided prediction, for example with the program for secondary structure prediction in GenomMax v.3.1 (InforMax, Inc.) based on the work of Qian (Qian N and Sejnowski T (1988) J Mol Biol 202: 865-884) and Sasgawa (Sasgawa F and Tajima K (1993) Cabios 9: 147-152).

Functional equivalents from other organisms, for example from plant organisms whose genomic sequence is known in whole or in part, such as, for example, from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum or Solanum tuberosum - can e.g. by database search in sequence databases such as GenBank or by screening gene or cDNA banks - e.g. using the sequence according to SEQ ID NO: 1 or a part thereof as a search sequence or probe. Mutations include substitutions, additions, deletions, inversions or insertions of one or more amino acid residues.

Said functional equivalents preferably have a homology of at least 50%, particularly preferably at least 65%, particularly preferably at least 80%, most preferably at least 90% of the protein with SEQ ID NO: 2. The homology extends over at least 30 amino acids , preferably at least 60 amino acids, particularly preferably at least 90 amino acids, most preferably over the entire length of the Tocen-1 polypeptide according to SEQ ID NO: 2.

Homology between two polypeptides means the identity of the amino acid sequence over the respective sequence length, which is determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following settings Parameter is calculated:

Gap Weight: 8 Length Weight: 2

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

By way of example, a sequence which has a homology of at least 80% on a protein basis with the sequence SEQ ID NO: 2 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 2 has a homology of at least 80% according to the above program algorithm with the above parameter set.

Functional equivalents also include those proteins which are encoded by nucleic acid sequences which have a homology of at least 50%, particularly preferably at least 65%, particularly preferably at least 80%, most preferably at least 90% to the nucleic acid sequence with SEQ ID NO: 1 , The homology extends over at least 100 bases, preferably at least 200 bases, particularly preferably at least 300 bases, most preferably over the entire length of the sequence according to 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 by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25: 3389ff) using the following parameters:

Gap Weight: 50 Length Weight: 3

Average Match: 10 Average Mismatch: 0

By way of example, a sequence which has a homology of at least 80% based on nucleic acids with the sequence SEQ ID NO: 1 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set Has at least 80% homology.

Functional equivalents also include those proteins which are encoded by nucleic acid sequences which, under standard conditions, have one of those described by SEQ ID NO: 1

Nucleic acid sequence that hybridize to this complementary nucleic acid sequence or parts of the aforementioned and have the essential properties of the protein described by SEQ ID NO: 2.

"Standard hybridization conditions" is to be understood broadly and means stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al. , in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. described.

By way of example, the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2X SSC at 50 ° C.) and those with high stringency (with approximately 0.2X SSC at 50 ° C., preferably at 65 ° C. C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). Furthermore, the temperature during the washing step can be raised from low stringent conditions at room temperature, about 22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can also be kept constant and only the other can be varied. Denaturing agents such as for example amide or SDS can also be used during the hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C. Some exemplary conditions for hybridization and washing step are given as a result:

(1) Hybridization conditions can be selected from the following conditions, for example:

a) 4X SSC at 65 ° C, b) 6X SSC at 45 ° C, c) 6X SSC, 100 μg / ml denatured, fragmented fish sperm DNA at 68 ° C, f) 50% formamide, 4X SSC at 42 ° C , h) 2X or 4X SSC at 50 ° C (weakly stringent condition), i) 30 to 40% formamide, 2X or 4X SSC at 42 ° C (weakly stringent condition).

(2) Washing steps can be selected, for example, from the following conditions:

a) 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% SDS at 50 ° C. b) 0.1X SSC at 65 ° C. c) 0.1X SSC, 0.5% SDS at 68 ° C. d) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C. e) 0.2X SSC, 0.1% SDS at 42 ° C. f) 2X SSC at 65 ° C (weakly stringent condition).

The invention further relates to transgenic expression constructs which encode a transgenic expression of the protein by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the abovementioned in a plant organism or a tissue, Can ensure organ, part or cell of said plant organism.

In said transgenic expression constructs, a nucleic acid molecule encoding a protein described by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the abovementioned preferably has a functional link to at least one genetic control element (for example a promoter) which is capable of transgenic expression a plant organism or a tissue, organ, part or cell of the same.

A functional link is understood to mean, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example the sequence according to SEQ ID NO: 1) and possibly other regulatory elements such as a terminator such that each of the regulatory elements has its function can meet 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, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed transgenically is positioned behind the sequence which acts as a promoter, so that both sequences are covalently linked to one another. The distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is preferably less than 200 base pairs, particularly 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 transgenic expression construct can be realized using common recombination 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), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and Gelvin et al. (1990) In: Plant Molecular Biology Manual. However, further sequences can also be positioned between the two sequences, which for example have the function of a linker with certain restriction enzyme interfaces or a signal peptide. The insertion of sequences for Expression of fusion proteins result. The transgenic expression construct, consisting of a linkage of promoter and nucleic acid sequence to be expressed, can preferably be integrated in a vector and inserted into a plant genome by, for example, transformation.

However, a transgenic expression construct should also be understood to mean constructions in which the nucleic acid sequence encoding the protein is encoded by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the abovementioned ones - for example by homologous recombination - after one is placed endogenous plant promoter that this ensures the transgenic expression of said nucleic acid sequence.

Plant-specific promoters basically means any promoter which can control the expression of genes, in particular foreign genes, in plants or parts of plants, lines, tissues or crops. The expression can be constitutive, inducible or development-dependent, for example.

Preferred are:

a) Constitutive promoters

“Constitutive” promoters mean those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development (Benfey et al. (1989) EMBO J 8: 2195-2202). In particular, a plant promoter or a plant virus-derived promoter is preferably used. Particularly preferred is the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shew aker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) or the 19S CaMV promoter (US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8: 2195-2202 ). Another suitable constitutive promoter is the "Rubisco small subunit (SSU)" promoter (US 4,962,028), the LeguminB promoter (GenBank Acc. No. X03677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (Octopin synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), the Ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18: 675-689 ; Bruce et al. (1989) Proc Natl Acad Sei USA 86: 9692-9696), S as promoter, the cinnamyl alcohol dehydrogenase promoter (US Pat. No. 5,683,439), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991), and further promoters of genes whose constitutive expression in plants is known to the person skilled in the art.

Tissue-specific promoters

Also preferred are promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds.

Seed-specific promoters such as the promoter of phaseoline (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53), of the 2S albumingen gene (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), legumin (Shirsat A et al. (1989) Mol Gen Genet 215 (2) .326-331), USP (unknown seed protein; Baumlein H et al. (1991) Mol Gen Genet 225 (3 ): 459-67), the Napin gene (US 5,608,152; Stalberg K et al. (1996) L Planta 199: 515-519), the sucrose binding protein (WO 00/26388) or the Legu in B4 promoter (LeB4; Baumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baumlein H et al. (1992) Plant J 2 (2): 233-9; Fiedler U et al. (1995) Biotechnology (NY) 13 (10): 1090f), the oleosin promoter from Arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980). Other suitable seed-specific promoters are those of the genes coding for "high molecular weight glutenin" (HMWG), gliadin, branching enzyme, ADP glucose pyrophosphate (AGPase) or starch synthase. Also preferred are promoters that allow seed-specific expression in monocots such as corn, barley, wheat, rye, rice, etc. The promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzine gene, etc.) can be used advantageously Prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the kasirin gene or the secalin gene). Further seed-specific promoters are described in WO 89/03887.

Tuber-, storage root- or root-specific promoters such as the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato.

Leaf-specific promoters such as a promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (s all subunit) of the Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the potato ST-LSI promoter (Stockhaus et al. (1989) EMBO J 8: 2445-2451).

Flower-specific promoters such as the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).

Anther-specific promoters such as the 5126 promoter (US 5,689,049, US 5,689,051), the glob-1 promoter and the γ-zein promoter.

c) Chemically inducible promoters

The transgenic expression constructs can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the exogenous gene in the plant is controlled at a specific point in time can. Such promoters, e.g. the PRPl promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), promoter inducible by salicylic acid (WO 95/19443), a promoter inducible by benzenesulfonamide (EP 0 388 186), one by Tetra - Cyclin-inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), an abscisic acid-inducible promoter (EP 0 335 528) or an ethanol- or cyclohexanone-inducible promoter (WO 93/21334) can also be used.

d) stress or pathogen inducible promoters

Also preferred are promoters that are induced by biotic or abiotic stress, such as the pathogen-inducible promoter of the PRPL gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (US 5,187,267), the cold-inducing alpha-amylase promoter from the potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinII promoter (EP375091).

Pathogen-inducible promoters include those of genes induced by pathogen attack such as genes from PR proteins, SAR proteins, β-1, 3-glucanase, chitinase etc. (e.g. Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes, et al. (1992) The Plant Cell 4: 645-656; Van Loon (1985) Plant Mol Viral 4: 111-116; Marineau et al. (1987) Plant Mol Biol 9: 335-342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2: 325-342; Somssich et al. (1986) Proc Natl Acad Sei USA 83: 2427-2430; Somssich et al. (1988) Mol Gen Genetics 2: 93-98; Chen et al. (1996) Plant J 10: 955-966; Zhang and Sing (1994) Proc Natl Acad Sei USA 91: 2507-2511; Warner, et al. (1993) Plant J 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968 (1989). Also included are wound-inducible promoters such as that of the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wunl and wun2 genes (US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin

(McGurl et al. (1992) Science 225: 1570-1573), the WIPl gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73- 76), the MPI gene (Corderok et al. (1994) The Plant J 6 (2): 141-150) and the like.

e) Development-dependent promoters

Further suitable promoters are, for example, fruit ripening-specific promoters, such as the fruit ripening-specific promoter from tomato (WO 94/21794, EP 409 625). Development-dependent promoters include

Divide the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.

Constitutive, seed-specific and leaf-specific promoters are particularly preferred.

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

The nucleic acid sequences contained in the transgenic expression constructs or transgenic expression vectors according to the invention can be functionally linked to further genetic control sequences in addition to a promoter. The term “genetic control sequences” is to be understood broadly and means all those sequences which have an influence on the formation or the function of a transgenic expression construct according to the invention. Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms. The transgenic expression constructs according to the invention preferably comprise a plant-specific promoter 5'-upstream of the respective transgenic nucleic acid sequence and 3'-downstream a terminator sequence as additional genetic Control sequence, as well as, where appropriate, other customary regulatory elements, in each case functionally linked to the nucleic acid sequence to be expressed transgenically.

Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. By means of genetic control sequences, for example, tissue-specific expression can additionally depend on certain stress factors. Corresponding elements are, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131 -17135) and heat stress (Schoffl F et al. (1989) Mol Gen Genetics 217 (2- 3): 246-53).

Further advantageous control sequences are, for example, in the gram-positive promoters amy and SP02, 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 advantageously.

Genetic control sequences also include the 5'-untranslated regions, introns or non-coding 3 'regions of genes such as the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes. An example of translation enhancers is the 5 'leader sequence from the tobacco mosaic virus (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. They can also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).

The transgenic expression construct can advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased transgenic expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the nucleic acid sequences to be expressed transgenically. The nucleic acid sequences to be expressed transgenically can be contained in one or more copies in the gene construct. Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J 3: 835 ff) or functional equivalents thereof. Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.

Control sequences are also to be understood as those which enable homologous recombination or insertion into the genome of a host organism or which allow removal from the genome. In homologous recombination, for example, the coding sequence of a specific endogenous gene can be specifically exchanged for the sequence coding for a dsRNA. Methods such as the cre / lox technology allow tissue-specific, possibly inducible removal of the transgenic expression construct 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 using the cre recombinase.

A transgenic expression construct and / or the transgenic expression 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 transgenic expression constructs according to the invention, the transgenic expression vectors or the transgenic organisms. Examples include, but are not limited to:

a) Selection markers which are resistant to a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleoycin, hygromycin, or phos- lend phinotricin etc. Particularly preferred selection markers are those which confer resistance to herbicides. Examples include: DNA sequences that code for phosphinothricin acetyltransferases (PAT) and inactivate glutamine synthase inhibitors (bar and pat gene), 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) that are resistant to Glyphosat® (N- (phosphon methyl) glycine), the gox gene coding for the glyphosate ^® degrading enzymes (glyphosate oxidoreductase), the deh gene (coding for a dehalogenase which inactivates dalapon), sulfonylurea and imidazolinone inactivating acetolactate synthases and bxn genes for bromoxynil encoding degrading nitrilase enzymes, the aasa gene conferring resistance to the antibiotic apectinomycin, the streptomycin phosphotransferase (SPT) gene conferring resistance to streptomycin, the neomycin phosphotransferase (NPTII) gene conferring resistance to kanamycin or geneticidin, the hygromycinphσsphotransferase (HPT) gene, which confers resistance to hygromycin, the acetolactate synthase gene (ALS), which confers resistance to sulfonylurea herbicides (for example mutated ALS variants with, for example, the S4 and / or Hra mutation).

b) Reports which encode easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an assessment of the transformation efficiency or the place or time of expression. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (l): 29-44) such as the "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784), the chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234: 856-859), the aequorin gene (Prasher et al. (1985) Biochem Biophys Res Coπtmun 126 (3): 1259-1268), the β-galactosidase, the β-glucuronidase being very particularly preferred (Jefferson et al. (1987) EMBO J 6: 3901-3907).

c) Origins of replication, which ensure an increase in the transgenic expression constructs or transgenic expression vectors according to the invention in, for example, E. coli. Examples are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al .: Molecular Cloning. A Laboratory Manual, ^2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

d) Elements which are required for an agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.

For the selection of successfully homologously recombined or also transformed cells, it is generally necessary to additionally introduce a selectable marker which gives the successfully recombined cells resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglucose. 6-phosphate (WO 98/45456) or an antibiotic. The selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5: 81-84). In addition, said transgenic expression constructs or transgenic expression vectors can contain nucleic acid sequences which do not code for the protein encoded by SEQ ID NO: 2, a functional equivalent thereof or a functionally equivalent part of the abovementioned, and their transgenic expression for an additional increase in vitamin E. Biosynthesis leads (as a result of pro-VitE). This additionally transgenically expressed pro-VitE nucleic acid sequence can be selected - by way of example but not by way of limitation - from nucleic acids coding for homogentisate phytyltransferase, 2-methyl-6-plastoquinone methyl transferase, tocopherol cyclase, γ-tocopherol methyltransferase, hydroxyphenylpyruvase dioxinase oxygenase. With the aforementioned, the desired effect is ensured by overexpression. However, a corresponding effect can also be achieved by expressing a pro-VitE nucleic acid sequence which, for example as antisense RNA or double-stranded RNA, suppresses the expression of certain genes. Suitable target genes would be - here, by way of example but not restrictive - the ho ogentisate dioxyase. Corresponding sequences are known to the person skilled in the art and are easily accessible from databases or corresponding cDNA banks of the respective plants. The person skilled in the art is aware that several different of the above-mentioned additional expressions of pro-VitE nucleic acid sequences can also be combined within the scope of the invention.

The introduction of a transgenic expression construct according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably in plants or plant-based cells, tissues, organs, parts or seeds) can advantageously be implemented using vectors in which the transgenic expression constructs are contained. Vectors can be, for example, plasmids, cosmids, phages, viruses or agri-bacteria. The transgenic expression construct can be introduced into the vector (preferably a plasmid vector) via a suitable restriction site. The resulting transgenic expression vector is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the combinant vector obtained using methods familiar to the person skilled in the art. Restriction analysis and sequencing can be used to check the cloning step. Preferred vectors are those which enable stable integration of the transgenic expression construct into the host genome.

The production of a transformed organism (or a transformed cell or tissue) requires that the corresponding DNA (eg the expression vector), RNA or protein is introduced into the corresponding host cell. A large number of methods are available for this process, which is referred to as transformation (or transduction or transfection) (Keown et al. (1990) Methods in Enzymology 185: 527-537). For example, the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles. The cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can get into the cell by diffusion. The DNA can also be obtained by protoplast fusion with other DNA-containing units 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. Corresponding methods are described (for example in Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol Gen Genet 228: 104-112; Guerche et al. (1987) Plant Science 52: 111-116; Neuhause et al. (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature 327: 70-73; Howell et al. (1980) Science 208: 1265; 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 for transient or stable transformation are used. Suitable methods include protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called "particle bombardment" method, electroporation, the incubation of dry embryos in DNA-containing solution and microinjection.

In addition to these "direct" transformation techniques, a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes. The Agrobacterium -mediated transformation is best suited for dicotyledonous plant cells. The methods are described, for example, by Horsch RB et al. (1985) Science 225: 1229f).

If Agrobacteria are used, the transgenic expression construct is to be integrated into special plasmids, either into a shuttle or intermediate vector or a binary vector. If a Ti or Ri plasmid is used for trans formation is to be used, at least the right border, but mostly the right and the left border of the Ti or Ri plasmid T-DNA as flanking region is connected to the transgenic expression construct to be introduced.

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

Further 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 Be 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, there are no special requirements for the plasmid used.

Simple plasmids such as the pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is necessary that there is an additional selectable marker gene on the plasmid.

Stably transformed cells, ie those which contain the inserted DNA integrated into the DNA of the host cell, can be selected from untransformed cells if a selectable marker is part of the inserted DNA. Any gene that 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 that express such a marker gene are able to survive in the presence of concentrations of an appropriate antibiotic or herbicide that kill an untransformed wild type. Examples are mentioned above and preferably comprise the bar gene which confers resistance to the herbicide phosphinotricin (Rathore KS et al. (1993) Plant Mol Biol 21 (5): 871-884), the nptll gene which confers resistance to kanamycin, the hpt gene, which confers resistance to hygromycin, or the EPSP gene, which confers resistance to the herbicide glyphosate. The selection marker allows the selection of transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5: 81-84). The plants obtained can be grown and crossed in a conventional manner. Two or more generations should be cultivated to ensure that genomic integration is stable and inheritable.

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

Once a transformed plant cell has been made, a whole plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown.

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

"Transgene" means - for example with respect to a nucleic acid sequence, an expression construct or an expression vector containing said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression construct or expression vector - all such by gene technical methods of constructions, in which either

a) the nucleic acid sequence coding for a Tocen-1 protein 5 according to SEQ ID NO: 2, a functional equivalent thereof or a functionally equivalent part of the aforementioned, or

b) a genetic control sequence functionally linked to said nucleic acid sequence under a), for example a

10 promoter, or

c) (a) and (b)

are not in their natural, genetic environment

15 or have been modified by genetic engineering methods, the modification being, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment means the natural chromosomal locus in the organism of origin

20 or being in a genomic library. In the case of a genomic library, the natural, genetic environment of the nucleic acid sequence is preferably at least partially preserved. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least

25 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, very particularly preferably at least 5000 bp. A naturally occurring expression construct - for example the naturally occurring combination of the promoter of a gene coding for a protein according to SEQ ID NO: 2 or

A functional equivalent thereof with its corresponding coding sequences becomes a transgenic expression construct if it is changed by non-natural, synthetic ("artificial") methods such as mutagenization. Appropriate procedures are

35 (US 5,565,350; WO 00/15815; see also above).

In relation to expression (“transgenic expression”), “transgene” preferably means all those expressions realized using a transgenic expression construct, transgenic expression vector or transgenic organism — according to the definitions given above.

Preferred host or starting organisms as transgenic organisms are, above all, plant organisms as defined above. Included within the scope of the invention are all genera and species of higher and lower plants in the plant kingdom, in particular plants which are intended for production oils such as rapeseed, sunflower, sesame, safflower, olive tree, soybean, corn, wheat and types of nuts are used. Also included are the mature plants, seeds, sprouts and seedlings, as well as parts, propagation material and cultures derived from them, for example cell cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.

The production of the transgenic organisms can be carried out using the processes described above for the transformation or transfection of organisms.

Another object of the invention relates to the use of the transgenic organisms according to the invention and the cells, cell cultures, parts derived from them - such as roots, leaves, etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits, for the production of food - or feed, pharmaceuticals or fine chemicals, especially of vitamin E or vitamin E derivatives such as vitamin E acetate.

sequences

1. SEQ ID NO: 1 nucleic acid sequence coding for Tocen-1

protein

22. SEQ ID NO: 2 protein sequence coding for Tocen-1

protein

23. SEQ ID NO: 3 oligonucleotide primers sll0832-5 ^v

24. SEQ ID NO: 4 oligonucleotide primers sll0832-3 ^v

25. SEQ ID NO: 5 nucleic acid sequence coding for the

Sall-PacI-ocs-HindIII sequence fragment

26. SEQ ID NO: 6 oligo sequence coding for the sense strand of a swal linker

27. SEQ ID NO: 7 oligo sequence coding for the antisense

Swal linker strand

28. SEQ ID NO: 8 nucleic acid sequence coding for the

Expression vector pSUN2-l

29. SEQ ID NO: 9 nucleic acid sequence coding for the rbcS

transit peptide

30. SEQ ID NO: 10 nucleic acid sequence coding for the

Expression vector pSUN2-2

31. SEQ ID NO: 11 oligonucleotide primer 0832exl-5 ^,

32. SEQ ID NO: 12 oligonucleotide primer 0832exl-3 '

33. SEQ ID NO: 13 nucleic acid sequence coding for the

A. thaliana nitrilase-1 promoter

34. SEQ ID NO: 14 nucleic acid sequence coding for the transgenic expression vector pSUN2 / NitP / sll0832 / ocs 35. SEQ ID NO: 15 nucleic acid sequence coding for the transgenic expression vector pSUN2 / NitP / rbcS / sll0832 / ocs

^ 36. SEQ ID NO: 16 nucleic acid sequence coding for the ÜSP-

Vicia faba promoter in pUC vector

37. SEQ ID NO: 17 oligonucleotide primer sll0832ex2-5 ^λ 0

38. SEQ ID NO: 18 oligonucleotide primer Sll0832ex2-3 ^λ

39. SEQ ID NO: 19 nucleic acid sequence coding for the transgenic expression vector 5 pSUN2-ÜSP-sll0832-catT

40. SEQ ID NO: 20 nucleic acid sequence coding for the rbcS

MCS-OCS fragment (rbs: rbs transit peptide; MCS: multiple cloning site; OCS: 0 termination signal of the octopine synthase gene.

41. SEQ ID NO: 21 nucleic acid sequence coding for the transgenic expression vector pSUN2 / USP / rbcS / sll0832 / ocs 5

pictures

Fig. 1: pCR-Script / sll0832 construct card

"A" represents the DNA fragment 0 coding for Tocen-1 (456 base pairs)

Fig. 2: Construct card from pCR-Script / sll0832:: tn903

Fragment A '(268 base pairs) contains the 5' region of Tocen-1, fragment B the transposon 903 (1286 base 5 pairs) and fragment 'A (194 base pairs) the 3 ^V region of Tocen-1. The kanamycin cassette of the tn903 inserted in pCR-Script / sll0832 in such a way that the transcription of the kanamycin resistance gene of the Tn903 runs in the opposite direction to the transcription of the open reading frame from 0 Tocen-1.

Fig. 3: Tocopherol production in Synechocystis / sll0832:: tn903 Two independent Tocen-1 knockout mutants (1 and 2) showed in comparison to the Synechocystis spec. PCC 6803 5 wild-type cells (WT) showed a significant reduction in tocopherol and tocotrienol production. 4: construct map of pSUN2 / NitP / sll0832 / ocs (SEQ ID No: 14) fragment "A" (1894 bp): nitrilase-1 promoter fragment "B" (456 bp): open reading frame of Tocen-1 fragment " C "(219 bp): termination signal of the octopine synthase gene.

Fig. 5: pSÜN2 / NitP / rbcS / sll0832 / ocs construct map

Fragment "A" (1894 bp nitrilase-1 promoter

Fragment "B" (167 bp) fragment coding for the rbcS transit peptide

Fragment "C" (456 bp) open reading frame of Tocen-1

Fragment "D" (219 bp) termination signal of the octopine synthase gene.

Fig. 6: construct card from pSUN2 / USP / sll0832 / 3 ^Λ cat

Fragment "A" (674 bp): promoter of the USP gene from Vicia faba fragment "B" (456 bp): open reading frame of Tocen-1 fragment "C" (229 bp): termination signal of the cathepsin D inhibitor gene.

Fig. 7: Construction card of pSUN2 / USP / rbcS / sll0832 / ocs

Fragment "A" (674 bp): promoter of the USP gene from Vicia faba Fragment "B" (174 bp): fragment coding for the rbcS

Transit peptide fragment C (459 bp): open reading frame of Tocen-1 fragment D (219 bp): termination signal of the octopine

Synthase gene.

Examples

Unless otherwise stated, all chemicals 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 made by Amersham-Pharmacia (Freiburg), Biometra (Göttingen), Röche (Mannheim), New England Biolabs (Schwalbach), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden), Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom). The 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 using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The Cloning steps carried out in the context of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of Phage and sequence analysis of recombinant DNA are carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules takes place with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sei USA 74: 5463- 5467).

Example 1: General procedures

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

a) Cultivation of Arabidopsis plants

The plants are grown either on Murashige-Skoog medium with 0.5% sucrose (Ogas et al. (1997) Science 277: 91-94) or on earth (Focks & Benning (1998) Plant Physiol 118: 91-101). In order to achieve uniform germination and flowering times, the seeds are stratified at 4 ° C for two days after plating or scattering on earth. After flowering, the pods are marked. According to the markings, pods with an age of 6 to

Harvested 20 days after flowering.

b) Cultivation of Synechocystis

The cells of Synechocystis sp. PCC 6803 are usually grown autotrophically in BGII medium. They have a diameter of 2.3 to 2.5 μm. In the investigations described here, the cyanobacterium Synechocystis sp. PCC 6803 strain from the collection of Prof. Dr. Peter Wölk and Prof. Dr. 'Lee Mclntosh (Plant Research Laboratory, Michigan State University, East Lansing,

Michigan, USA). This is glucose tolerant, which means that it can also be heterotrophic in the dark with just a few minutes dim blue light lighting a day, grow. These culture conditions were developed by Anderson and Mcintosh (Anderson and Mcintosh (1991) J Bacteriol 173: 2761-2767) and called "Light-activated heterotrophic growth" (LAHG). This makes it possible to cultivate these cyanobacteria without ongoing photosynthesis and thus without the production of oxygen.

BG 11 culture medium for Synechocystis stock solution 100 x BGll:

NaN0 ₃ 1.76 M = 149.58 g

MgSO ₄ x 7 HO 30.4 mM = 7.49 g

CaCl ₂ x 2 H ₂ 0 24.5 mM = 3.6 g

Citric acid 3.12 M = 0.6 g Na EDTA pH 8 0.279 mM = 0.104 g

The weighed substances are dissolved in 900 ml H0 and made up to 1000 ml with 100 ml of the "Trace metal mix stock" 100,000. This obtained solution serves as a stock solution.

Trace metal mix stock lOOOx:

H ₃ B0 ₃ 46.3 mM = 2.86 g / 1

MnCl ₂ x 4 H ₂ 0 4.15 mM = 1.81 g / 1

ZnS0 ₄ x 7 H ₂ 0 0.77mM = 0.222 g / 1 Na ₂ Mo0 ₄ x 2 H ₂ 0 1.61 mM = 0.39 g / 1

CuS0 ₄ x 5 H ₂ 0 0.32 mM = 0.079 g / 1

Co (N0 ₃ ) ₂ x 6 H ₂ 0 0.17 mM = 0.0494 g / 1

The following solutions are required for 1 liter of BGll culture solution:

1. 10 ml of the stock solution 100 x BG 11

2. 1 ml Na ₂ CO ₃ (189 mM)

3.5 ml TES (1 M, pH 8)

4.1 ml K ₂ P0 ₄ (175 mM)

While solution 2 and 3 should be sterile filtered, solution 4 must be autoclaved. All of the BGII culture solution must be autoclaved before use and then 1 ml of iron ammonium citrate (6 mg / ml), which has previously been sterile filtered, must be added. Under no circumstances should the iron ammonium citrate be autoclaved. For agar plates, 1.5% (w / v) Bactoagar per liter of BGII medium is added. Example 2: Amplification and cloning of Tocen-1 from Synechocystis spec. PCC 6803

The DNA coding for Tocen-1 was by means of "Polymerase Chain 5 Reaction" (PCR) from Synechocystis spec. PCC 6803 according to the method according to Crispin A. Howitt (Howitt CA (1996) BioTechniques 21: 32-34) using a sense-specific primer (sll0832-5 '; SEQ ID NO: 3) and an antisense-specific primer (sll0832-3 '; SEQ IDNO: 4) amplified. 10

Primer sll0832-5 ':

5 '-GGATCCATGGGACGTTGGCCCACTGG-3' (SEQ ID NO: 3)

Primer sll0832-3 ': 15 5' -GTCGACCTAGCAACGGCCGCTATCC-3 '(SEQ ID NO: 4)

The PCR was carried out in a 50 μl reaction mixture which contained:

20 5 μl of a Synechocystis spec. PCC 6803 cell suspension

0.2mM dATP, dTTP, dGTP, dCTP

1.5 mM Mg (0Ac) ₂

5 μg bovine serum albumin

40 p ol oligonucleotide primer sll0832-5 '

25 40 pmol oligonucleotide primer sll0832-3 '

15 μl 3.3X rTth DNA Polymerase XL buffer (PE Applied Biosystems)

5U rTth DNA Polymerase XL (PE Applied Biosystems)

30 The PCR was carried out under the following cycle conditions:

Step 1: 5 minutes 94 ° C (denaturation)

Step 2: 3 seconds at 94 ° C

Step 3: 1 minute 52 ° C (annealing)

Step 4: 1 minute 72 ° C (elongation) 35 35 repetitions of steps 2 to 4

Step 5: 10 minutes 72 ° C (post-elongation)

Step 6: 4 ° C (holding pattern)

The amplificate (468 base pairs) was cloned into the PCR cloning vector pCR-Script (Stratagene) using 40 standard methods. The construct is called pCR-Script / sll0832. The identity of the amplicon generated was confirmed by sequencing using the T3 and T7 primers (cf. SEQ ID NO: 1). Figure 1 illustrates pCR-45 Script / sll0832, where "A" represents the Tocen-1 coding DNA fragment (456 base pairs). Example 2: Generation of a Tocen-1 knock out mutant

A DNA construct for the generation of a deletion mutant of Tocen-1 in Synechocystis spec. PCC 6803 was created using standard cloning techniques. The vector pCR-Script / sll0832 was partially digested using the restriction enzyme Ncol and the protruding ends of the restriction digest were blunt-ended using standard methods. The aminoglycoside-3 'phosphotransferase of the transposon Tn903 was cloned into the blunt-ended Ncol site of the open reading frame of Tocen-1. For this purpose, the Tn903 was isolated as an EcoRI fragment from the vector pUC4K (Vieira J and Messing J (1982) Gene 19: 259-268), the protruding ends of the restriction digest were converted into smooth ends according to standard methods and the vector pCR-Script / sll0832 ligated (Ncol with smooth ends). The ligation approach was used to transform E. coli Xll Blue cells. Transformants were selected using kanamycin and ampicillin. A recombinant plasmid (pCR-Script / sll0832:: tn903; see Fig. 2) was isolated and used to transform Synechocystis spec. PCC 6803 used according to the Williams method (Williams (1987) Methods Enzymol 167: 776-778).

The kanamycin cassette of the tn903 inserted in pCR-Script / sll0832 in such a way that the transcription of the kanamycin resistance gene of the

Tn903 runs in the opposite direction to the transcription of the open reading frame of Tocen-1.

In FIG. 2 Fragment A includes' (268 base pairs) of the 5 'region of Tocen-1, fragment B, the transposon 903 (1286 base pairs) and fragment' A (194 base pairs) the 3 ^λ range of Tocen-1.

Synechocystis spec. PCC 6803 transformants were selected on Kanamycin-containing (km) BG-11 solid medium (Castenholz (1988) Methods in Enzymology page 68-93) at 28 ° C. and 30 μmol photons * (m ² * s) - ¹ . Eight independent knockout mutants were generated after five rounds of selection (passages from individual colonies to fresh BG-11km medium). The complete loss of the Tocen-1 endogen or the exchange for the recombinant Tocen-1:: tn903 DNA was confirmed by PCR analysis.

Example 3: Tocopherol production in Synechocystis spec. PCC 6803 wild-type cells and the Tocen-1 knockout mutants

The cells cultured on the BG-llkm agar medium from two independent Synechocystis spec. PCC 6803 Tocen-1 knockout mutants and untransformed wild-type cells (on BGll agar medium without kanamycin) were used to inoculate liquid cultures. For this purpose, cells of the mutant or the wild type Synechocystis spec. Transfer PCC 6803 from plate to 10 ml of liquid culture. These cultures were cultivated at 28 ° C. and 30 μmol photons * (m ² * s) - ¹ (30 μE) for approx. 3 days. To

Determination of the OD ₃ Q of the individual cultures, the OD ₃ o of all cultures was synchronized by appropriate dilutions with BG-11 (wild types) or BG-11km (mutants). These cultures, which were synchronized to cell density, were used to inoculate three cultures of the mutant or the wild-type control. The biochemical analyzes could thus be carried out using three independently grown cultures of a mutant and the corresponding wild types. The cultures were grown to an optical density of OD ₇₃ o = 0.3.

The medium of the cell culture was removed by centrifugation twice at 14000 rp in an Eppendorf table centrifuge. The subsequent digestion of the cells and extraction of the tocopherols and tocotrienols was carried out by incubating twice in an Eppendorf shaker at 30 ° C., 100 ° C. in 100% methanol for 15 minutes, the supernatants obtained in each case being combined. Further incubation steps resulted in no further release of tocopherols or tocotrienols.

In order to avoid oxidation, the extracts obtained were analyzed immediately after extraction using a Waters Allience 2690 HPLC system. Tocopherols and tocotrienols were separated on a reverse phase column (ProntoSil 200-3-C30, Bischoff) with a mobile phase of 100% methanol and identified using standards (Merck). The fluorescence of the substances (excitation 295 μm, emission 320 nm) was used as the detection system, which was detected with the aid of a Jasco FP 920 fluorescence detector.

The Tocen-1 knockout mutants surprisingly showed in comparison to the Synechocystis spec. PCC 6803 wild type cells lost tocopherol and tocotrienol production (Fig. 3).

Example 4: Manipulation of the tocopherol biosynthesis in Nicotiana tahacum Samsun NN, Arabidopsis thaliana and Brassica napus by transgenic expression of Tocen-1

Transgenic plants are generated which contain Tocen-1 on the one hand under the control of the constitutive promoter of the nitrilase-1 (nitl) gene from A. thaliana (GenBank Acc. -No.: Y07648.2, nucleotides 2456-4340, Hillebrand et al. (1996) Gene 170: 197-200) and secondly under the control of the USP's seed-specific promoter (unknown seed protein) gene from Vicia faba (Bäumlein et. al.

(1991) Mol Gen Genet 225: 459-467). In addition, Tocen-1 is expressed as a fusion protein with the transit peptide of the rbcS protein (Guerineau et al. (1988) Nucl Acid Res 16: 11380), both constitutively and seed-specifically. Derivatives of the pSUN vector (WO 02/00900) serve as the expression vector.

For constitutive expression, the vector pSUN2 was modified using standard methods such that the ocs terminator (Gielen et al. (1984) EMBO J 3: 835-846) as a Sall PacI-ocs HindIII fragment (sequence ID No: 5) is introduced. By introducing a linker, a swal interface is also introduced into the polylinker (SEQ ID No: 6 and SEQ ID No: 7). The resulting plasmid is designated pSUN2-1 (Sequence ID NO: 8).

For expression as a fusion protein with the rbcS transit peptide, the DNA fragment coding for the rbcS transit peptide is amplified as a SacI-Swal fragment (sequence ID No: 9) and introduced into the pSUN2-1. The vector is called pSUN2-2 (Sequence ID NO: 10).

For expression of Tocen-1 under constitutive control of the ORF is ^λ as SwaI-PacI fragment using standard methods with the aid of primers 0832exl-5 and 0832exl-3 (SEQ ID No. 11 and SEQ ID No. 12) ^λ amplified. The resulting 462 bp fragment is cloned into the pCR4Blunt-T0P0 (Invitrogen) according to the manufacturer's instructions and sequenced using the T3 and T7 primers.

A correct clone is cut with Swal-Pacl and under

Use standard methods ligated into a Swal-Pacl cut pSUN2-l. In the resulting construct, which is cut with Ecll36ll, the nitrilase promoter is cloned as an Xhol fragment (SEQ ID No: 13), the overhanging ends of which are filled in with Klenow polymerase according to standard methods. This plasmid is designated pSUN2 / NitP / sll0832 / ocs (SEQ ID No: 14) and used to generate transgenic plants (cf. FIG. 4).

To construct a plasmid that expresses Tocen-1 as a fusion protein with the transit peptide of the rbcS protein under the control of the constitutive promoter NitP, pSUN2-2 is cut with Swal and Pacl. The vector cut in this way is ligated with the Tocen-1 fragment which, after digestion with Swal and Pacl, is isolated from pCR-Script / sll0832. In the resulting plasmid, which is cut with Ecll36II, the nitrilase promoter (SEQ ID NO: 13) as Xhol fragment, the overhanging ends of which are filled in with Klenow polymerase according to standard methods, ligated. This cloning allows the expression of a fusion protein which contains the transit peptide of the rbcS protein and Tocen-1 (see FIG. 5, SEQ ID No. 15).

To generate a plasmid which enables the seed-specific expression of Tocen-1 in plants, the seed-specific promoter of the USP gene from Vici faba is used, which is present as an EcoRI-Ncol fragment in a pUC derivative (SEQ ID NO: 16).

For the cloning under the control of the USP promoter, the open reading frame of Tocen-1 is amplified using standard PCR methods in such a way that a Bbsl interface is generated at the 5 'end and an EcoRV interface at the 3' end , The following oligonucleotide primers are used for this:

sll0832ex2-5 ': (SEQ ID NO: 17) and

S110832ex2-3 ': (SEQ ID No: 18)

The amplificate is cloned in pCR4Blunt-TOPO and sequenced.

The PCR product is ligated as a BbsI-EcoRV fragment in the vector cut with Ncol (overhanging ends compatible with 5 'overhang of the PCR product) and EcoRV. The expression construct is ligated as Pmel and Afel fragments in pSUN2 cut with EcoRV. The resulting plasmid is called pSun2-USP-sll0832-3 ^, cat (see FIG. 6, SEQ ID NO: 19).

For the construction of a plasmid which allows seed-specific expression as a fusion protein with the rbcS transit peptide, the rbcS fragment is amplified together with the ocs terminator as an Ncol-HindIII fragment (SEQ ID No: 20) from pSUN2-2 and into the Cloned pUC derivative containing the USP promoter. Promoters, rbcS and ocs are cloned as Pmel-Afel fragments in pSUN2 cut with EcoRV. Tocen-1 is cloned into this expression vector as a Swal-Pacl fragment. The resulting plasmid is called pSUN2 / USP / rbcS / sll0832 / ocs (Fig. 7, SEQ ID No: 21).

Example 5: Production of transgenic A. thaliana plants

Wild-type A. thaliana plants (Columbia) are based on the Agrobacterium tumefaciens strain (EHA105) based on a modified method (Steve Clough and Andrew Bent (1998) Plant J 16 (6): 735-743) according to the vacuum infiltration method Bechtold et al. (Bechtold N et al. (1993) CRAcad Sei Paris 1144 (2) .204-212).

The A. tumefaciens cells used are pre-loaded with the plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSUN2 / USP / sll0832 / catT (SEQ ID NO: 19) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21).

Seeds of the Agrobacterium-transformed primary transformants are selected on the basis of antibiotic resistance. Antibiotic-resistant seedlings are planted in soil and used as fully developed plants for biochemical analysis.

Example 6: Production of transgenic Brassica napus plants

The production of transgenic oilseed rape plants is based on a protocol from Bade JB and Damm B (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38), in which also shows the composition of the media and buffers used.

The transformations are carried out with the Agrobacterium tumefaciens strains EHA105 and GV3101. The plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSUN2 / USP / sll0832 / catT (SEQ ID NO: 19 ) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21).

Brassica napus var. Westar seeds are surface-sterilized with 70% ethanol (v / v), washed in water for 10 minutes at 55 ° C, in 1% hypochlorite solution (25% v / v tea pol, 0.1% v / v Tween 20) incubated for 20 minutes and washed six times with sterile water for 20 minutes each. The seeds are dried on filter paper for three days and 10 to 15 seeds are germinated in a glass flask with 15 ml of germination medium. The roots and apices are removed from several seedlings (approx. 10 cm in size) and the remaining hypocotyls are cut into pieces approx. 6 mm long. The approximately 600 explants obtained in this way are washed for 30 minutes with 50 ml of basal medium and transferred to a 300 ml flask. After adding 100 ml of callus induction medium, the cultures were incubated for 24 hours at 100 rpm.

From the individual with the plasmids pSUN2 / NitP / sll0832 / ocs

(SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSUN2 / USP / sll0832 / catT (SEQ ID NO: 19) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21) transformed Agrobacteria strains, an overnight culture is set up at 29 ° C. in Luria Broth medium with kanamycin (20 mg / 1), of which 2 ml in 50 ml Luria Broth medium without kanamycin for 4 Incubated for hours at 29 ° C to an OD600 of 0.4 to 0.5. After pelleting the culture at 2000 rpm for 25 min, the cell pellet is resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution is adjusted to an OD _ß oo of 0.3 by adding further basal medium.

The callus induction medium is removed from the oilseed rape explants using sterile pipettes, 50 ml of Agrobacterium solution are added, mixed gently and incubated for 20 min. The agrobacterial suspension is removed, the oilseed rape explant is washed for 1 min with 50 ml of callus induction medium and then 100 ml of callus induction medium is added. The co-cultivation is carried out for 24 h on a rotary shaker at 100 rpm. The co-cultivation is stopped by removing the callus induction medium and the explants are washed twice for 1 min with 25 ml and twice for 60 min with 100 ml of washing medium at 100 rpm. The washing medium with the explants is transferred to 15 cm petri dishes and the medium is removed with sterile pipettes.

20 to 30 explants in 90 mm each are used for regeneration

Transfer Petri dishes containing 25 ml shoot induction medium with kanamycin. The Petri dishes are closed with 2 layers of Leukopor and incubated at 25 ° C and 2000 lux with photoperiods of 16 hours light / 8 hours dark. Every 12 days, the developing calli are transferred to fresh petri dishes with shoot induction medium. All further steps for the regeneration of whole plants are described as from Bade JB and Damm B (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38) carried out.

Example 7: Production of transgenic Nicotiana tabacum plants

10 ml of YEB medium with antibiotic (5 g / 1 beef extract, 1 g / 1 yeast extract, 5 g / 1 peptone, 5 g / 1 sucrose and 2 mM MgS0 _4. ) With a colony of the individual with the Plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSU 2 / USP / sll0832 / catT (SEQ ID NO: 19) or Inoculated pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21) transformed Agrobacterium tumefaciens strains and cultured overnight at 28 ° C. The cells are pelleted in a table centrifuge for 20 min at 4 ° C., 3500 rpm and then in resuspended fresh YEB medium without antibiotics under sterile conditions. The cell suspension is used for the transformation.

The wild-type plants from sterile culture are obtained by vegetative replication. To do this, only the tip of the plant is cut off and transferred to fresh 2MS medium in a sterile mason jar. The hair on the top of the leaf and the central ribs of the leaves are removed from the rest of the plant. The leaves are cut into approximately 1 cm ² large pieces with a razor blade. The agrobacterial culture is transferred to a small petri dish (diameter 2 cm). The leaf pieces are briefly drawn through this solution and the underside of the leaf is placed on 2MS medium in Petri dishes (diameter 9 cm) so that they touch the medium. After two days in the dark at 25 ° C, the explants are transferred to plates with callus induction medium and heated to 28 ° C in the climatic chamber. The medium must be changed every 7 to 10 days. As soon as calli were formed, the explants were placed in sterile mason jars on shoot induction medium with Claforan (0.6% BiTec agar (w / v), 2.0 mg / 1 zeatin ribose,

0.02 mg / 1 naphthylacetic acid, 0.02 mg / 1 gibberelic acid, 0.25 g / ml claforan, 1.6% glucose (w / v) and 50 mg / 1 kanamycin). After about a month, organogenesis occurs and the shoots formed can be cut off. The shoots are cultivated on 2MS medium with Claforan and a selection marker. As soon as a strong root ball has formed, the plants can be potted in prickly potting soil.

Example 8: Characterization of the transgenic plants

In order to confirm that the expression of Tocen-1 affects the vitamin E biosynthesis in the transgenic plants, the tocopherol and tocotrienol contents in leaves and seeds of the plants transformed with the described constructs (Arabidopsis. Thaliana, Brassica napus and Nicotiana tabacum) were analyzed. For this purpose, the transgenic plants are cultivated in the greenhouse and plants which express the gene coding for Tocen-1 are identified at the Northern level. The tocopherol content and the tocotrienol content are determined in the leaves and seeds of these plants. In all cases, the tocopherol or tocotrienol concentration is increased compared to non-transformed plants.

Claims

claims

1. A method for increasing the vitamin E content in a plant organism or a tissue, organ, part, cell or propagation material thereof, comprising

a) transgenic expression of a protein with SEQ ID NO: 2 or a functional equivalent thereof with an identity of at least 60% to the sequence with SEQ ID NO: 2, and

b) Selection of plant organisms in which - in contrast to or compared to the starting organism - the vitamin E content in the said plant organism or in a tissue, organ, part, cell or propagation material of the same is increased.

2. The method of claim 1, wherein the plant is an oil plant.

3. A transgenic expression construct comprising a nucleic acid sequence coding for a protein with the SEQ ID NO: 2 or a functional equivalent thereof with an identity of at least 60% under the control of a promoter which is functional in a plant organism or a tissue, organ, part or cell the sequence with SEQ ID NO: 2.

4. The transgenic expression construct according to claim 3, wherein the nucleic acid sequence is described by

a) a sequence with SEQ ID NO: 1 or

b) a sequence which is derived from a sequence with SEQ ID NO: 1 in accordance with the degenerate genetic code, or

c) a sequence which is at least 60% identical to the sequence with SEQ ID NO: 1.

5. Transgenic expression construct according to one of claims 3 or 4, wherein the promoter is a constitutive or a seed-specific promoter.

6. Transgenic expression vector containing a transgenic expression construct according to one of claims 3 to 5.

7. Transgenic plant organism or tissue, organ, part, cell or propagation material thereof, containing a transgenic expression construct according to one of claims 3 to 5 or a transgenic expression vector according to claim 6.

8. Transgenic plant organism according to claim 7, wherein the plant organism is selected from the group of oil plants consisting of Borago officinalis, Brassica campestris, Brassica napus, Brassica rapa, Cannabis sativa, Carthamus tinctorius, Cocos nucifera, Crambe abyssinica, Cuphea species, Elaeis guineensis, Ekeis oleiferu, Glycine max, Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum, Helianthus annus, Linum usitatissimum, Oenothera biennis, Ozea europea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticum species and Maizeel species, Zea species

9. Use of a transgenic plant organism or tissue, organ, part, cell or propagation material of the same according to one of claims 8 or 9 for the production of vitamin E, oils, fats, free fatty acids or derivatives of the aforementioned.