WO2000008169A1

WO2000008169A1 - Dna sequence coding for a 1-deoxy-d-xylulose-5-phosphate synthase and the overproduction thereof in plants

Info

Publication number: WO2000008169A1
Application number: PCT/EP1999/005467
Authority: WO
Inventors: Andreas Reindl; Patricia Leon Mejia; Juan Manuel Esteves Palmas; Maria Araceli Cantero Gracia; Marcus Ebneth; Karin Herbers
Original assignee: Sungene Gmbh & Co.Kgaa
Priority date: 1998-08-05
Filing date: 1999-07-30
Publication date: 2000-02-17
Also published as: WO2000008169A8; AU757440B2; CA2339519A1; AU5415799A; JP2002525034A; EP1102852A1

Abstract

Method for the production of plants with enhanced vitamin E biosynthesis efficiency by overproduction of a 1-deoxy-D-xylulose-5-phosphate synthase gene from Arabidopsis or E. coli.

Description

DNA sequence coding for an l-deoxy-D-xylulose-5-phosphate synthase and its overproduction in plants

description

The present invention relates to the use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) for the production of plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content, specifically the use of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a DNA sequence hybridizing with this, the use of a DNA sequence SEQ ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ-ID No. 5 or with these hybridizing DNA sequences coding for an l-deoxy-D-xylulose-5-phosphate synthase (DOXS) and a p-hydroxyphenylpyruvate dioxygenase (HPPD) for the production of plants with an increased tocopherol content, vitamin K, Chlorophylls and / or carotenoids, the use of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ ID No. 7 or with these hybridizing DNA sequences coding for an l-deoxy-D-xylulose-5-phosphate synthase (DOXS) and a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) for the production of plants with an increased content of tocopherols, vitamin K, chlorophylls and / or carotenoids, the use of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3, a DNA sequence SEQ-ID No. 5 and a DNA sequence SEQ-ID No. 7 or with these hybridizing DNA sequences coding for an l-deoxy-D-xylulose-5-phosphate synthase (DOXS), a hydroxyphenylpyruvate dioxygenase (HPPD) and a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) for the production of plants with an increased content of tocopherols, vitamin K, chlorophylls and / or carotenoids, process for the production of plants with an increased tocopherol, vitamin K, chlorophyll and / or carotenoid content, containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 or a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5 and SEQ-ID No. 7, the plants themselves, and the use of SEQ-ID No. 1 or SEQ-ID No. 3 for the production of a test system for the identification of inhibitors of DOXS.

An important goal of plant molecular genetic work so far has been the production of plants with an increased content of sugars, enzymes and amino acids. However, the development of plants with an increased vitamin content, such as, for example, increasing the tocopherol content, is also of economic interest. 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 first group (la-d) is derived from tocopherol, the second group consists of derivatives of tocotrienol (2ad):

la, α-tocopherol: Ri R ² = R ³ = CH-; Ib, β-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]: R ¹ = R ² = H, R ³ = CH ₃

Α-Tocopherol is of great economic importance.

There are limits to the development of crops with increased tocopherol content through tissue culture or seed mutagenesis and natural selection. On the one hand, the tocopherol content must already be detectable in tissue culture and, on the other hand, only those plants can be manipulated using tissue culture techniques whose regeneration to whole plants from cell cultures is successful. In addition, after mutagenesis and selection, crop plants can show undesirable properties which have to be eliminated by backcrossing, in some cases repeatedly. The increase in the tocopherol content by crossing would also be restricted to plants of the same type. For these reasons, the genetic engineering procedure to isolate an essential biosynthetic gene coding for the tocopherol synthesis performance and to transfer it specifically in crop plants is superior to the classic breeding method. This method assumes that biosynthesis and its regulation are known and that genes that influence biosynthesis performance are identified.

Isoprenoids or terpenoids consist of different classes of lipid-soluble molecules and are partially or completely formed from Cs-isoprene units. Pure prenyl lipids (e.g. carotenoids) consist of C skeletons, which are exclusively based on isoprene units, while mixed prenyl lipids (e.g. chlorophyll) have an isoprenoid side chain which is linked to an aromatic nucleus.

The starting point for the biosynthesis of prenyl lipids are 3 x acetyl-CoA units, which are converted via ß-hydroxymethylglutaryl-CoA (HMG-CoA) and mevalonate into the starting isoprene unit (C ₅ ), the isopentenyl pyrophosphate (IPP). It has recently been shown by in vivo feeding experiments with C ¹³ that in various eubacteria, green algae and plant chloroplasts a Mevalonate-independent path to the formation of IPP is followed:

Glyceraldehyde-3-phosphate + pyruvate

DOXS

1-Deoxy-D-xylulose 5 - phosphate

10

Carotenoids

PPP

25

Tocopherols

30

Hydroxyethylthiamine, which is formed by decarboxylation of pyruvate, and glyceraldehyde-3-phosphate (3-GAP) are mediated in a l-deoxy-D-xylulose-5-phosphate synthase

35 "Transketolase" reaction first converted to 1-deoxy-D-xylulose-5-phosphate (Schwender et al., FEBS Lett. 414 (1), 129-134 (1997); Arigoni et al., Proc. Natl. Acad. Sci. USA 94 (2), 10600-10605 (1997); Lange et al., Proc. Natl. Acad. Sci. USA 95 (5), 2100-2104 (1998); Lichtenthaler et al., FEBS Lett. 400 (3),

40 271-274 (1997). This is then converted into IPP by an intramolecular rearrangement (Arigoni et al., 1997). Biochemical data indicate that the mevalonate route operates in the cytosol and leads to the formation of phytosterols. The antibiotic mevinolin, a specific inhibitor of mevalonate formation, only leads

45 for the inhibition of sterol biosynthesis in the cytoplasm, while the prenyl lipid formation in the plastids is unaffected (Bach and Lichtenthaler, Physiol. Plant 59 (1983), 50-60. Der Mevalonat- The independent path, on the other hand, is localized plastidically and leads primarily to the formation of carotenoids and plastidic prenyl lipids (Schwender et al., 1997; Arigoni et al, 1997).

IPP is in equilibrium with its isomer, dimethylallyl pyrophosphate (DMAPP). A condensation of IPP with DMAPP in head-tail attachment gives the monoterpene (Cio) geranyl pyrophosphate (GPP). The addition of further IPP units leads to the sesquiterpene (Cι ₅ ) Farnesy pyrophosphate (FPP) and to the diterpene (C ₂₀ ) geranyl-geranyl pyrophosphate (GGPP). Linking two GGPP molecules leads to the formation of the C ₄₀ precursors for carotenoids. GGPP is converted to phytyl pyrophosphate (PPP) by a prenyl chain hydrogenase, the starting material for the further formation of tocopherols.

The ring structures of the mixed prenyl lipids that lead to the formation of vitamins E and K are quinones, the starting metabolites of which come from the Shikimate pathway. The aromatic amino acids phenylalanine and tyrosine are converted into hydroxyphenyl pyruvate, which is converted into homogenisic acid by dioxygenation. This is bound to PPP to form the precursor of α-tocopherol and α-tocoquinone, 2-methyl-6-phytylquinol. Methylation steps with S-adenosylmethionine as the methyl group donor initially produce 2,3-dimethyl-6-phytylquinol, then cyclization γ-tocopherol and repeated methylation of α-tocopherol (Richter, Biochemie der Pflanzen, Georg Thieme Verlag Stuttgart , 1996).

In the literature there are examples which show that the manipulation of an enzyme can have a directional influence on the metabolite flow. In experiments with a modified expression of phytoene synthase, which links two GGPP molecules to 15-cis-phytoene, a direct influence on the carotenoid amounts of these transgenic tomato plants could be measured (Fray and Grierson, Plant Mol. Biol .22 ( 4), 589-602 (1993), Fray et al., Plant J., 8, 693-701 (1995) As expected, transgenic tobacco plants with reduced amounts of phenylalanine ammonium lyase show reduced amounts of phenylpropanoid Phenylalanine ammonium lyase catalyzes the breakdown of phenylalanine, i.e. it removes it from phenylpropanoid biosynthesis (Bäte et al., Proc. Natl. Acad. Sei USA 91 (16): 7608-7612 (1994); Howles et al., Plant Physiol. 112. 1617-1624 (1996).

On increasing the metabolite flow to increase the tocopherol content in plants through overexpression of individuals

Little is known about biosynthetic genes. Only WO 97/27285 describes a modification of the tocopherol content by increased expression or by down-regulation of the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD).

The object of the present invention was to develop a transgenic plant with an increased content of tocopherols, vitamin K, chlorophylls and carotenoids.

The task was surprisingly achieved by overexpressing a 1-deoxy-D-xylulose-5-phosphate synthesis (DOXS) gene in the plants.

In order to increase the metabolite flow from the primary metabolism into the isopronide metabolism, the formation of IPP as a general starting substrate for all plastidic isoprenoids was increased. For this purpose, the activity of DOXS in plants was increased by overexpression of the homologous gene (gene from organism of the same type). This can also be achieved by expressing a heterologous gene (gene from distant organisms). Nucleotide sequences are described from Arabidopsis thaliana DOXS (Acc. No. U 27099), rice (Acc. No. AF024512) and peppermint (Acc. No. AF019383).

In an exemplary embodiment 1, the DOXS gene from Arabidopsis thaliana (SEQ ID No.:1; Mandel et al, Plant J. 9, 649-658 (1996); Acc. No. U27099) is expressed to a greater extent in transgenic plants. Plastid localization is ensured by the transit signal sequence contained in the gene sequence. A DNA sequence which codes for a DOXS gene which is identified by SEQ-ID No. 1 hybridizes and that comes from other organisms such as E. coli (SEQ-ID No. 3) or preferably from other plants.

The GGPP, which is now increasingly available, is being further converted towards tocopherols and carotenoids.

The efficient formation of carotenoids is essential for photosynthesis, and in addition to chlorophylls they serve as "light collector complexes" for better use of photon energy (Heldt, Plant Biochemistry. Spectrum Academic Publishing House Heidelberg Berlin Oxford, 1996). In addition, carotenoids perform important protective functions against oxygen radicals, such as singlet oxygen, which they can return to the ground state (Asada, 1994; Demming-Adams and Adams, Trends in Plant Sciences 1; 21-26 (1996) 1-Deoxy-D-xyllos-5-phosphate synthase defective Arabidopsis thaliana mutant isolated, which shows an "albino phenotype" (Mandel et al, 1996). From this it can be deduced that a reduced amount of carotenoids in the plastids has negative effects on the plant.

The problem was also solved by overexpressing an l-deoxy-D-xylulose-5-phosphate synthase (DOXS) gene and a p-hydroxyphenylpyruvate dioxygenase (HPPD) gene in the plants, see Figure 1.

In order to increase the metabolite flow from the primary metabolism to the isoprenoid metabolism, the formation of IPP as a general starting substrate for all plastidic isoprenoids was increased. For this purpose, the activity of the DOXS in transgenic tobacco and rapeseed plants was increased by overexpression of the DOXS from E. coli. This can be achieved by expressing homologous or other heterologous genes.

The now increasingly available D-1-deoxy-xylulose-5-phosphate is further converted towards tocopherols and carotenoids.

In addition, the formation of homogentisic acid further increases the metabolite flow in the direction of phytylquinones and thus tocopherol, see Figure 1. Homogentisic acid is formed from p-hydroxyphenyl pyruvate by the enzyme p-hydroxyphenyl pyruvate dioxygenase (HPPD). cDNAs coding for this enzyme have been described from various organisms such as, for example, from microorganisms, from plants and from humans.

In embodiment 11, the HPPD gene from Streptomyces avermitilis (Denoya et al., J. Bacteriol. 176 (1994),

5312-5319; SEQ ID No. 5) together with the DOXS from E.coli SEQ-ID No. 3 overexpressed in plants and plant plastids.

The increase in plastid IPP formation leads to the increased formation of all plastid isoprenoids. The increased provision of homogentisic acid ensures that sufficient substrate is available for the formation of tocopherols in the plastids. This now increasingly available homogenate can in turn be implemented in the transgenic plants with the increased amount of phytyl diphosphate (PPP) due to the overexpression of the DOXS. PPP plays a key role in this, as it serves on the one hand as a starting substrate for chlorophylls and phylloquinones and on the other hand for tocopherols.

The transgenic plants are produced by transforming the plants with a construct containing the DOXS and HPPD genes. As model plants for the production of Tocopherols, vitamin K, chlorophylls and carotenoids were used in tobacco and rapeseed.

The invention also relates to the use of the DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5, which code for a DOXS or HPPD or their functional equivalents, for producing a plant with an increased tocopherol, vitamin K, chlorophyll and / or carotenoid content. The nucleic acid sequences can e.g. DNA or cDNA sequences. Coding sequences suitable for insertion into an expression cassette are, for example, those which code for a DOXS or HPPD and which give the host the ability to overproduce tocopherol.

The expression cassettes also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and optionally further regulatory elements which are operatively linked to the intermediate coding sequence for the DOXS or HPPD gene.

An expression cassette is produced by fusing a suitable promoter with a suitable DOXS or HPPD DNA sequence and preferably a DNA inserted between the promoter and DOXS or HPPD DNA sequence which codes for a chloroplast-specific transit peptide, and a polyadenyly - Signaling according to common recombination and cloning techniques, as described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

Expression cassettes whose DNA sequence codes for a DOXS or HPPD fusion protein can also be used, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide. Preferred transit peptides are preferred for the chloroplasts, which are cleaved enzymatically from the DOXS or HPPD part after translocation of the DOXS or HPPD gene into the chloroplasts. The transit peptide which is derived from plastid transketolase (TK) is particularly preferred. or a functional equivalent of this transit peptide (e.g. the transit peptide of the Rubisco small subunit or the

Ferredoxin NADP oxidoreductase) is derived.

The fused expression cassette, which codes for a DOXS gene and an HPPD gene, is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens.

Another object of the invention relates to the use of an expression cassette containing DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5 or with these hybridizing DNA sequences for transforming plants, cells, tissues or parts of plants. The aim of the use is preferably to increase the tocopherol, vitamin K, chlorophyll and carotenoid content of the plant.

Depending on the choice of the promoter, the expression can take place specifically in the leaves, in the seeds or in other parts of the plant. Such transgenic plants, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention.

The invention also relates to transgenic plants, transformed with an expression cassette containing the sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5 or hybridizing with these DNA sequences, as well as transgenic cells, tissues, parts and propagation material of such plants. Transgenic crop plants, such as e.g. Barley, wheat, rye, corn, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, AI falfa, lettuce and the various tree, nut and wine species.

Further objects of the invention are:

Process for transforming a plant, characterized in that expression cassettes containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ-ID No. 5 or with these hybridizing DNA sequences in a plant cell, in callus tissue, an entire plant or protoplasts of plants.

Use of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5 or with these hybridizing DNA sequences for the production of plants with increased tocopherol, vitamin min K, chlorophyll and / or carotenoid content by expression of a DOXS and an HPPD DNA sequence in plants.

The problem was also solved by overexpressing an l-deoxy-D-xylulose-5-phosphate synthase (DOXS) gene and a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) gene in the plants, see Figure 1.

In order to convert the increasingly available GGPP towards tocopherols and carotenoids, the activity of the enzyme geranylgeranyl pyrophosphate oxidoreductase is additionally increased in a further step of the invention by overexpression of a corresponding gene. Through this measure, an increased formation of phytyl pyrophosphate is achieved by an increased conversion of geranylgeranyl pyrophosphate to phytyl pyrophosphate.

For this purpose, for example, the GGPPOR gene from Arabidopsis thaliana (SEQ-ID No. 7) is increasingly expressed in transgenic plants. In order to ensure plastid localization, the Arabidopsis GGPPOR is preceded by a transit signal sequence. Also suitable as an expression cassette is a DNA sequence which codes for a GGPPOR gene which is identified by SEQ-ID No. 7 hybridizes and that comes from other organisms or from other plants.

Exemplary embodiment 15 describes the cloning of the GGPPOR gene from Arabidopsis thaliana.

The increase in plastid l-deoxy-D-xylulose-5-phosphate and phytyl pyrophosphate formation leads to the increased formation of all plastid isoprenoids, so that there is sufficient substrate for the formation of tocopherols, chlorophylls, vitamin K and phylloquinones in the plastids.

The transgenic plants are produced by transforming the plants with a construct containing the DOXS and GGPPOR genes. As model plants for the production of Tocopherols, vitamin K, chlorophylls and carotenoids were used in tobacco and rapeseed.

The invention relates to the use of the DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 which code for a DOXS or GGPPOR or their functional equivalents, for the production of a plant with an increased tocopherol, vitamin K, chlorophyll and / or carotenoid content. The nucleic acid sequences can e.g. DNA or cDNA sequences. Coding sequences suitable for insertion into an expression cassette are, for example, those which code for a DOXS or GGPPOR and which give the host the ability to overproduce tocopherol.

The expression cassettes also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and, if appropriate, further regulatory elements which are operatively linked to the intervening coding sequence for the DOXS or GGPPOR gene. An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence. The sequences preferred but not limited to the operative linkage are targeting sequences to ensure the sub-cellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or other compartments and Translation enhancers such as the 5 'guiding sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).

As an example, the plant expression cassette can be installed in the tobacco transformation vector pBinAR-Hyg. Fig. 2 shows the tobacco transformation vectors pBinAR-Hyg with 35S promoter (A) or pBinAR-Hyg with seed-specific promoter Phaseolin 796 (B):

HPT: hygromycin phosphotransferase OCS: octopine synthase terminator PNOS: nopalin synthase promoter - in addition, restriction sites are shown that only cut the vector once. In principle, any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette. A plant promoter or a promoter which originates from a plant virus is preferably used. The CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980), 285-294). As is known, this promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette can also contain a chemically inducible promoter, by means of which the expression of the exogenous DOXS or GGPPOR gene in the plant can be controlled at a specific point in time. Such promoters as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/19443), one inducible by benzenesulfonamide (EP-A 388186), one inducible by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), one inducible by abscisic acid (EP-A 335528) or inducible by ethanol or cyclohexanone ( WO 93/21334) promoters can include be used.

Furthermore, promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Promoters that ensure leaf-specific expression should be mentioned in particular. The promoter of the cytosolic FBPase from potatoes or the ST-LSI promoter from potatoes should be mentioned (Stockhaus et al., EMBO J. 8 (1989), 2445-245).

An expression cassette is produced by fusing a suitable promoter with a suitable DOXS or GGPPOR DNA sequence and preferably a DNA which is inserted between the promoter and DOXS or GGPPOR DNA sequence and which codes for a chloroplast-specific transit peptide and one Polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987). Expression cassettes can also be used, the DNA sequence of which codes for a DOXS or GGPPOR fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide. Preferred transit peptides are preferred for the chloroplasts, which are cleaved enzymatically from the DOXS or GGPPOR part after translocation of the DOXS or GGPPOR gene into the chloroplasts. Particularly preferred is the transit peptide derived from plastid transketolase (TK) or a functional equivalent of this transit peptide (eg the transit peptide of the small subunit of the

Rubisco or the ferredoxin NADP oxidoreductase) is derived.

The fused expression cassette, which codes for a DOXS gene or a GGPPOR gene, is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens.

Another object of the invention relates to the use of an expression cassette containing DNA sequences SEQ ID No. 1 or SEQ ID No. 3, SEQ ID No. 7 or with these hybridizing DNA sequences for transforming plants, cells, tissues or parts of plants. The aim of the use is preferably to increase the tocopherol, vitamin K, chlorophyll and carotenoid content of the plant.

The invention also relates to transgenic plants transformed with an expression cassette containing the sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 or DNA sequences hybridizing with these, as well as transgenic cells, tissues, parts and propagation material of such plants. Transgenic crop plants, such as e.g. Barley, wheat, rye, corn, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, AI falfa, lettuce and the various tree, nut and wine species.

Further objects of the invention are:

Process for transforming a plant is characterized in that expression cassettes containing a DNA sequence SEQ-ID No. 1 or a DNA sequence SEQ-ID No. 3 and a SEQ ID No. 7 or DNA sequences hybridizing with these zen into a plant cell, into callus tissue, an entire plant or protoplasts of plants.

Use of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 or with these hybridizing DNA sequences for the production of plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content by expression of a DOXS and a GGPPOR DNA sequence in plants.

The problem was also solved by overexpressing an l-deoxy-D-xylulose-5-phosphate synthase (DOXS) gene, a p-hydroxyphenylpyruvate dioxygenase (HPPD) gene and a geranylgeranylpyrophosphate oxidoreductase (GGPPOR) gene in the Plants, see Figure 1.

In order to increase the metabolite flow from the primary metabolism to the isoprenoid metabolism, the formation of IPP as a general starting substrate for all plastidic isoprenoids was increased. For this purpose, the activity of the DOXS in transgenic tobacco and rapeseed plants was increased by overexpression of the DOXS from E. coli. This can also be done by expressing homologous or other heterologous DOXS genes - such as a DNA sequence SEQ-ID No. 1 - can be achieved.

The now increasingly available D-1-deoxy-xylulose-5-phosphate is further converted in the direction of geranylgeranyl pyrophosphate.

In order to convert the increasingly available GGPP towards tocopherols and carotenoids, the activity of the enzyme geranylgeranyl pyrophosphate oxidoreductase is additionally increased by overexpression of a corresponding homologous or heterologous gene in a further step which is essential to the invention. Through this measure, an increased formation of phytyl pyrophosphate is achieved by an increased conversion of geranylgeranyl pyrophosphate to phytyl pyrophosphate.

For this purpose, for example, the GGPPOR gene from Arabidopsis thaliana (SEQ-ID No. 7) is increasingly expressed in transgenic plants. In order to ensure plastid localization, the Arabidopsis GGPPOR is preceded by a transit signal sequence. Also suitable as an expression cassette is a DNA sequence which codes for a GGPPOR gene which is identified by SEQ-ID No. 7 hybridizes and that comes from other organisms or from other plants. Exemplary embodiment 15 describes the cloning of the GGPPOR gene from Arabidopsis thaliana.

In order to convert the increasingly available PPP towards tocopherols and carotenoids, the activity of the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) is additionally increased in a further step of the invention by overexpression of a corresponding homologous or heterologous gene. This measure leads to an increased formation of homogentisic acid by an increased conversion of hydroxyphenylpyruvate into homogentisic acid.

cDNAs coding for this enzyme have been described from various organisms such as, for example, from microorganisms, from plants and from humans.

In embodiment 10, the cloning of the HPPD gene from Streptomyces avermitilis is described (Denoya et al., J. Bacteriol. 176 (1994), 5312-5319; SEQ ID No. 5). In order to ensure plastid localization, the HPPD from Streptomyces is preceded by a transit signal sequence. Also suitable as an expression cassette is a DNA sequence which codes for an HPPD gene which is identified by SEQ-ID No. 5 hybridizes and that comes from other organisms or plants.

The increase in the D-1-deoxy-xylulose-5-phosphate, the phytyl pyrophosphate and the homogentisic acid formation leads to the increased formation of all plastid isoprenoids. The increased availability of these precursors ensures that there is sufficient substrate available for the formation of tocopherols, chlorophylls, vitamin K and phylloquinones in the plastids.

The transgenic plants according to the invention are produced by transforming the plants with a construct containing the DOXS, the HPPD gene and the GGPPOR gene (Example 17). Tobacco and rapeseed were used as model plants for the production of tocopherols, vitamin K, chlorophylls and carotenoids.

The invention relates to the use of the DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7, which code for a DOXS, an HPPD and a GGPPOR or their functional equivalents, for the production of a plant with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content. The nucleic acid sequences can be, for example, DNA or cDNA sequences. Coding sequences suitable for insertion into an expression cassette are, for example, those which encode for a DOXS, an HPPD and a GGPPOR and which give the host the ability to overproduce tocopherol.

The expression cassettes also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and optionally further regulatory elements which are operatively linked to the intermediate coding sequence for the DOXS, the HPPD or the GGPPOR gene. An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements such that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence. The sequences preferred but not limited to the operative linkage are targeting sequences to ensure the subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or others Compartments and translation enhancers such as the 5 'guiding sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).

HPT: hygromycin phosphotransferase OCS: octopine synthase terminator PNOS: nopalin synthase promoter - in addition, restriction sites are shown that only cut the vector once.

In principle, any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette. In particular, a plant promoter or a plant virus-derived promoter is preferably used. The CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980), 285-294). As is known, this promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette can also contain a chemically inducible promoter, by means of which the expression of the exogenous DOXS, HPPD or GGPPOR gene in the plant can be controlled at a specific point in time. Such promoters as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzene-sulfonamide-inducible promoter

(EP-A 388186), one that can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), one that can be induced by abscisic acid (EP-A 335528) or that by ethanol or cyclohexanone-inducible (WO 93/21334) promoter can include be used.

Furthermore, promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Promoters that ensure leaf-specific expression should be mentioned in particular. The promoter of the cytosolic FBPase from potato or the ST-LSI promoter from potato are to be mentioned (Stockhaus et al., EMBO J. 8 (1989), 2445-245).

An expression cassette is produced by fusing a suitable promoter with a suitable DOXS, HPPD or GGPPOR DNA sequence and preferably a DNA inserted between promoter and DOXS, HPPD or GGPPOR DNA sequence, which is suitable for a chloroplast-specific transit peptide encoded, and a polyadenylation signal according to common recombination and cloning techniques, such as those described in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Inter-science (1987).

Expression cassettes whose DNA sequence codes for a DOXS, HPPD or GGPPOR fusion protein can also be used, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide. Preferred transit peptides are preferred for the chloroplasts, which are cleaved enzymatically from the DOXS, HPPD or GGPPOR part after translocation of the DOXS, HPPD or GGPPOR gene into the chloroplast.

The transit peptide which is derived from plastid transketolase (TK) or a functional equivalent is particularly preferred. This transit peptide (for example the transit peptide of the small subunit of the Rubisco or the ferredoxin NADP oxidoreductase) is derived.

The fused expression cassette, which codes for a DOXS gene, an HPPD gene and a GGPPOR gene, is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens.

Another object of the invention relates to the use of an expression cassette containing DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7 or with these hybridizing DNA sequences for transforming plants, cells, tissues or parts of plants. The aim of the use is preferably to increase the tocopherol, vitamin K, chlorophyll and carotenoid content of the plant.

The invention also relates to transgenic plants, transformed with an expression cassette containing the sequence SEQ-ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7 or DNA sequences hybridizing with these, as well as transgenic cells, tissues, parts and propagation material of such plants. Transgenic crop plants, such as e.g. Barley, wheat, rye, corn, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species.

Further objects of the invention are:

Process for transforming a plant, characterized in that expression cassettes containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3, a DNA sequence SEQ-ID No. 5 and a DNA sequence SEQ-ID No. 7 or with these hybridizing DNA sequences in a plant cell, in callus tissue, an entire plant or protoplasts of plants.

- Use of the DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7 or DNA sequences hybridizing with these for the production of plants with increased toco pherol, vitamin K, chlorophyll and / or carotenoid content by expression of a DOXS, an HPPD and a GGPPOR DNA sequence in plants.

An additional object of the present invention was therefore

Development of a test system for the identification of inhibitors of DOXS.

This object was achieved by the expression of a DOXS gene from Arabidopsis or E. coli or DNA sequences hybridizing therewith and subsequent testing of chemicals for inhibition of DOXS enzyme activity.

The transgenic plants are produced by transforming the plants with a construct containing the DOXS gene. Arabidopsis and oilseed rape were used as model plants for the production of tocopherols, vitamin K, chlorophylls and carotenoids.

The complete DOXS gene from Arabidopsis is cloned by isolating the cDNA specific for the DOXS gene (SEQ-ID No. 1).

The invention relates to the use of the DNA sequence SEQ ID No. 1 or SEQ-ID No. 3 which codes for a DOXS or its functional equivalent, for the production of a plant with an increased tocopherol, vitamin K, chlorophyll and / or carotenoid content. The nucleic acid sequence can e.g. be a DNA or a cDNA sequence. Coding sequences suitable for insertion into an expression cassette are, for example, those which code for a DOXS and which give the host the ability to overproduce tocopherol.

The expression cassettes also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, an expression cassette comprises upstream, ie at the 5 'end of the coding sequence, a promoter and downstream, ie at the 3' end, a polyadenylation signal and, if appropriate, further regulatory elements which match the coding sequence for the DOXS gene in between are operationally linked. An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence. The sequences preferred but not limited to the operative linkage are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil bodies or other compartments and translation enhancers like the 5 'guiding sequence from tobacco Mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987) 8693-8711).

As an example, the plant expression cassette can be installed in the tobacco transformation vector pBinAR-Hyg. Fig. Shows the tobacco transformation vectors pBinAR-Hyg with 35S promoter (A) or pBinAR-Hyg with seed-specific promoter Phaseolin 796 (B):

HPT: hygromycin phosphotransferase - OCS: octopine synthase terminator PNOS: nopalin synthase promoter also such restriction sites are shown that only cut the vector once.

In principle, any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette. In particular, a plant promoter or a promoter derived from a plant virus is preferably used. The CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980), 285-294). As is known, this promoter contains different recognition sequences for transcriptional effectors which, in their entirety, lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette can also contain a chemically inducible promoter, by means of which the expression of the exogenous DOXS gene in the plant can be controlled at a specific point in time. Such promoters as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/19443), one inducible by benzenesulfonamide (EP-A 388186 ), one that can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), one that can be induced by abscisic acid (EP-A 335528) or one that can be induced by ethanol or cyclohexanone (WO 93 / 21334) Promoter can include be used.

Furthermore, promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Promoters that are leaf-specific are particularly noteworthy Ensure expression. The promoter of the cytosolic FBPase from potatoes or the ST-LSI promoter from potatoes should be mentioned (Stockhaus et al., EMBO J. 8 (1989) 2445-245).

With the help of a seed-specific promoter, a foreign protein could be stably expressed up to a proportion of 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio / Technology 10 (1995), 1090-1094). The expression cassette can therefore, for example, be a seed-specific promoter (preferably the phaseolin

Promoter (US 5504200), the USP- (Baumlein, H. et al. Mol. Gen. Genet. (1991) 225 (3), 459-467) or LEB4 promoter (Fiedler and Conrad, 1995)), the LEB4 Signal peptide containing the gene to be expressed and an ER retention signal. The structure of such a cassette is shown schematically as an example in Figure 2.

An expression cassette is produced by fusing a suitable promoter with a suitable DOXS-DNA sequence and preferably a DNA inserted between promoter and DOXS-DNA sequence, which codes for a chloroplast-specific transit peptide, and a polyadenylation signal according to common recombination and cloning techniques, such as those for example in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al. , Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

Sequences are particularly preferred which ensure targeting in the apoplasts, in plastids, in the vacuole, in the mitochondrion, in the endoplasmic reticulum (ER) or in the absence of corresponding operative sequences to ensure that they remain in the compartment of formation, the cytosol (Kermode, Crit. Rev. Plant Sei. 15, 4 (1996), 285-423). Localization in the ER has proven to be particularly beneficial for the amount of protein accumulation in transgenic plants (Schouten et al., Plant Mol. Biol. 30 (1996), 781-792).

Expression cassettes can also be used, the DNA sequence of which codes for a DOXS fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide. Particularly preferred for the chloroplasts are specific transit peptides which, after translocation of the DOXS gene into the chloroplasts, are enzymatically removed from the DOXS part. will split. The transit peptide which is derived from the plastidic transketolase (TK) or a functional equivalent of this transit peptide (for example the transit peptide of the small subunit of the Rubisco or the ferredoxin NADP oxidoreductase) is particularly preferred.

The inserted nucleotide sequence coding for a DOXS can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components, as well as consist of different heterologous DOXS gene sections of different organisms. In general, synthetic nucleotide sequences with codons are generated which are preferred by plants. These codons preferred by plants can be determined from codons with the highest protein frequency, which are expressed in most interesting plant species. When preparing an expression cassette, various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.

The promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. In general, the linker has a size of less than 100 bp within the regulatory areas, often less than 60 bp, but at least 5 bp. The promoter can be native or homologous as well as foreign or heterologous to the host plant. The expression cassette contains in the 5 '-3' transcription direction the promoter, a DNA sequence which codes for a DOXS gene and a region for the transcriptional termination. Different termination areas are interchangeable.

Manipulations which provide suitable restriction sites or which remove unnecessary DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as transitions and trans versions are possible, in vitro mutagenesis, "primer pair", restriction or ligation can be used. With suitable manipulations, such as restriction, "chewing-back" or replenishment len of overhangs for "bluntends", complementary ends of the fragments can be provided for the ligation.

Of importance for the success according to the invention can i.a. the attachment of the specific ER retention signal SEKDEL (Schouten, A. et al. Plant Mol. Biol. 30 (1996), 781-792), the average expression level is thus tripled to quadrupled. Other retention signals, which occur naturally in plant and animal proteins located in the ER, can also be used to construct the cassette.

Preferred polyadenylation signals 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 pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents.

An expression cassette can contain, for example, a constitutive promoter (preferably the CaMV 35 S promoter), the LeB4 signal peptide, the gene to be expressed and the ER retention signal. The amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as the ER retention signal.

The fused expression cassette which codes for a DOXS gene is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens. Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as e.g. of tobacco plants can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media. The transformation of plants by agrobacteria is known, among other things, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38. From the transformed cells of the wounded leaves or leaf pieces, transgenic plants can be regenerated in a known manner which contain a gene integrated into the expression cassette for the expression of a DOXS gene contain.

To transform a host plant with a DNA coding for a DOXS, an expression cassette is inserted as an insert into a recombinant vector, the vector DNA of which contains additional functional regulatory signals, for example Contains sequences for replication or integration. Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119 (1993).

Using the recombination and cloning techniques cited above, the expression cassettes can be cloned into suitable vectors that allow their proliferation, for example in E. coli. Suitable cloning vectors include pBR332, pUC series, Ml3mp series and pACYC184. Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.

Another object of the invention relates to the use of an expression cassette containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 or a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5 and SEQ-ID No. 7, or with these hybridizing DNA sequences for the transformation of plants, cells, tissues or parts of plants. The aim of the use is preferably to increase the tocopherol, vitamin K, chlorophyll and carotenoid content of the plant.

The expression cassette can also be used to transform bacteria, cyanobacteria, yeast, filamentous fungi and algae with the aim of increasing tocopherol, vitamin K, chlorophyll and / or carotenoid production.

The transfer of foreign genes into the genome of a plant is called transformation. 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 are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene cannon - the so-called particle bombardment method, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the gene transfer mediated by Agrobacterium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agro acteriu / n tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

Agrobacteria transformed with an expression cassette can also be used in a known manner to transform plants, in particular crop plants, such as cereals, maize, oats, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes, rape, Alfalfa, lettuce and the various tree, nut and wine species can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.

Functionally equivalent sequences which code for a DOXS gene are those sequences which, despite a different nucleotide sequence, still have the desired functions. Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. Artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.

A functional equivalent is also understood to mean, in particular, natural or artificial mutations in an originally isolated sequence coding for a DOXS, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, for example, the present invention also encompasses those nucleotide sequences which are obtained by modifying the DOXS nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also be the insertion of further restriction enzyme interfaces.

Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.

In addition, artificial DNA sequences are suitable as long as they have the desired property, for example, of increasing the tocopherol content in the plant, as described above

Mediate overexpression of the DOXS gene in crop plants. Such artificial DNA sequences can be Setting of proteins constructed using molecular modeling, which have DOXS activity or are determined by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable. The specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.

Other suitable equivalent nucleic acid sequences are sequences which code for fusion proteins, part of the fusion protein being a plant DOXS polypeptide or a functionally equivalent part thereof. The second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect DOXS expression (e.g. myc-tag or his-tag). However, this is preferably a regulatory protein sequence, such as e.g. a signal or transit peptide that directs the DOXS protein to the desired site of action.

However, the invention also relates to the expression products and fusion proteins produced according to the invention from a transit peptide and a polypeptide with DOXS activity.

In the context of the present invention, increasing the tocopherol, vitamin K, chlorophyll and / or carotenoid content means the artificially acquired ability to increase the biosynthetic capacity of these compounds by functional overexpression of the DOXS gene in the plant compared to the non-genetically modified plant for the duration of at least one generation of plants.

The tocopherol biosynthesis site is generally the leaf tissue, so that leaf-specific expression of the DOXS gene is useful. However, it is obvious that the tocopherol biosynthesis need not be restricted to the leaf tissue, but can also be tissue-specific in all other parts of the plant - for example in fatty seeds.

In addition, constitutive expression of the exogenous DOXS gene is advantageous. On the other hand, inducible expression may also appear desirable.

The effectiveness of the expression of the transgenically expressed DOXS gene can be determined, for example, in vitro by proliferation of the shoot meristem. In addition, a change in the type and level of expression of the DOXS gene and its effect on the tocopherol Biosynthetic performance can be tested on test plants in greenhouse experiments.

The invention also relates to transgenic plants transformed with an expression cassette containing the

Sequence SEQ-ID No. 1 or SEQ-ID No. 3; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-No. 5; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 or a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 5 and SEQ-ID No. 7, or DNA sequences hybridizing with these, as well as transgenic cells, tissues, parts and propagation material of such plants. Transgenic crop plants, such as e.g. Barley, wheat, rye, corn, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rape, alfalfa, lettuce and the various tree, nut and wine species.

Plants in the sense of the invention are mono- and dicotyledonous plants or algae.

In order to find efficient inhibitors of DOXS, it is necessary to provide suitable test systems with which inhibitor-enzyme binding studies can be carried out. For this purpose, for example, the complete cDNA sequence of the Arabidopsis DOXS is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.

The DOXS protein expressed with the aid of the expression cassette is particularly suitable for the detection of inhibitors specific for the DOXS.

For this purpose, the DOXS can be used, for example, in an enzyme test in which the activity of the DOXS is determined in the presence and absence of the active substance to be tested. By comparing the two activity determinations, a qualitative and quantitative statement can be made about the inhibitory behavior of the active substance to be tested. Methods for determining the activity of DOXS are described (Putra et. Al., Tetrahedron Letters 39 (1998), 23-26; Sprenger et al., PNAS 94 (1997), 12857-12862).

With the help of the test system according to the invention, a large number of chemical compounds can be checked quickly and easily for inhibitory properties. The method makes it possible to selectively reproducibly select those with great potency from a large number of substances in order to subsequently carry out further in-depth tests known to the person skilled in the art with these substances. By overexpression of the gene sequence coding for a DOXS SEQ-ID No. 1 or SEQ-ID No. 3 In principle, increased resistance to DOXS inhibitors can be achieved in a plant. The transgenic plants produced in this way are also the subject of the invention.

Further objects of the invention are:

Process for transforming a plant is characterized in that an expression cassette containing a

DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or introduces a hybridizing DNA sequence into a plant cell, into callus tissue, an entire plant or plant protoplasts.

Using a plant to make plant DOXS.

Use of the expression cassette containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a hybridizing DNA sequence for the production of plants with increased resistance to inhibitors of DOXS by increased expression of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a DNA sequence hybridizing with this.

- Use of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a hybridizing with this DNA sequence for the production of plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content by expression of a DOXS DNA sequence in plants.

Use of the expression cassette containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a DNA sequence hybridizing with this to produce a test system for identifying inhibitors of DOXS.

The invention is illustrated by the following examples, but is not limited to these:

General cloning procedures

The cloning steps carried out in the context of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria Multiplication of phages and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).

The bacterial strains used below (E. coli, XL-I Blue) were obtained from Stratagene. The agrobacterial strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850 can) was developed by Deblaere et al. in (Nucl. Acids Res. 13 (1985) 4777). Alternatively, the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used. For cloning, the vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711-8720) and pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990), 221-230).

Sequence analysis of recombinant DNA

The sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from Licor (sold by MWG Biotech, Ebersbach) according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463 - 5467).

example 1

Production of Arabidopsis thaliana DOXS transformation constructs

The Arabisopsis thaliana DOXS gene was described in Mandel et al. (1996) described as a complete cDNA cloned into the vector pBluescript KS- (Stratagene).

To produce overexpression constructs, a 2.3 kb fragment (labeled F-23-C) was isolated via the pBluescript KS-Hincll (blunt-end) and Sacl interfaces. This sequence contains the complete DOXS cDNA including chloroplast transit peptide from the ATG start codon to an EcoRI interface that is 80 bp downstream of the stop codon. This fragment was cloned via the Smal (blunt-end) and SacI interfaces into the pBIN 19 3X35S vector (Figure 3) (Bevan et al., 1980), which is the 35S promoter of the Cauliflower Mosaic Virus (Franck et al., Cell 21 (1), 285-294 (1980)) three times in a row.

To produce antisense constructs, a region of the 3 'end of the cDNA (designated F-23-C antisense) was cloned into the pBIN19-3X35S vector mentioned above. Part of the 5 'area of the DOXS cDNA in pBluescript KS- was via Hincll and the DOXS- digested internal Bglll interface and removed the resulting fragment. (Figure 4). The BglII interface was filled in via the Klenow fill-in reaction (Klenow polymerase; Röche; after reaction according to the manufacturer's protocol), so that a "blunt end" occurs. The now compatible ends (Bglll - "blunt-end" and in were ligated. Now the 3 'region of the DOXS cDNA was via Kpnl and Xbal (both interfaces are in the polylinker of pBluescript KS-5'- and 3'- der DOXS cDNA) in antisense orientation was cloned into the pBIN19 vector described above in antisense orientation.

The transformations of Arabidopsis thaliana plants with the constructs described above were carried out with Agrobacterium tumefaciens using the vacuum infiltration method (Bent et al., Science 265 (1994), 1856-1860). Several independent transformants were isolated per construct. Each letter (see Table 1) means an independent transformed line. From the Tl generation obtained from this, plants were examined for homo- or heterozygosity. Several plants from each line were crossed to perform a segregation analysis. The number in Table 1 corresponds to the individual plant that was selected for further analysis. Both homo- and heterozygote lines were obtained. The segregation analysis of the lines obtained is shown in Table 1 below:

Table 1. Segregation analysis of the transgenic DOXS-T2 plants

Example 2

Isolation of genomic DNA from the bacterium Escherichia coli XL1 Blue 5

A culture of Escherichia coli XL1 Blue was grown in 300 ml Luria Broth medium for 12 hours at 37 ° C. The genomic DNA of the bacterium was isolated from this culture by first pelleting at 5000 revolutions in a Sorvall RC50 fugue

Turned 10. The pellet was then resuspended in 1/30 volume of the original culture lysis buffer (25 mM EDTA, 0.5% SDS; 50 mM Tris HC1, pH 8.0). An equal volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added and incubated at 70 degrees for 10 minutes. Subsequently, in a Heraeus under-table

15 centrifuge at 3500 U for 15 minutes the aqueous phase separated from the phenolic. The aqueous supernatant was mixed with 2.5 volumes of ethanol and 1/10 volumes of 8 M lithium chloride and the nucleic acids were precipitated at room temperature for 10 minutes. The pellet was then taken up in 400 ul TE / RNAse and at

20 37 degrees incubated for 10 minutes. The solution was again shaken with a volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) and the supernatant was precipitated with 2.5 volumes of ethanol and 1/10 volumes of 8 M lithium chloride. The pellet was then washed with 80% ethanol and taken up in 400 μl TE / RNAse.

25

Example 3

Isolation of the DOXS from E. coli

30 For the PCR, oligonucleotides were derived from the DNA sequence of the DOXS (Acc. Number AF035440), to which a BamHI at the 5 'end and an Xbal or another BamHI restriction site were added at the 3' end. The oligonucleotide at the 5 'end comprises the sequence 5' -A1GGATCCATGAGTTTT-GATATTGCCAAATAC-3 '

35 (nucleotides 1-24 of the DNA sequence; written in italics) starting with the ATG start codon of the gene, the oligonucleotide at the 3 'end comprises the sequence 5' - ATTCT AG ATTATGCCAGCCAGGCCTTG- 3 'or 5'-ATG-GATCCTTATGCCAGCCAGGCCTTG -3 '(nucleotides 1845-1863 of the reverse complementary DNA sequence; written in italics) starting with the

40 stop codon of the gene. The PCR reaction with the two BamHI-containing oligonucleotides was carried out with the Pfu polymerase (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions. 500 ng of the genomic DNA from E. coli were used as template. The PCR program was:

45

5 cycles: 4 sec 94 ° C, 30 sec 52 ° C, 2 min 72 ° C; 5 cycles: 4 sec 94 ° C, 30 sec 48 ° C, 2 min 72 ° C; 25 cycles: 4 sec 94 ° C, 30 sec 44 ° C, 2 min 72 ° C

The fragment was cleaned using a gene clean kit (Dianova GmbH, Hilden) and cloned into the vector PCR script (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions. The correctness of the sequence was determined by sequencing. The BamHI fragment was isolated from the PCR script vector and ligated into a correspondingly cut Bin19 vector which additionally contains the transit peptide of the potato transketolase behind the CaMV 35S promoter. The transit peptide ensures plastid localization. The constructs are shown in Figures 5 and 6 and the fragments have the following meaning:

Fragment A (529 bp) contains the 35S promoter of the Cauliflower Mosaic Virus (nucleotides 6909 to 7437 of the Cauliflower Mosaic Virus). Fragment B (259 bp) contains the transit peptide of the transketolase. Fragment E contains the gene of the DOXS. Fragment D (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination.

The PCR reaction with the oligonucleotides containing 5 '-BamHI and 3' -Xbal was carried out using Taq polymerase (Takara, Sosei Co., Ltd.) according to the manufacturer's instructions. 500 ng of the genomic DNA from E. coli were used as template. The PCR program was:

5 cycles: 4 sec 94 ° C, 4 sec 50 ° C, 2 min 30 ° C 5 cycles: 4 sec 94 ° C, 30 sec 46 ° C, 2 min 68 ° C 25 cycles: 4 sec 94 ° C, 30 sec 42 ° C, 2 min 68 ° C

The fragment was purified using the gene clean kit and ligated into the vector pGemT (Promega GmbH, Mannheim). It was cloned as a BamHI / Xbal fragment into a correspondingly cut pBinl9AR vector behind the CaMV 35S promoter. The sequence was checked by sequencing (SEQ-ID No. 3) and two non-conservative base changes were found, which lead to a change in amino acid 152 (asparagine) in valine and amino acid 330 (cysteine) in tryptophan compared to the published sequence.

Example 4

Detection of increased DOXS RNA levels in transgenic plants

Total RNA from 15 day old seedlings of different transgenic lines, which have the DOXS overexpression construct, was determined according to the method of Logeman et al., Anal. Biochem. 163, 16-20 (1987) extracted, separated in a 1.2% agarose gel, transferred to filters and hybridized with a 2.1 kb long DOXS fragment as a probe (Figure 7).

Example 5

Detection of increased DOXS protein levels in transgenic plants

Whole protein (Figure 8) from 15 day old seedlings of different, independent transgenic plants, which have the DOXS overexpression construct, was isolated and detected with a polyclonal anti-DOXS antibody (IgG) in a Western analysis (Figure 9).

Example 6

Measurement of the carotenoid and chlorophyll content

The total carotenoid and chlorophyll amounts were determined as described in Lichtenthaler and Wellburn (1983) with 100% acetone extracts. The results of the multiple measurements of the transgenic lines which have the DOXS overexpression construct are shown in Table 2 below.

Table 2: Total carotenoid and chlorophyll content of the transgenic DOXS lines

Example 7

Rapeseed transformation

The production of the transgenic rape plants is based on a protocol by Bade, JB and Damm, B (in Gene, Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38 ), which also gives the composition of the media used. The transformations were carried out with the Agrobacterium strain LBA4404 (Clontech). The pBIN19 constructs already described above with the entire DOXS cDNA were used as binary vectors. In these pBIN vectors, the NOS terminator sequence was replaced by the OCR terminator sequence. Brassica napus seeds were sterilized with 70% (v / v) ethanol, washed for 10 min in 55 ° CH ₂ 0, in 1% hypochlorite solution (25% v / v Teepol, 0.1% v / v Twenn 20 ) incubated for 20 min and washed six times with sterile H0 for 20 min each. The seeds were dried on filter paper for three days and 10-15 seeds were germinated in a glass flask with 15 ml of germination medium. The roots and apices were removed from several seedlings (approx. 10 cm in size) and the remaining hypocotyls were cut into pieces approx. 6 mm long. The approx. 600 explants obtained in this way are washed with 50 ml of basal medium for 30 min and transferred to a 300 ml flask. After addition of 100 ml callus induction medium, the cultures were incubated for 24 h at 100 rpm.

An overnight culture of the Agrobacterium strain was set up at 29 ° C. in LB with kanamycin (20 mg / l), of which 2 ml in 50 ml LB without kanamycin for 4 h at 29 ° C. up to an OD ₆₀ o of 0.4- 0.5 incubated. After pelleting the culture at 2000 rpm for 25 min, the cell pellet was resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution was adjusted to an OD600 of 0.3 by adding further basal medium.

The callus induction medium was removed from the oilseed rape explants using sterile pipettes, 50 ml of Agrobacterium solution were added, mixed gently and incubated for 20 min. The Agrobacteria suspension was removed, the oilseed rape explants were washed for 1 min with 50 ml callus induction medium and then 100 ml callus induction medium was added. The co-cultivation was carried out on a rotary shaker at 100 rpm for 24 h. The co-cultivation was stopped by removing the callus induction medium and the explants were washed twice for 1 min with 25 ml and twice for 60 min with 100 ml washing medium at 100 rpm. The washing medium with the explants was transferred to 15 cm petri dishes and the medium was removed using sterile pipettes. distant. For regeneration, 20-30 explants were transferred to 90 mm petri dishes containing 25 ml shoot induction medium with kanamycin. The petri dishes were closed with two layers of leucopore and incubated at 25 ° C and 2000 lux for 16/8 H photoperiods. Every 12 days, the developing calli was transferred to fresh petri dishes with sprout induction medium. All further steps for the regeneration of whole plants were carried out as by Bade, JB and Damm, B. (in Gene Transfer to Plants, Potrycus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30- 38).

Example 8

Increase in tocopherol biosynthesis in oilseed rape

The cDNA of DOXS (SEQ-ID No. 1) was provided with a CaMV35S promoter and overexpressed in rapeseed using the 35S promoter. In parallel, the seed-specific promoter of the phaseolin gene was used to specifically increase the tocopherol content in the rapeseed. Rapeseed plants transformed with the appropriate constructs were grown in the greenhouse. The α-tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the α-tocopherol concentration was increased compared to the non-transformed plant.

Example 9

Evidence of the expression of DOXS from E. coli in transgenic tobacco plants

Leaf disks with a diameter of 0.9 cm were taken from completely unfolded leaves from plants which contained the construct pBinAR HPPD-DOXS and were frozen in liquid nitrogen. The leaf material was homogenized in a HEPES-KOH buffer which contained proteinase inhibitors and the protein concentration was determined from the extract using the Bio-Rad protein assay according to the manufacturer's instructions. 45 μg protein of each extract were mixed with a volume of application buffer (Laemmli, 1970) and incubated for 5 min at 95 ° C. The proteins were then separated on a 12.5 percent SDS-PAGE gel. The proteins were then transferred to Porablot membrane (Machery and Nagel) using semi-dry electroblots. The DOXS protein was detected by means of an antibody against the E. coli DOXS from rabbits. The color reaction is based on the binding of a secondary antibody and an alkaline phosphatase, which converts NBT / BCIP into a dye. Secondary antibody and alkaline Phosphatase are from Pierce and were carried out according to the manufacturer's instructions.

Figure 10 shows the detection of the DOXS protein in leaves of 5 transgenic plants. 1: marker; 2: plant 10; 3:62; 4: 63; 5: 69; 7:71; 8: 112; 9: 113; 10: 116; 11: WT1; 12: WT2; 13: 100 recombinant protein; 14:50ng recombinant protein; 15:10 ng recombinant protein.

10 Example 10

Cloning of an HPPD gene from Streptomyces avermitilis U11864

15 Isolation of genomic DNA of the bacterium Streptomyces avermitilis U11864:

A culture of Streptomyces avermitilis U11864 was made in 300 ml of YEME medium (5 g malt extract, 2 g yeast extract, 2 g glucose)

20 96 h at 28 ° C. The genomic DNA of the bacterium was isolated from this culture by first pelleting it at 5000 U in a Sorvall RC5C fugue. The pellet was then resuspended in 1/30 volume of lysis buffer (25 mM EDTA, 0.5% SDS, 50 mM Tris-HCl, pH 8.0). An equal volume of phenol /

25 chloroform / isoamyl alcohol (25: 24: 1) was added and incubated at 70 ° C for 10 minutes. The aqueous phase was then separated from the phenolic in a Heraeus under-table centrifuge at 3500 U for 15 minutes. The aqueous supernatant was mixed with 2.5 volumes of ethanol and 1/10 volumes of 8 M lithium chloride and the

30 nucleic acids precipitated at room temperature for 10 minutes. The pellet was then taken up in 400 μl TE / RNAse and incubated at 37 degrees for 10 minutes. The solution was shaken again with a volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) and the supernatant was precipitated with 2.5 volumes of ethanol and 1/10 volume.

35 men 8 M lithium chloride. The pellet was then washed with 80% ethanol and taken up in 400 ul TE / RNAse.

From the DNA sequence of the HPPD from Streptomyces avermitilis (Denoya et al, 1994; Acc. Number U11864), oligo-

40 nucleotides were derived, to which a BamHI had been added at the 5 'end and an Xbal restriction site at the 3' end. The oligonucleotide at the 5 'end comprises the sequence 5' -GGATCCAGCGGA - CAAGCCAAC-3 '(37 to 55 bases from the ATG in 5' direction; written in italics), the oligonucleotide at the 3 'end comprises the sequence

45 Sequence 5 '-TCTAGA TTATGCCAGCCAGGCCTTG-3' (nucleotides 1845-1863 of the reverse complementary DNA sequence; written in italics). The PCR reaction was carried out with Pfu polymerase (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions. 400 ng of the genomic DNA were used as template. The PCR program was:

5 5 cycles: 4 sec 94 ° C, 30 sec 54 ° C, 2 min 72 ° C 5 cycles: 4 sec 94 ° C, 30 sec 52 ° C, 2 min 72 ° C 25 cycles: 4 sec 94 ° C, 30 sec 50 ° C, 2 min 72 ° C

The fragment was purified using a gene clean kit (Dianova GmbH, Hilden) 10 and cloned into the vector PCR script (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions. The correctness of the sequence was checked by sequencing. It was found that the isolated gene codes for an additional amino acid. It contains the three bases TAC (coding for tyrosine), 15 before the nucleotide N429 of the cited sequence (Denoya et al., 1994).

The fragment was isolated from the vector with a BamHI and Xbal digest and into a correspondingly cut Bin19AR vector

Ligated 20 behind the CaMV 35S promoter, for expression of the gene in the cytosol. The gene was isolated as a BamHI fragment from the same PCR script vector and ligated into a correspondingly cut pBinl9 vector, which additionally exits the transit peptide of the plastid transketolase behind the CaMV 35S promoter

Contains 25 potatoes. The transit peptide ensures plastid localization. The constructs are shown in Figures 11 and 12 and the fragments have the following meaning:

Fragment A (529 bp) contains the 35S promoter of the Cauliflower 30 mosaic virus (nucleotides 6909 to 7437 of the Cauliflower Mosaic virus). Fragment B (259 bp) contains the transit peptide of the transketolase. Fragment C contains the HPPD gene. Fragment D (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen, J. et al., EMBO J. 3 (1984), 35 835-846) for transcription termination.

Example 11

Production of constructs for plant transformation with DOXS 40 and HPPD-DNA sequences

For the production of plants that are transgenic for the DOXS and the HPPD, a binary vector was prepared that contains both gene sequences (Figure 13). The gene sequences of the DOXS 45 and the HPPD were each cloned as BamHI fragments as described in Examples 3 and 10. The vector pBinAR-Hyg contains the 35S promoter of the cauliflower mosaic virus and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination. The pBinAR-Hyg vector mediates resistance to the antibiotic hygromycin in plants and is therefore suitable for superinfecting plants with kanamycin resistance.

To clone the HPPD into vectors which additionally contain another cDNA, oligonucleotides were derived for a PCR, to which a BamHI restriction site had been added at the 5 'end and at the 3' end. The oligonucleotide at the 5 'end comprises the sequence 5' -GGATCCTCCAGCGGACAAGCCAAC-3 '(nucleotides 37 to 55 from the ATG in 5' direction; italics), the oligonucleotide at the 3 'end comprises the sequence 5'-ATGGATC- CCGCGCCGCCTACAGGTTG-3 '(ending with base pair 1140 of the coding sequence, starting 8 base pairs 3' of the TAG stop codon; written in italics). The PCR reaction was carried out using Tli polymerase (Promega GmbH, Mannheim) according to the manufacturer's instructions. 10 ng of the plasmid pBinAR-HPPD were used as template. The PCR program was:

5 cycles: 94 ° C 4 sec, 68 ° C 30 sec, 72 ° C 2 min 5 cycles: 94 ° C 4 sec, 64 ° C 30 sec, 72 ° C 2 min 25 cycles: 94 ° C 4 sec, 60 ° C 30 sec, 72 ° C 2 min

The fragment was cleaned using a gene clean kit (Dianova GmbH, Hilden) and cloned into the vector PCR script (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions. The correctness of the sequence was checked by sequencing. It was cut out of the vector PCR script as a BamHI fragment and ligated into a correspondingly cut pBinAR vector, which additionally contains the transit peptide of transketolase, for introducing the gene product into the plastids. The plasmid pBinAR-TP-HPPD was formed (Figure 12).

For cloning, the plasmid pBinAR-TP-HPPD was used to convert the 35S promoter, the transketolase transit peptide, the HPPD gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) isolated for transcription termination by means of PCR. A HindIII interface was added to each of the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which is attached to the 5 'region of the promoter (italics) is 5'-ATAAGCTT-CATGGAGTCAAA-GATTCAAATAGA-3', that of the oligonucleotide which is attached to the termination sequence (italics) is 5 '-ATAAGCTTGGACAATCAGTAAATTGAACGGAG-3'. The fragment obtained was purified using a gene clean kit (Dianova GmbH, Hilden) and according to the manufacturer's instructions in the vector PCR script (Stratagene GmbH, Heidelberg) cloned. The correctness of the sequence was checked by sequencing (SEQ-ID No. 5). From this PCR script vector, it was transferred as a Hindlll fragment into the correspondingly cut vector pBinl9 (Bevan, 1984, Nucleic 5 Acids Res. 12, 8711-8721).

The plasmid pBinAR-TP-DOXS was used to convert the 35S promoter, the trans - ketolase transit peptide, the DOXS gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al.,

10 1984) for transcription termination by means of PCR. The

An EcoRI site was added to each of the oligonucleotides for the promoter and the terminator sequence. The sequence of the oligonucleotide that attaches to the promoter (italics) is 5 '-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3', that of the

15 oligonucleotide, which is attached to the terminator sequence (italics) is 5 '-ATGAATTCGGACAATCAGTAAATTGAA-CGGA-G-3'. The fragment was purified using a gene clean kit (Dianova GmbH, Hilden) and cloned into the vector PCR script (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions. The accuracy

20 of the sequence was checked by sequencing (SEQ-ID No. 3). From the PCR script vector, it was transferred as an EcoRI fragment into the correspondingly cut vector pBin19 (Bevan, 1984).

From the PCR script vector, it was transferred as an Xbal fragment into the correspondingly cut vector, which, as described above, already contained the sequence of the HPPD. The construct pBinAR-HPPD-DOXS (Figure 13) was created, the fragments of which have the following meaning:

Fragment A (529 bp) contains the 35S promoter of the Cauliflower mosaic virus (nucleotides 6909 to 7437). Fragment B contains the transit peptide of plastid transketolase. Fragment C contains the gene of the HPPD. Fragment D contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al.,

35 1984) for transcription termination. Fragment E contains the gene of the DOXS.

Example 12

40 Production of transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN)

For the production of transgenic tobacco plants that have a modified prenyl lipid content, tobacco leaf disks were transformed with 45 sequences of the DOXS and the HPPD. To transform tobacco plants, 10 ml of an overnight culture of Agrobacterium tumefaciens grown under selection were centrifuged, the supernatant was discarded and the bacteria were resuspended in the same volume of antibiotic-free medium. Leaf disks of sterile plants (diameter approx. 1 cm) were bathed in this bacterial suspension in a sterile petri dish. The leaf disks were then placed in Petri dishes on MS medium (Murashige and Skoog, Physiol. Plant (1962) 15, 473) with 2% sucrose and 0.8% Bacto agar. After 2 days incubation in the dark at 25 ° C, they were on MS medium with 100mg / l kanamycin, 500mg / l claforan, 1mg / l benzylaminopurine (BAP), 0.2mg / l naphthylacetic acid (NAA), 1.6% glucose and 0.8 % Bacto agar transferred and cultivation continued (16 hours light / 8 hours dark). Growing shoots were transferred to hormone-free MS medium with 2% sucrose, 250 mg / l Claforan and 0.8% Bacto agar.

Example 13

Production of transgenic oilseed rape plants (Brassica napus)

The production of the transgenic rapeseed plants, which have a changed prenyl lipid content, was based on a protocol by Bade, J.B. and Damm, B. (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38), in which the compositions of the media and buffers used are also given .

The transformations were carried out with the Agrobacterium tumefaciens strain LBA4404 (Clontech GmbH, Heidelberg). The binary constructs already described above with the entire cDNAs of the DOXS and the HPPD were used as binary vectors. In all binary vectors used here, the NOS terminator sequence was replaced by the polyadenylation signal of gene 3 of the T-DNA of the ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination. Brassica napus seeds were sterilized with 70% (v / v) ethanol, washed for 10 min at 55 ° C in H0, in 1% hypochlorite solution (25% v / v Teepol, 0.1% v / v Tween 20 ) incubated for 20 min and washed six times with sterile H ₂ 0 for 20 min each. The seeds were dried on filter paper for three days and 10-15 seeds were germinated in a glass flask with 15 ml of germination medium. The roots and apices were removed from several seedlings (approx. 10 cm in size) and the remaining hypocotyls were cut into pieces approx. 6 mm long. The approx. 600 explants obtained in this way are washed with 50 ml of basal medium for 30 min and transferred to a 300 ml flask. After addition of 100 ml callus induction medium, the cultures were incubated for 24 h at 100 rpm. An overnight culture of the Agrobacterium strain was 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 h at 29 ° C. up to an OD ₆ oo ^on 0.4 to 0.5 incubated. After pelleting the culture at 2000 rpm for 25 min, the cell pellet was resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution was adjusted to an ODgoo of 0.3 by adding further basal medium.

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

Transfer petri dishes and remove the medium with sterile pipettes.

For regeneration, 20-30 explants each were transferred to 90 mm petri dishes which contained 25 ml shoot induction medium with kanamycin. The petri dishes were closed with 2 layers of leucopor and incubated at 25 ° C. and 2000 lux with photoperiods of 16 hours light / 8 hours dark. Every 12 days, the developing calli were transferred to fresh petri dishes with shoot induction medium. All further steps for the regeneration of whole plants were carried out as described by Bade, J.B. and Damm, B. (described in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38).

Example 14

Increase in tocopherol biosynthesis in oilseed rape

The cDNA of DOXS (SEQ-ID No. 3) and HPPD (SEQ-ID No. 5) was provided with a CaMV35S promoter and overexpressed in rapeseed using the 35S promoter. In parallel, the seed-specific promoter of the phaseolin gene was used to specifically increase the tocopherol content in the rapeseed. Rapeseed plants transformed with the appropriate constructs were grown in the greenhouse. The α-tocopherol content of the whole plant or the seeds of the plant was then determined. In all In some cases, the α-tocopherol concentration was increased compared to the non-transformed plant.

Example 15

Cloning of a GGPPOR gene from Arabidopsis thaliana

Isolation of total RNA from fully unfolded Arabidopsis thaliana leaves:

Fully unfolded Arabidopsis thaliana leaves were harvested and frozen in liquid nitrogen. The material was then pulverized in a mortar and taken up in Z6 buffer (8 M guanidium hydrochloride, 20 mM MES, 20 mM EDTA pH 7.0). The suspension was transferred to reaction vessels and shaken with a volume of phenol / chloroform / isoamyl alcohol 25: 24: 1. After centrifugation at 15,000 rpm for 10 minutes, the supernatant was transferred to a new reaction vessel and the RNA was precipitated with 1/20 volume IN acetic acid and 0.7 volume ethanol (absolute). After centrifugation again, the pellet was first washed with 3M sodium acetate solution and after a further centrifugation in 70% ethanol. The pellet was then dissolved in DEPC water and the RNA concentration was determined photometrically.

Production of cDNA from RNA of fully unfolded leaves of A. thaliana:

20 μg of total RNA were first mixed with 3.3 μl of 3M sodium acetate solution and 2 μl of IM magnesium sulfate solution and made up to 100 μl of final volume with DEPC water. 1 μl of RNase-free DNase (Boehringer Mannheim) was added and incubated at 37 ° C. for 45 min. After removing the enzyme by shaking with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was taken up in 100 μl DEPC water. 2.5 μg RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco, Life Technologies).

From the DNA sequence of geranylgeranyl pyrophosphate oxidoreductase (Keller et al, Eur. J. Biochem. (1998) 251 (1-2), 413-417); Accession Number Y14044) were derived for a PCR oligonucleotides to which a BamHI had been added at the 5 'end and a Sall restriction site at the 3' end. The oligonucleotide at the 5 'end comprises the sequence 5' -ATGGATCCATGGCGACGACGGTTACAC C-3 'starting with the first codon of the cDNA (printed in italics), the oligonucleotide at the 3' end comprises the sequence 5 '-ATGTCGACGTGATGA- TAGATTACTAACAGAC-3 'starting with base pair 1494 of the cDNA sequence (printed in italics).

The PCR reaction was carried out using Pfu polymerase from Strategagene GmbH, Heidelberg, according to the manufacturer's instructions. 1/8 volume of the cDNA was used as template (corresponds to 0.3 μg RNA). The PCR program was:

5 cycles: 94 ° C for 4 sec, 48 ° C for 30 sec, 72 ° C for 2 min 5 cycles: 94 ° C for 4 sec, 46 ° C for 30 sec, 72 ° C for 2 min 25 cycles: 94 ° C for 4 sec, 44 ° C for 30 sec, 72 ° C for 2 min

The fragment was purified using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of the fragment was checked by sequencing (SEQ ID No. 7). The gene was cloned as a BamHI / SalI fragment into the correspondingly cut vector BinAR-Hyg by means of the restriction cut sections added to the sequence by the primers. This contains the 35S promoter of the cauliflower mosaic virus and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., EMBO J. 3 (1984), 835-846) for transcription termination. The plasmid imparts resistance to the antibiotic hygromycin in plants and is thus suitable for superinfecting plants with kanamycin resistance. Since the plastid transit peptide is

GGPPOR was also cloned, the protein should be transported to the plastids in transgenic plants. The construct is shown in Figure 14. The fragments have the following meaning:

Fragment A (529 bp) contains the 35S promoter of the Cauliflower Mosaic Virus (nucleotides 6909 to 7437 of the Cauliflower Mosaic Virus). Fragment D contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination. Fragment F contains the gene of the GGPPOR including the intrinsic plastid transit sequence.

Example 16

Production of constructs for plant transformation with DOXS and GGPPOR sequences

For the production of plants that are transgenic for the DOXS and the GGPPOR, a binary vector was prepared that contains both gene sequences (Figure 15). The GGPPOR gene with the intrinsic plastid localization sequence was (as described in Example 15) as a BamHI / Sall fragment in the corresponding cloned vector pBinAR-Hyg cloned. The gene of the DOXS was cloned as a BamHI fragment as described in Example 3. The vector pBinAR-Hyg contains the 35S promoter of the cauliflower mosaic virus and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination. This plasmid mediates resistance to the antibiotic hygromycin in plants and is therefore suitable for superinfecting plants with kanamycin resistance.

The plasmid pBinAR-TP-DOXS became the 35S promoter, the trans-ketolase transit peptide, the DOXS gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination isolated by PCR. An EcoRI site was added to each of the oligonucleotides for the promoter and the terminator sequence. The sequence of the oligonucleotide which is attached to the promoter (italics) is 5 '-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3', that of the oligonucleotide which is attached to the terminator sequence (italics) is 5 '-ATGAATTCGGACAATCAGTAAATTGAACGGA-. The fragment was cleaned using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR script vector as an EcoRI fragment into the correspondingly cut vector pBin19 (Bevan, Nucleic Acids Res. 12 (1984), 8711-8721).

The 35S promoter, the GGPPOR gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) were isolated from the plasmid pBinARHyg-GGPPOR for PCR transcription termination. An Xbal interface was added to each of the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which is attached to the promoter (italics) is 5 '-ATTCTAGACÄTG- GAGTCAAA-GATTCAAATAGA-3', that of the oligonucleotide which is attached to the terminator sequence (italics) is 5 '-ATTCTAGAGGACAA-TCAGGAGAATT 3 '. The fragment was purified using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR script vector as an Xbal fragment into the correspondingly cut vector, which already contained the sequence of the DOXS as described above. The construct pBinAR-DOXS-GGPPOR (Figure 15) was created, the fragments of which have the following meaning: Fragment A (529 bp) contains the 35S promoter of the Cauliflower Mosaic Virus (nucleotides 6909 to 7437 of the Cauliflower Mosaic Virus). Fragment B contains the transit peptide of the plastid transketolase. Fragment E contains the gene of the DOXS. Fragment D 5 contains the polyadenylation signal of gene 3 of the T-DNA of the ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination. Fragment F contains the gene of the GGPPOR including the intrinsic plastid transit sequence.

10 Example 17

Production of constructs for plant transformation with DOXS, GGPPOR and HPPD DNA sequences

15 For the production of plants that are transgenic for the DOXS, the GGPPOR and the HPPD, a binary vector was prepared that contains all three gene sequences (Figure 16). The GGPPOR gene was tagged with the intrinsic plastid localization sequence (as described in Example 15). The pBinAR-Hyg Vek-

20 tor mediates resistance to the antibiotic hygromycin in plants and is therefore suitable for superinfecting plants with kanamycin resistance.

To clone the HPPD into vectors which additionally contain another 25 cDNA, oligonucleotides were derived for a PCR, to which a BamHI restriction site had been added at the 5 'end and at the 3' end. The oligonucleotide at the 5 'end comprises the sequence 5' -GGATCCTCCAGCGGACAAGCCAAC-3 '(nucleotides 37 to 55 from the ATG in 5' direction; written in italics), the 30 oligonucleotide at the 3 'end comprises the sequence 5'-ATGGATC -

CCGCGCCGCCTACAGGTTG-3 '(ending with base pair 1140 of the coding sequence, starting 8 base pairs 3' of the TAG stop codon; written in italics). The PCR reaction was carried out using Tli polymerase from Promega GmbH, Mannheim, according to the manufacturer's instructions. 10 ng of the plasmid pBinAR-HPPD were used as the 35 template. The PCR program was:

5 cycles: 94 ° C 4 sec, 68 ° C 30 sec, 72 ° C 2 min

5 cycles: 94 ° C 4 sec, 64 ° C 30 sec, 72 ° C 2 min

40 25 cycles: 94 ° C 4 sec, 60 ° C 30 sec, 72 ° C 2 min

The fragment was purified using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of sequence 45 was checked by sequencing. It was cut out as a BamHI fragment from the vector PCR script and ligated into a correspondingly cut pBinAR vector which additionally contains the Transketolase transit peptide contains, for the introduction of the gene product in the plastids. The plasmid pBinAR-TP-p-HPPD was formed.

For cloning, the plasmid pBinAR-TP-HPPD was used to convert the 35S promoter, the transketolase transit peptide, the p-HPPD gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid PTIACH5 (Gielen et al. 1984 ) for transcription termination by means of PCR. A HindIII interface was added to each of the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which is attached to the 5 'region of the promoter (italics) is 5'-ATAAGCTT-CATGGAGTCAAA-GATTCAAATAGA-3', that of the oligonucleotide which is attached to the termination sequence (italics) is 5 ' -ATAAGCTTGGAC-AATCAGTAAATTGAACGGAG-3 '. The fragment obtained was purified using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. From this PCR script vector it was transferred as a HindIII fragment into the correspondingly cut vector pBin19 (Bevan, 1984, Nucleic Acids Res. 12, 8711-8721).

The plasmid pBinAR-TP-DOXS became the 35S promoter, the trans - ketolase transit peptide, the DOXS gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination isolated by PCR. An EcoRI site was added to each of the oligonucleotides for the promoter and the terminator sequence. The sequence of the oligonucleotide, which is attached to the promoter (italics) is 5'-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3 ', that of the oligonucleotide, which is attached to the terminator sequence (italics) is 5' -ATGAATTCGGACAATCÄGA- GTAAATTGAAC . The fragment was cleaned using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR script vector as an EcoRI fragment into the correspondingly cut vector, which already contained the sequence of the HPPD as described above.

The 35S promoter, the GGPPOR gene and the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) were isolated from the plasmid pBinARHyg-GGPPOR for transcription termination by means of PCR. An Xbal interface was added to each of the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide, which is located at the The promoter (written in italics) is 5 '-ATTCTAGACATG-GAGTCAAA-GATTCAAATAGA-3', that of the oligonucleotide which is attached to the terminator sequence (italics) is 5'-ATTCTAGAGGACAA-TCAGTAAATTGAACGGAG-3 '. The fragment was purified using a gene clean kit (Dianova GmbH, Hilden) and cloned according to the manufacturer's instructions into the vector PCR script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR script vector as an Xbal fragment into the correspondingly cut vector which already contained the sequences of the HPPD and the DOXS as described above. The construct pBinAR-DOXS-GGPPOR-HPPD (Figure 16) was created, the fragments of which have the following meaning:

Fragment A (529 bp) contains the 35S promoter of the Cauliflower Mosaic Virus (nucleotides 6909 to 7437 of the Cauliflower Mosaic Virus). Fragment B contains the transit peptide of the plastid transketolase. Fragment C contains the gene of the HPPD. Fragment D contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for transcription termination.

Fragment E contains the gene of the DOXS. Fragment F contains the gene of the GGPPOR including the intrinsic plastid transit sequence.

Example 18

Increase in tocopherol biosynthesis in oilseed rape

The cDNA of DOXS (SEQ-ID No. 3) and GGPPOR (SEQ-ID No. 7) was provided with a CaMV35S promoter and overexpressed in rapeseed using the 35S promoter. In parallel, the seed-specific promoter of the phaseolin gene was used to specifically increase the tocopherol content in the rapeseed. Rapeseed plants transformed with the appropriate constructs were grown in the greenhouse. The α-tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the α-tocopherol concentration was increased compared to the non-transformed plant.

Example 19

Increase in tocopherol biosynthesis in oilseed rape

The cDNA of DOXS (SEQ-ID No. 3), HPPD (SEQ-ID No. 5) and GGPPOR (SEQ-ID No. 7) was provided with a CaMV35S promoter and in rapeseed using the 35S promoter overexpressed. In parallel, the seed-specific promoter of the phaseolin gene was used to specifically determine the tocopherol content in the rapeseed to increase. Rapeseed plants transformed with the appropriate constructs were grown in the greenhouse. The α-tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the α-tocopherol concentration was increased compared to the non-transformed plant.

Claims

claims

1. Use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) for the production of

Plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content.

2. Use of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a hybridizing with this DNA sequence coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) for the production of plants with an increased content of tocopherols, vitamin K, chlorophylls and / or carotenoids.

3. Use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) and coding for a p-hydroxyphenylpyruvate dioxygenase (HPPD) for the production of plants with increased tocopherol, vitamin K , Chlorophyll and / or carotenoid content.

4. Use of a DNA sequence SEQ ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ-ID No. 5 or hybridizing with these DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) and a p-hydroxyphenylpyruvate dioxygenase for the production of plants with an increased content of tocopherols, vitamin K, chlorophylls and / or carotenoids.

5. Use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) and coding for a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) for the production of plants with increased tocopherol, vitamin K , Chlorophyll and / or carotenoid content.

6. Use of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ ID No. 7 or with these hybridizing DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) and a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) for the production of plants with an increased tocopherol content, vitamin K, Chlorophylls and / or carotenoids.

7. Use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS), coding for a hydroxyphenylpyruvate dioxygenase (HPPD) and coding for a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) for Her- Position of plants with increased tocopherol, vitamin K,

Chlorophyll and / or carotenoid content.

8. Use of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3, 5 of a DNA sequence SEQ-ID No. 5 and a DNA sequence SEQ-ID

No. 7 or with these hybridizing DNA sequences coding for an l-deoxy-D-xylulose-5-phosphate synthase (DOXS), a hydroxyphenylpyruvate dioxygenase (HPPD) and a geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) for the production of 10 plants with increased content on tocopherols, vitamin K, chlorophylls and / or carotenoids.

9. Process for the production of plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content,

15 characterized in that a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a DNA sequence hybridizing with this is expressed in plants.

10. Process for the production of plants with increased toco

20 pherol, vitamin K, chlorophyll and / or carotenoid content, characterized in that a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ-ID No. 5 or with these hybridizing DNA sequences are expressed in plants.

25

11. A process for the production of plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content, characterized in that a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 and a DNA sequence SEQ-ID No. 7 or with

30 these hybridizing DNA sequences are expressed in plants.

12. Process for the production of plants with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content,

35 characterized in that DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7 or with these hybridizing DNA sequences are expressed in plants.

13. A process for transforming a plant is characterized by drawing an expression cassette containing one

Promoter and a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 in a plant cell, in callus tissue, an entire plant or protoplasts of plant cells.

45 14. A method for transforming a plant, characterized in that an expression cassette containing a promoter and DNA sequences SEQ-ID No. 1 or SEQ-ID No. 3 and SEQ ID No. 5 in a plant cell, in callus tissue, an entire plant or protoplasts of plant cells.

15. A method for transforming a plant is characterized in that one contains an expression cassette

Promoter and DNA Sequences SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7- in a plant cell, in callus tissue, an entire plant or protoplasts of plant cells.

16. A method for transforming a plant, characterized in that an expression cassette containing a promoter and DNA sequences SEQ ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7 in a plant cell, in callus tissue, an entire plant or protoplasts of plant

15 cells.

17. A method for transforming plants according to claim 13-16, characterized in that the transformation with the help of the strain Agrobacterium tumefaciens, the electro

20 poration or the particle bombardment method.

18. Plant with increased tocopherol, vitamin K, chlorophyll and / or carotenoid content containing an expression cassette according to claim 13-16.

25

19. Plant according to claim, selected from the group of soybeans, canola, barley, oats, wheat, rapeseed, maize or sunflower.

20. Use of SEQ-ID No. 1 or SEQ-ID No. 3 for the production 30 of a test system for the identification of inhibitors of the

DOXS.

21. Test system based on the expression of an expression cassette according to claim 13 for the identification of

35 DOXS inhibitors.

22. Use of a plant containing a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a hybridizing with this DNA sequence for the production of plant and bacterial

40 DOXS.

45