EP1299413A2 - Homogentisatphytyl transferase - Google Patents

Homogentisatphytyl transferase

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Publication number
EP1299413A2
EP1299413A2 EP01923581A EP01923581A EP1299413A2 EP 1299413 A2 EP1299413 A2 EP 1299413A2 EP 01923581 A EP01923581 A EP 01923581A EP 01923581 A EP01923581 A EP 01923581A EP 1299413 A2 EP1299413 A2 EP 1299413A2
Authority
EP
European Patent Office
Prior art keywords
derivatives
nucleic acid
organism
homogentisate
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP01923581A
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German (de)
English (en)
Inventor
Karin Herbers
Ralf Badur
Irene Kunze
Susanne Sommer
Rainer Lemke
Michael Geiger
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SunGene GmbH
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SunGene GmbH
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Publication date
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Publication of EP1299413A2 publication Critical patent/EP1299413A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/70—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with two hydrocarbon radicals attached in position 2 and elements other than carbon and hydrogen in position 6
    • C07D311/72—3,4-Dihydro derivatives having in position 2 at least one methyl radical and in position 6 one oxygen atom, e.g. tocopherols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • the invention relates to nucleic acid sequences encoding a protein with homogentisate phytyltransferase activity, the use of the nucleic acids for the production of transgenic organisms, such as, for example, transgenic plants with an increased content of tocopherols and / or tocotrienols, a process for the production of plants with an increased content of tocopherols and tocotrienols , as well as the transgenic organisms, such as transgenic plants themselves.
  • the eight naturally occurring compounds with vitamin E activity are derivatives of 6-chromanol (Ulimann's Encyclopedia of Industrial Kitchen, Vol. A 27 (1996), VCH Verlagsgesellschaft, Chapter 4., 478-488, Vitamin E) ,
  • the group of tocopherols (la-d) has a saturated side chain
  • the group of tocotrienols (2a-d) has an unsaturated side chain:
  • vitamin E is understood to mean all eight tocopherols and tocotrienols with vitamin E activity mentioned above.
  • Vitamin E compounds are important natural fat-soluble antioxidants. A lack of vitamin E leads to pathophysiological situations in humans and animals. Vitamin E compounds are therefore of great economic value as additives in the food and feed sectors, in pharmaceutical formulations and in cosmetic applications.
  • Particularly economical processes are biotechnological processes that use proteins and biosynthetic genes from tocopherol or tocotrienol biosynthesis from organisms that produce vitamin E.
  • Figure 5 shows a biosynthetic scheme of tocopherols and tocotrienols.
  • homogentisic acid is bound to phytyl pyrophosphate (PPP) or geranylgeranyl pyrophosphate in order to form the precursors of oc-tocopherol and ⁇ -tocotrienol, the 2-methyl-phytylhydroquinone and the 2-methyl-geranylgeranylhydro.
  • PPP phytyl pyrophosphate
  • geranylgeranyl pyrophosphate in order to form the precursors of oc-tocopherol and ⁇ -tocotrienol, the 2-methyl-phytylhydroquinone and the 2-methyl-geranylgeranylhydro.
  • WO 97/27285 describes a modification of the tocopherol content by increased expression or by downregulation of the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD).
  • HPPD p-hydroxyphenylpyruvate dioxygenase
  • WO 99/04622 describes gene sequences coding for a ⁇ -tocopherolmethyltransferase from Synechocystis PCC6803 and Arabidopsis thaliana and their incorporation into transgenic plants.
  • WO 99/23231 shows that the expression of a geranylgeranyl reductase in transgenic plants results in an increased tocopherol biosynthesis.
  • the invention was based on the object of providing a further biosynthetic gene of the vitamin E biosynthetic pathway and thus further advantageous transgenic plants with an increased content of tocopherols and tocotrienols.
  • the object was achieved by finding nucleic acid sequences coding for a homogentisate phytyltransferase and by overexpressing the homogentisate phytyltransferase gene in plants.
  • the present invention relates to proteins which have the activity of a homogentisate phytyltransferase (HGPT), that is to say the ability to bind phytyl pyrophosphate to homogentisate, for example an enzymatic activity for
  • HGPT homogentisate phytyltransferase
  • Preferred 2-methyl-phytyl hydroquinones are 2-methyl-6-phytyl hydroquinone or 2-methyl-5-phytyl hydroquinone.
  • Homogentisate phytyltransferases are understood below to mean the proteins according to the invention.
  • Preferred proteins have the enzymatic activity for converting homogenate and phytyl pyrophosphate into 2-methyl-phytyl hydroquinone and contain the amino acid sequence SEQ ID NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has a homology of at least 20%, preferably 40%, preferably at least 60%, more preferably at least 80%, particularly preferably at least 90% at the amino acid level with the sequence SEQ ID NO. 2 has.
  • proteins according to the invention can be found, for example, from various organisms whose genomic sequence is known, for example from Arabidopsis thaliana, by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SEQ ID. NO. 2 easy to find.
  • the proteins according to the invention can be used as homogentisate phytyl transferases.
  • the preferred proteins are preferred for all uses of the proteins according to the invention.
  • Substitution means the replacement of one or more amino acids by one or more amino acids. So-called conservative exchanges are preferably carried out, in which the replaced amino acid has a similar property to the original amino acid, for example replacement of Glu by Asp, Gin by Asn, Val by Ile, Leu by Ile, Ser by Thr.
  • Deletion is the replacement of an amino acid with a direct link.
  • Preferred positions for deletions are the termini of the polypeptide and the links between the individual protein domains.
  • Inserts are insertions of amino acids into the polypeptide chain, whereby a direct bond is formally replaced by one or more amino acids.
  • Homology between two proteins means the identity of the amino acids over the respective total protein length, which is calculated by comparison with the aid of the program algorithm GAP (UWGCG, University of Wisconsin, Genetic Computer Group) with the following parameters:
  • a protein which has a homology of at least 20% at the amino acid level with the sequence SEQ ID.NO.2 is accordingly understood to be a protein which, when its sequence is compared with the sequence SEQ'-ID NO.2 according to the above program algorithm
  • the above parameter set has a homology of at least 20%.
  • the homogentisate phytyltransferases according to the invention are capable of converting ho ogentisate derivatives and phytyl pyrophosphate derivatives into 2-methyl-phytyl hydroquinone derivatives and / or homogentisate derivatives and geranyl-geranyl pyrophosphate derivatives into 2-methyl-geranyl-geranyl hydroquinone derivatives.
  • Homogentisate derivatives are understood to mean homogentisate and homogentisate compounds derived therefrom, which are accepted as substrates by the homogentisate phytyltransferases according to the invention.
  • Phytyl pyrophosphate derivatives are understood to mean phytyl pyrophosphate and phytyl pyrophosphate compounds derived therefrom, which are accepted as substrates by the homogeneous phytyl transferases according to the invention.
  • 2-methyl-phytylhydroquinone derivatives are understood to mean the resulting compounds of the enzymatic conversion, such as, for example, 2-methyl-phytylhydroquinone and the corresponding derived compounds.
  • Preferred 2-methyl-phytyl hydroquinone derivatives are derivatives of 2-methyl-6-phytyl hydroquinone or 2-methyl-5-phytyll hydroquinone.
  • Geranyl-geranyl pyrophosphate derivatives include geranyl-geranyl pyrophosphate and derivatives thereof. Geranyl-Geranyl-pyrophosphate compounds understood, the-i ⁇ pn the Homogentisatphytyltransferasen according to the invention as ⁇ ; Substrate are accepted.
  • 2-methyl-geranylgeranyl hydroquinone derivatives are understood to mean the resulting compounds of the enzymatic reaction, such as, for example, 2 ethyl-geranylgeranyl hydro ⁇ hinone and the corresponding derived compounds.
  • Preferred 2-methyl-geranylgeranyl hydroquinones are 2-methyl-6-geranylgeranyl hydroquinone or 2-methyl-5-geranylgeranyl hydroquinone.
  • Preferred 2-methyl-geranylgeranyl hydroquinone derivatives are derivatives of 2-methyl-6-geranylgeranylhydroquinone or 2-methyl-5-geranylgeranylhydroquinone.
  • the invention relates to a "process for biotransformation, characterized in that homogentisate derivatives and phytyl pyrophosphate derivatives in 2-methyl-phytylhydroquinone derivatives or homogentisate derivatives.
  • Geranyl-geranyl pyrophosphate derivatives in 2-methyl-geranyl ge anylhydroquinone Transferred in the presence of a homogentisate phytyl transferase according to the invention.
  • the biotransformation can in principle be carried out with whole cells which express the enzyme HGPT or cell extracts from these cells or with purified or highly pure HGPT.
  • the homogentisate phytyl transferase can also be present in free or in immobilized form.
  • the homogentisate phytyltransferases according to the invention can also be used for the production of vitamin E.
  • the enzymatic biosynthesis step of the homogeneous phytyl transferases can be carried out in vitro or, as described below, in vivo, for example in transgenic organisms, for example in transgenic plants.
  • the invention relates to a process for the preparation of vitamin E, characterized in that homogenate derivatives and phytyl pyrophosphate derivatives. converted into 2-methyl-phytylhydroquinone derivatives or homogentisate derivatives and geranyl-geranyl pyrophosphate derivatives into 2-methyl-geranylgeranylhydroquinone derivatives in the presence of homogentisate phytyl transferase according to the invention.
  • the vitamin E biosynthetic pathway continues to provide target enzymes for the development of inhibitors. Since, according to the current state of the art, there is no enzyme identical or similar to the Synechocystis HGPT in human and animal organisms, it can be assumed that inhibitors have a very specific effect on plants.
  • the invention therefore also relates to the use of the homogentisate phytyltransferase according to the invention as a herbicidal target for finding inhibitors of homogentisate phytyltransferase.
  • the HGPT is a target for herbicides.
  • the complete cDNA sequence of the HGPT from Synechocystis is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.
  • the HGPT protein expressed with the aid of the expression cassette according to the invention is particularly suitable for the detection of inhibitors specific for the HGPT.
  • the invention relates to a method for finding inhibitors of homogeneous phytyltransferase, characterized in that the enzymatic activity of the homogeneous measures tisate phytyl transferase in the presence of a chemical compound and, when the enzymatic activity is reduced compared to the uninhibited activity, the chemical compound is an inhibitor.
  • the HGPT can be used, for example, in an enzyme test in which the activity of the HGPT 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.
  • test system With the help of the test system according to the invention, a large number of chemical compounds can be checked quickly and easily for herbicidal 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.
  • the invention therefore furthermore relates to herbicidal active ingredients which can be identified using the test system described above.
  • homogentisate phytyltransferases according to the invention can be produced from natural or genetically modified organisms, as described below, by gene expression of the corresponding nucleic acids which encode these proteins.
  • the invention furthermore relates to nucleic acids, hereinafter called homogentisate phytyltransferase genes (HPGT genes), which encode the proteins according to the invention described above.
  • HPGT genes homogentisate phytyltransferase genes
  • the nucleic acid sequence can be, for example, an RNA, DNA or cDNA sequence. Coding sequences suitable for insertion into a nucleic acid construct, such as, for example, an expression cassette, are, for example, those which code for an HGPT and which give the host the ability to overproduce tocopherols and / or tocotrienols.
  • Suitable nucleic acid sequences can be obtained by back-translating the polypeptide sequence according to the genetic code.
  • codons are preferably used for this that are frequently used according to the organism-specific codon usage.
  • the codon usage can be based on computer evaluations easily identify other known genes of the organism in question.
  • the protein is to be expressed in a plant, it is often advantageous to use the plant's codon usage for the back translation.
  • Preferred nucleic acids encode a vegetable homogenate phytyltransferase or a homogenate phytyltransferase from cyanobacteria.
  • the sequence SEQ ID NO has a particularly preferred nucleic acid. 1.
  • This nucleic acid is a prokaryotic genomic DNA from the cyanobacterium Synechocystis sp. PCC6803, which the Homogentisatphytyltransferase of the sequence SEQ ID NO. 2 coded.
  • All of the above-mentioned homogentisate phytyltransferase genes can be prepared in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the attachment of synthetic oligonucleotides and the filling of gaps using the Klenow fragment of DNA polymerase and ligation reactions as well as general ionization methods are described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
  • the invention further relates to the use of the HGPT according to the invention or the HGPT genes according to the invention for the production of antibodies.
  • the invention further relates to nucleic acid constructs containing one of the above-described homogentisate phytyltransferase genes which are functionally linked to one or more regulation signals which ensure transcription and translation in prokaryotic or eukaryotic organisms.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences can be carried out before the actual structural genes are still present and may have been genetically modified so that natural regulation has been switched off and the expression of the genes has been increased.
  • the nucleic acid construct can also have a simpler structure, that is to say no additional regulation signals are inserted in front of the above-mentioned homogentisate phytyltransferase genes and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased.
  • These modified promoters can also be placed in front of the natural genes to increase activity.
  • the nucleic acid construct can also advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences.
  • the above-mentioned homogentisate phytyltransferase genes can be contained in one or more copies in the gene construct.
  • Nucleic acid constructs are preferably used which enable the expression of the homogentisate phytyltransferase gene according to the invention in a host cell, hereinafter also called the expression cassette.
  • the expression cassettes contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell.
  • 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 functionally linked to the intervening coding sequence for the homogentisate phytyltransferase gene.
  • a functional link means the sequential arrangement of promoter, 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 mitochondrial, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or other compartments and translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).
  • Advantageous regulatory sequences for the nucleic acid constructs according to the invention, for the method for producing vitamin E described below and for the genetically modified organisms described below are found, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac-, lpp-lac-, laclq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, 1-PR- or in the 1-PL promoter, which advantageously in gram-negative bacteria Find application.
  • promoters such as cos, tac, trp, tet, trp-tet, lpp, lac-, lpp-lac-, laclq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, 1-PR- or in the 1-PL promoter, which advantageously in gram-negative bacteria Find application.
  • promoters ay and SP02 are found, for example, in the gram-positive promoters ay and SP02, in the yeast or fungal promoters ADCl, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al., 1980, Cell 21: 285-294], PRP1 [Ward et al. , Plans. Mol. Biol. 22 (1993)], SSU, OCS, leb4, usp, STLSl, B33, nos or in the ubiquitin or phaseolin promoter.
  • any promoter which can control the expression of foreign genes in plants is in principle suitable as a promoter of the expression cassette.
  • a plant promoter or a plant virus-derived promoter is preferably used.
  • the CaMV 35S promoter from the Bluenkohl Mosaic Virus is particularly preferred (Franck et al., Cell 21 (1980), 285-294).
  • 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 pathogen or chemically inducible promoter, by means of which the expression of the exogenous homogentisate phytyltransferase gene in the plant can be controlled at a specific point in time.
  • Such promoters as for example the PRPL promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/19443), a by benzene sulfonamide-inducible (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 by ethanol - or cyclohexanone-inducible (WO 93/21334) promoter can be used for example.
  • promoters are particularly preferred which ensure expression in tissues or parts of plants in which, for example, the biosynthesis of tocopherol or its precursors takes place or in which the products are advantageously accumulated.
  • promoters for the whole plant based on constitutive expression such as the CaMV promoter, the OCS promoter from Agrobacterium (octopine synthase), the NOS promoter from Agrobacterium (nopalin synthase), the ubiquitin promoter, promoters of vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (wheat WO 9113991)
  • Promoters which ensure leaf-specific expression are also to be mentioned in particular. These include the promoter of the cytosolic FBPase from potatoes (WO9705900), the SSU promoter (small subunit) from Rubisco (ribulose-1, 5-bisphosphate carboylase) or the ST-LSI promoter from potatoes (Stockhaus et al., EMBO J. 8: 2445-245 (1989).
  • patatin promoter class I B33
  • GBSS1 starch synthase
  • fruit-specific promoters such as the fruit-specific promoter from tomato (EP409625),
  • fruit ripening-specific promoters such as the fruit ripening-specific promoter from tomato (WO 9421794),
  • flower-specific promoters such as the phytoene synthase promoter (W09216635) or the promoter of the P-rr gene (W09822593) or
  • RNA polymerase promoter W09706250
  • the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max see also Genbank Accession number U87999
  • another node-specific promoter as in EP 249676 can also be used advantageously.
  • the plant expression cassette can be installed in a derivative of the transformation vector pBin-19 with 35s promoter (Bevan, M., Nucleic Acids Research 12: 8711-8721 (1984)).
  • Figure 2 shows a derivative of the transformation vector pBin -19 with seed-specific legu in B4 promoter.
  • the expression cassette can be, for example, a seed-specific promoter (preferably the phaseolin promoter (US 5504200), the USP- (Baumlein, H. et al., Mol. Gen. Genet. (1991) 225 (3), 459-467) Bce4 gene promoter from Brassica (WO 9113980) or LEB4 promoter (Fiedler and Conrad, 1995)), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
  • a seed-specific promoter preferably the phaseolin promoter (US 5504200), the USP- (Baumlein, H. et al., Mol. Gen. Genet. (1991) 225 (3), 459-467) Bce4 gene promoter from Brassica (WO 9113980) or LEB4 promoter (Fiedler and Conrad, 1995)
  • the LEB4 signal peptide the gene to be expressed and an ER retention signal.
  • An expression cassette is produced, for example, by fusing a suitable promoter with a suitable HGPT-DNA sequence and preferably a DNA inserted between the promoter and HGPT-DNA sequence, which codes for a chloroplast-specific transit peptide, and a polyadenylation signal according to common recombination methods. 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.
  • Expression cassettes can also be used, the DNA sequence of which codes for an HGPT fusion protein, for example, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
  • Preferred transit peptides are preferred for the chloroplasts. location of the HGPT gene in the chloroplasts from the HGPT part are enzymatically cleaved.
  • the transit peptide which is derived from the plastid Nicotiana tabacum Transketolase or another transit peptide (for example the transit peptide of the small subunit of Rubisco or the ferredoxin NADP oxidoreductase as well as the isopentenyl pyrophosphate isomerase-2) or its functional equivalent is particularly preferred.
  • DNA sequences of three cassettes of the plastid transit peptide of plastid transketolase from tobacco in three reading frames are particularly preferred as Kpnl / BamHI fragments with an ATG codon in the Ncol interface:
  • the inserted nucleotide sequence coding for an HGPT can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components, as well as consist of different heterologous HGPT gene sections of different organisms.
  • 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.
  • 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.
  • adapters or linkers can be attached to the fragments.
  • the promoter and the terminator regions can expediently in the transcription direction with a linker or polylinker which has one or more restriction sites for the
  • 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, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter can be native or homologous as well as foreign or heterologous to the host plant.
  • the expression cassette contains the promoter in the 5 '-3' transcription direction, a DNA sequence which codes for an HGPT gene and a region for the transcriptional termination. Different termination areas are interchangeable.
  • 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.
  • the fused expression cassette which codes for an HGPT 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, for example, tobacco plants, for example by wounded leaves or Leaf pieces are bathed in an agrobacterial solution and then cultivated in suitable media.
  • the transformation of plants by agrobacteria is known, inter alia, from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press, 1993, pp. 15-38.
  • transgenic plants can be regenerated from the transformed cells of the wounded leaves or leaf pieces, which plants contain a gene integrated into the expression cassette for the expression of an HGPT gene.
  • the nucleic acid constructs according to the invention can be used for the production of genetically modified organisms.
  • the genetically modified organisms are produced by transforming the host organisms, hereinafter also referred to as parent organisms, with a construct containing the HGPT gene.
  • Starting or host organisms are understood to mean prokaryotic or eukaryotic organisms, such as, for example, microorganisms, mosses or plants.
  • Preferred microorganisms are bacteria, yeast, algae or fungi.
  • Preferred bacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or cyano-bacteria of the genus Synechocystis.
  • Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.
  • Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6 described mushrooms.
  • Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.
  • the invention relates to a genetically modified organism, the genetic modification being the gene expression of a nucleic acid according to the invention compared to a wild type
  • the transgenic organisms containing the HGPT gene according to the invention are capable of converting homogentisate derivatives and phytylpyrophosphate derivatives into 2-methyl-phytylhydroquinone derivatives and / or homogentisate derivatives and geranyl-geranyl pyrophosphate derivatives into 2-methyl-geranylgeranylhydroquinone derivatives.
  • Transgenic organisms containing an exogenous HGPT gene according to the invention which already have the biosynthesis genes for the production of vitamin E as starting organisms, such as plants or other photosynthetically active organisms such as cyanobacteria, mosses or algae, have an increased content of tocopherols and / or tocotrienols compared to the respective wild type.
  • the invention therefore relates to such a genetically modified organism according to the invention which has an increased vitamin E content compared to the wild type.
  • the present invention further relates to the use of the HGPT or the HGPT genes according to the invention for the production of vitamin E in transgenic organisms.
  • Genetically modified organisms according to the invention preferably plants which can have an increased vitamin E content compared to the wild type, can be used to produce vitamin E.
  • the present invention therefore also relates to processes for the production of vitamin E by cultivating a genetically modified organism according to the invention, preferably a genetically modified plant according to the invention, which has an increased vitamin E content compared to the wild type
  • Organism is harvested and the vitamin E compounds are then isolated from the organism.
  • Genetically modified plants according to the invention with increased vitamin E content that can be consumed by humans and animals can also be used, for example, directly or after preparation known per se as food or feed.
  • plants are used to produce organisms with an increased vitamin E content (tocopherols and / or tocotrienols) compared to the wild type used as starting organisms and, accordingly, as genetically modified organisms.
  • vitamin E content tocopherols and / or tocotrienols
  • Preferred plants are, for example, tagetes, sunflower, arabidopsis, tobacco, red pepper, soy, tomato, eggplant, paprika, carrot, carrot, potato, corn, salads and cabbages, cereals, alfalfa, oats, barley, rye, wheat, triticale, millet , Rice, alfalfa, flax, cotton, hemp, brassicaca such as rape or canola, sugar beet, sugar cane, nut and wine species or woody plants such as aspen or yew.
  • Arabidopsis thaliana Tagetes erecta, Brassica napus, Nicotiana tabacum, canola, potatoes and oilseeds such as soybeans are particularly preferred.
  • the invention further relates to a method for producing genetically modified organisms in which a nucleic acid or a nucleic acid construct according to the invention is introduced into the genome of the starting organism.
  • an expression cassette is inserted as an insert into a recombinant vector, the vector DNA of which may preferably contain additional functional regulatory signals, for example sequences for replication or integration.
  • Suitable vectors for plants are inter alia in "Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Chap. 6/7, pp. 71-119 (1993).
  • 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, M13mp 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 a DNA sequence hybridizing therewith for transforming plants, cells, tissues or parts of plants.
  • the aim of the use is preferably to increase the tocopherols and / or tocotrienols content of the plant.
  • the expression can take place specifically in the leaves, in the seeds, petals or 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 expression cassette can also be used to transform bacteria, cyanobacteria, yeast, filamentous fungi, mosses and algae with the aim of increasing the tocopherol and / or tocotrienol content.
  • transformation 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, published by S.D. Kung and R. Wu, Academic Press (1993), 128-143 and in Potrykus, Annu. Rev.
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium 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, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • Increasing the content of tocopherols or tocotrienols in the context of the present invention means the artificially acquired ability of an increased biosynthetic performance of these compounds by functional overexpression of an HGPT gene according to the invention in the plant compared to the non-genetically modified plant.
  • the content of tocopherols or tocotrienols can be increased.
  • the tocopherol content is preferably increased. But it is also possible under certain conditions to preferably increase the tocotrienol content.
  • the biosynthesis site of tocopherols is the leaf tissue, so that leaf-specific expression of the HGPT gene is useful.
  • the tocopherol biosynthesis need not be restricted to the leaf tissue, but can also be tissue-specific in all other parts of the plant - especially in fatty seeds.
  • constitutive expression of the exogenous HGPT gene is advantageous.
  • inducible expression may also appear desirable.
  • the effectiveness of the expression of the transgenically expressed HGPT gene can be determined, for example, in vitro by multiplication of the shoot meristem.
  • a change in the type and level of expression of the HGPT gene and its effect on the tocopherol biosynthesis performance on test plants can be tested in greenhouse experiments.
  • the invention also relates to transgenic plants, transformed with an expression cassette containing an HGPT gene according to the invention, and transgenic cells, tissues, parts and propagation material of such plants.
  • transgenic plants such as, for example, tagetes, sunflower, arabidopsis, tobacco,
  • Plants in the sense of the invention are mono- and dicotyledonous plants.
  • the invention further relates to further photosynthetically active organisms transformed with an expression cassette containing an HGPT gene according to the invention.
  • the invention therefore relates to a genetically modified organism according to the invention, preferably a plant which is genetically modified according to the invention and which is resistant to inhibitors of homogeneous phytyltransferase.
  • the present invention succeeds, for example, in increasing the activity of homogentisate phytyltransferase (HGPT) in transgenic plants by overexpressing the HGPT gene according to the invention. In principle, this can be achieved by expressing homologous or heterologous HGPT genes.
  • HGPT homogentisate phytyltransferase
  • Example 1 the cloning of an HGPT DNA sequence (SEQ ID No. 1) from Synechocystis spec. PCC 6803. To ensure plastid localization, the HGPT nucleotide sequence from Synechocystis is preceded by a transit signal sequence (Fig. 1-4).
  • HGPT gene according to the invention for the production of plants with an increased content of tocopherols and / or tocotrienols by expression of a HGPT DNA sequence in plants.
  • the DNA coding for the ORF slrl736 was obtained using polymerase chain reaction (PCR) from Synechocystis spec. PCC 6803 according to the method according to Crispin A. Howitt (BioTechniques 21: 32-34, July 1996) using a sense-specific primer (slrl7365 ' Figure 8, SEQ ID NO. 3) and an antisense-specific primer (slrl7363', Figure 9 , SEQ ID NO. 4).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • Step 1 5 minutes 94 ° C (denaturation) Step 2 3 seconds 94 ° C
  • Step 3 2 minutes 48 ° C (annealing)
  • Step 4 2 minutes 72 ° C (elongation) 35 repetitions of steps 2-4
  • Step 5 10 minutes 72 ° C (post-elongation)
  • Step 6 4 ° C (hold)
  • the amplicon was cloned into the PCR cloning vector pGEM-T (Promega) using standard methods. The identity of the amplicon generated was confirmed by sequencing using the M13F (-40) primer.
  • the vector pGEM-T / slrl736 was digested using the restriction enzyme Hpal. This digest deletes a 348 bp internal fragment of the slrl736.
  • the aminoglycoside-3 'phosphotransferase of the transposon Tn903 was then cloned into the Hpal cleavage sites.
  • the Tn903 was isolated as an EcoRI fragment from the vector pUC4k (Vieira, J and Messing, J., Gene: 19, 259-268, 1982), the protruding ends of the restriction digest were converted into blunt ends by standard methods and into the Hpal cut vector pGEM-T / slrl736 ligated.
  • the ligation approach was used to transform E. coli Xll blue cells. Transformants were selected using kanamycin and ampicillin. A recombinant plasmid (pGEM-T / slrl736:: tn903, see Fig. 6) was isolated and used to transform Synechocystis spec. PCC 6803 used according to the Williams method (Methods Enzymol. 167: 776-778, 1987).
  • Figure 6 shows a construct for knock-out mutagense of ORF slrl736 in Synechocystis spec. PCC 6803.
  • Synechocystis spec. PCC 6803 transformants were selected on (km) BG-11 solid medium containing kanamycin (Castenholz, Methods in Enzymology, 68-93, 1988) at 28 ° C. and 30 ⁇ mol photons x (m 2 xs) -1 .
  • Four independent knock out mutants were generated after five rounds of selection (passages from individual colonies to fresh BG-11 km medium).
  • the biochemical analyzes could thus be carried out using three independently grown cultures of a mutant and the corresponding wild types.
  • the medium of the cell culture was removed by centrifugation twice at 14000 rpm in an Eppendorf table centrifuge.
  • the subsequent digestion of the cells was carried out by four incubations in an Eppendorf shaker at 30 ° C., 1000 rpm in 100% methanol for 15 minutes, the supernatants obtained in each case being combined. Further incubation steps resulted in no further release of tocopherols or tocotrienols.
  • the extracts obtained were analyzed immediately after extraction using a Waters Allience 2690 HPLC system. Tocopherols and tocotrienols were separated on a reverse phase column (ProntoSil 00-3-C30, Bischoff) with a mobile phase of 100% methanol and identified using standards (Merck). The fluorescence of the substances (excitation 295 nm, emission 320 nm) was used as the detection system, which was detected with the aid of a Jasco FP 920 fluorescence detector.
  • the hypothetical protein slr1736 from Synechocystis spec. PCC 6803 was identified by functional expression in E. coli as homogentisate phytyltransferase.
  • the from Synechocystis spec. PCC 6803 amplified gene slrl736 was subcloned in the correct reading frame into the expression vector pQE-30 (Qiagen).
  • the amplification of OFR slrl736 from Synechocystis spec. PCC 6803 primers slrl7365 'and slrl7363' (SEQ. ID. Nos.
  • the slrl736 fragment was isolated from the recombinant plasmid pGEM-T / slrl736 using these flanking BamHI restriction sites and ligated into a BamHI cut pQE-30 using standard methods.
  • the ligation batch was used to transform Ml5 E. coli cells and kanamycin and ampicillin resistant transformants were analyzed. Kanamycin resistance is mediated by the pREP-4 plasmid contained in the Ml5 cells.
  • a recombinant plasmid (pQE-30 / slrl736) which carried the slrl736 fragment in the correct orientation was isolated. The identity and orientation of the insert was confirmed by sequencing.
  • the recombinant plasmid pQE-30 / slrl736 was used to transform M15 E. coli cells to produce recombinant slrl736 protein.
  • an overnight culture in Luria Broth Medium was inoculated with 200 ⁇ g / ml ampicillin (Amp) and 50 ⁇ g / ml kanamycin (Km).
  • Amp ampicillin
  • Km 50 ⁇ g / ml kanamycin
  • a 100 ml Luria Broth culture (Amp / Km) was inoculated the next morning. This culture was incubated at 28 ° C. on a shaking incubator until an ODgoo: 0, 35-0, 4 was reached.
  • the production of the recombinant protein was then induced by adding 0.4 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the pellet was resuspended in 600 ⁇ l lysis buffer (approx. 1-1.5 ml / g pellet wet weight, 10 mM HEPES KOH pH 7.8, 5 mM dithiothreinol (DTT), 0.24 M sorbitol). Then PMSF (phenyl methyl sulfonate) was added to a final concentration of 0.15 mM and the mixture was placed on ice for 10 minutes. The cells were disrupted by a 10-second ultrasound pulse using an ultrasound rod. After adding Triton X100 (final concentration 0.1%), the cell suspension was incubated on ice for 30 minutes. The mixture was then centrifuged at 25,000 xg for 30 minutes and the supernatant was used for the assay. The activity of the homogentisate phytyltransferase is determined by detecting radioactively labeled 2-methyl-phytylhydroquinone as a reaction product.
  • PMSF phenyl methyl sulfonate
  • 235 ⁇ l of the enzyme (approx. 300-600 ⁇ g) together with 35 ⁇ l phytyl pyrophosphate and 50 ⁇ l (1.2 nmol) 3 H-homogentisic acid were added in the following reaction buffer: 100 ⁇ l (250mM) tricine-NaOH pH 7.6 , 100 ⁇ l (1.25 mM) sorbitol, 10 ⁇ l (50 mM) MgCl 2 and 20 ⁇ l (250 mM) ascorbate for 4 hours at 25 ° C.
  • the tritium-labeled homogentisic acid is present in an ethanolic solution with 1 mg ascorbate / ml. 50 ⁇ l of this are concentrated and the buffer, the enzyme and the phytyl pyrophosphate are added.
  • the reaction was stopped by extracting the mixture twice with ethyl acetate.
  • the ethyl acetate phases were concentrated and the residues were taken up in methanol and applied to a thin-layer plate for chromatographic separation of the substances (solid phase: HPTLC plates: silica gel 60 F 254 (Merk), liquid phase: toluene).
  • the radioactively labeled reaction product is detected by using a phospho-agent.
  • PCC 6803 encoded protein is a homogentisate phytyltransferase because it has the enzymatic activity to form 2-methyl-phytylhydroquinone from homogentisates and phytyl pyrophosphate.
  • Transgenic plants were generated which contain the homogeneous phytyltransferase from Synechocystis spec.
  • PCC 6803 on the one hand under the control of the constitutive 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., Cell 21: 285-294, 1980) and on the other hand under the control of the seed-specific promoter of the legumin gene from Vicia faba (Kafatos et al., Nuc. Acid. Res., 14 (6): 2707-2720, 1986).
  • Plasmid generated by PCC 6803 was pBinAR-TkTp-9 (Ralf Badur, Dissertration University of Göttingen, 1998).
  • This vector is a derivative of the pBinAR (Höfgen and Willmitzer, Plant Sei. 66: 221-230, 1990) and contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., 1980) the termination signal of the octopine synthase gene ( Gielen et al., EMBO J. 3: 835-846, 1984) and the DNA encoding the transit peptide of Nicotiana tabacum plastidic transketolase Sequence.
  • CaMV cauliflower mosaic virus
  • the gene slrl736 was isolated from the plasmid pGEM-T / slrl736 using the flanking BamHI restriction sites. This fragment was ligated into a BamHI cut pBinAR-TkTp-9 using standard methods (see Figure 1). This plasmid (pBinAR-TkTp-9 / slrl736) was used to generate transgenic Nicotiana tabacum plants.
  • Fragment A (529 bp) in Figure 1 contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus), fragment B (245 bp) fragment encodes the transit peptide of the Nicotiana tabacum transketolase, fragment C (944Bp) encodes ORF slrl736 from Synechocystis spec. PCC 6803, fragment D (219 bp) codes for the termination signal of the octopine synthase gene.
  • the seed-specific promoter of the Legumin B4 gene (Kafatos et al., Nuc. Acid. Res., 14 (6): 2707-2720, 1986) was used.
  • the 2.7 Kb fragment of the legumin B4 gene promoter was isolated from the plasmid pCR-Script / lePOCS using the EcoRI and the 3 'flanking Kpnl cleavage sites.
  • the plasmid pBinAR-TkTp-9 / slrl736 was also treated with the restriction enzymes EcoRI and Kpnl. As a result, the CaMV 35S promoter was separated from this plasmid. The promoter of the legumin gene was then cloned into this vector as an EcoRI / Kpnl fragment, whereby a plasmid was generated which put the expression of the gene slrl736 under the control of this seed-specific promoter, see Figure 2.
  • This plasmid (pBinARleP-TkTp-9 / slrl736) was used to generate transgenic Nicotiana tabacum plants.
  • Fragment A (2700 bp) in Figure 2 contains the promoter of the legumin B4 gene from Vicia faba, fragment B (245bp) encodes the transit peptide of the Nicotiana tabacum transketolase, fragment C (944Bp) encodes the ORF slrl736 from Synechocystis spec. PCC 6803, fragment D (219 bp) for the termination signal of the octopine synthase gene. Generation of DNA constructs for the expression of the homogenate phytyltransferase from Synechocystis spec. PCC 6803 in A. thaliana and B. napus.
  • chimeric DNA constructs for the production of transgenic A. thaliana or B.napus plants, which the Homogentisatphytyltransferase from Synechocystis spec. Express PCC 6803, the vectors pPTVkan35S-IPP-Tp-90CS and pPTVkanLeP-IPP-Tp-IONOS were used. These vectors are derivatives of pGPTVkan (D.Becker, E. Kemper, J. Schell, R. Masterson. Plant Molecular Biology 20: 1195-1197, 1992) to which the uidA gene has been deleted.
  • the pPTVkan35S-IPP-Tp-90CS contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., 1980), the sequence coding for the transit peptide of A. thaliana plastid-specific isopentenyl pyrophosphate isomerase-2 (IPP-2) (Badur, unpublished) and the termination signal of the octopine synthase gene (Gielen et al., 1984)
  • the vector pPTVkanLeP-IPP-Tp-lOnos contains the seed-specific promoter of the Legumin B4 gene (Kafatos et al., Nuc. Acid.
  • Fragment A (529 bp) in Figure 3 contains the 35S ⁇ promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus).
  • Fragment B (205 bp) fragment coding for the transit peptide of A. thaliana isopentenyl pyrophosphate isomerase-2.
  • Fragment C (205 bp) fragment coding for the transit peptide of A. thaliana isopentenyl pyrophosphate isomerase-2.
  • Fragment A (2700 bp) in Figure 4 contains the promoter of the legumin B4 gene from Vicia faba, fragment B (206 bp) fragment coding for the transit peptide of A. thaliana isopentenylpyrophosphate isomerase-2.
  • Fragment C (944Bp) encodes the ORF slrl736 from Synechocystis spec. PCC 6803.
  • Fragment D (272 bp) for the termination signal of the nopaline synthase gene.
  • Wild-type Arabidopsis thaliana plants (Columbia) were transformed with the Agrabacterium tumefaciens strain (GV3101 [pMP90]) on the basis of a modified vacuum filtration method (Steve Clough and Andrew Bent. Floral dip: a simplified method for Agrobacterium mediated transformation of A. thaliana. Plant J 16 (6): 735-43, 1998; der Bechtold, N. Ellis, J. and Pelltier, G., in: Planta Agrobacterium-ediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CRAcad Sei Paris, 1993, 1144 (2 ): 204-212).
  • the Agrobacterium tumefaciens cells used had previously been transformed with the plas iden pPTVkan35SIPP-Tp9 / slrl736 or pPTVkanLePIPP-Tp9 / slrl736 ( Figures 3 and 4).
  • Seeds of the primary transformants were selected based on antibiotic resistance. Antibiotic-resistant seedlings were planted in soil and used as fully developed plants for biochemical analysis.
  • transgenic oilseed rape plants were based on a protocol by Bade, J.B. and dam, B. (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer
  • the transformations were carried out with the Agrobacterium tumefaciens strain GV3101 [pMP90].
  • the plasmids pPTVkan35SIPP-Tp9 / slrl736 and pPTVkanLePIPP-TplO / slrl736 were used for the transformation ( Figures 3 and 4).
  • Brassica napus var. Westar seeds were surface sterilized with 70% ethanol (v / v), washed in water for 10 minutes at 55 ° C, in 1% hypochlorite solution (25% v / v tea pol, 0.1% v / v Tween 20) incubated for 20 minutes and washed six times with sterile water for 20 minutes each.
  • the seeds 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 approximately 600 explants obtained in this way were washed with 50 ml of basal medium for 30 minutes transferred to a 300 ml flask. After adding 100 ml of callus induction medium, the cultures were incubated for 24 hours 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 of Luria Broth medium without kanamycin for 4 hours at 29 ° C. until an ODßoo of Incubated 0.4-0.5. After pelleting the culture at 2000 rpm for 25 min, the cell pellet was resuspended in 10 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 15 sterile pipettes, 50 ml of Agrobacterium solution were added, mixed gently and incubated for 20 min. The agrobacterial suspension was removed, the oilseed rape explant was washed for 1 min with 50 ml of callus induction medium and then 100 ml of callus induction medium was added.
  • the co-cultivation 20 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 25 was transferred to 15 cm petri dishes and the medium was removed with sterile pipettes.
  • 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. The developing calli were transferred to fresh petri dishes with shoot induction medium every 12 days. All further
  • the wild-type plants from sterile culture were obtained by vegetative replication. For this purpose, only the tip of the plant was cut off and transferred to fresh 2MS medium in a sterile mason jar. The hair on the top of the leaf and the midribs of the leaves were removed from the rest of the plant. The leaves were cut into 2 large pieces with a razor blade. The agrobacterial culture was transferred to a small petri dish (2 cm in diameter). The leaf pieces were briefly drawn through this solution and the underside of the leaf was placed on 2MS medium in Petri dishes (diameter 9 cm) so that they touched the medium. After two days in the dark at 25 ° C, the explants were transferred to plates with callus induction medium and heated to 28 ° C in the climatic chamber.
  • the medium had to be changed every 7-10 days. As soon as calli formed, the explants were placed in sterile mason jars on shoot induction medium with Claforan (0.6% BiTec agar (g / v), 2.0 mg / 1 zeatin ribose,
  • Organogenesis occurred after about a month and the shoots formed could be cut off.
  • the shoots were cultivated on 2MS medium with Claforan and a selection marker. As soon as a strong root ball had formed, the plants could be potted in prickly soil.
  • the vitamin E biosynthesis is influenced in the transgenic plants
  • the tocopherol and tocotrienol contents in leaves and seeds of the plants transformed with the described constructs are analyzed.
  • the transgenic plants are cultivated in the greenhouse and plants which code the gene for the homogenate phytyltransferase from Synechocystis spec.
  • PCC 6803 express analyzed at Northern level. The tocopherol content and the tocotrienol content are determined in the leaves and seeds of these plants. In all cases, the tocopherol or tocotrienol concentration in transgenic plants which additionally express a nucleic acid according to the invention is increased in comparison to plants which have not been transformed.

Abstract

La présente invention concerne des séquences d'acide nucléique codant pour une protéine ayant une activité d'homogentisatphytyl transférase, l'utilisation des acides nucléiques pour produire des organismes transgéniques tels que des végétaux transgéniques ayant une teneur élevée en tocophérols et tocotriénols, un procédé de production de végétaux transgéniques ayant une teneur élevée en tocophérols et/ou tocotriénols, ainsi que les végétaux transgéniques eux-mêmes.
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WO2000068393A1 (fr) 1999-05-07 2000-11-16 Pioneer Hi-Bred International, Inc. Acides nucleiques de transferase phytyle/prenyle, polypeptides, et leur utilisation
US6872815B1 (en) 2000-10-14 2005-03-29 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
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US7161061B2 (en) 2001-05-09 2007-01-09 Monsanto Technology Llc Metabolite transporters
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WO2003016482A2 (fr) 2001-08-17 2003-02-27 Monsanto Technology Llc Genes de methyltransferase et leurs utilisations
DE60235252D1 (de) 2001-10-25 2010-03-18 Monsanto Technology Llc Aromatische methyltransferasen und ihre verwendung
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