WO2000008169A1 - Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes - Google Patents

Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes Download PDF

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WO2000008169A1
WO2000008169A1 PCT/EP1999/005467 EP9905467W WO0008169A1 WO 2000008169 A1 WO2000008169 A1 WO 2000008169A1 EP 9905467 W EP9905467 W EP 9905467W WO 0008169 A1 WO0008169 A1 WO 0008169A1
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seq
plants
doxs
plant
dna sequence
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PCT/EP1999/005467
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German (de)
English (en)
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WO2000008169A8 (fr
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Andreas Reindl
Patricia Leon Mejia
Juan Manuel Esteves Palmas
Maria Araceli Cantero Gracia
Marcus Ebneth
Karin Herbers
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Sungene Gmbh & Co.Kgaa
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Priority claimed from DE1998135219 external-priority patent/DE19835219A1/de
Priority claimed from DE1998145224 external-priority patent/DE19845224A1/de
Priority claimed from DE1998145216 external-priority patent/DE19845216A1/de
Priority claimed from DE1998145231 external-priority patent/DE19845231A1/de
Application filed by Sungene Gmbh & Co.Kgaa filed Critical Sungene Gmbh & Co.Kgaa
Priority to EP99940083A priority Critical patent/EP1102852A1/fr
Priority to JP2000563793A priority patent/JP2002525034A/ja
Priority to CA002339519A priority patent/CA2339519A1/fr
Priority to AU54157/99A priority patent/AU757440B2/en
Publication of WO2000008169A1 publication Critical patent/WO2000008169A1/fr
Publication of WO2000008169A8 publication Critical patent/WO2000008169A8/fr

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    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)

Definitions

  • 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.
  • DOXS 1-deoxy-D-xylulose-5-phosphate synthase
  • 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.
  • ⁇ -Tocopherol is of great economic importance.
  • 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
  • C skeletons which are exclusively based on isoprene units
  • mixed prenyl lipids e.g. chlorophyll
  • 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:
  • 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
  • IPP is in equilibrium with its isomer, dimethylallyl pyrophosphate (DMAPP).
  • DMAPP dimethylallyl pyrophosphate
  • GPP monoterpene
  • FPP Farnesy pyrophosphate
  • GGPP geranyl-geranyl pyrophosphate
  • 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 ⁇ -tocopherol and ⁇ -tocoquinone, 2-methyl-6-phytylquinol.
  • 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.
  • DOXS 1-deoxy-D-xylulose-5-phosphate synthesis
  • IPP 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.
  • 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).
  • 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.
  • 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.
  • DOXS l-deoxy-D-xylulose-5-phosphate synthase
  • HPPD p-hydroxyphenylpyruvate dioxygenase
  • the formation of IPP as a general starting substrate for all plastidic isoprenoids was increased.
  • 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.
  • D-1-deoxy-xylulose-5-phosphate is further converted towards tocopherols and carotenoids.
  • 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).
  • HPPD p-hydroxyphenyl pyruvate dioxygenase
  • the HPPD gene from Streptomyces avermitilis (Denoya et al., J. Bacteriol. 176 (1994),
  • 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.
  • an expression cassette also 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 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.
  • Transit peptides 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.
  • 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.
  • DOXS l-deoxy-D-xylulose-5-phosphate synthase
  • GGPPOR geranylgeranyl pyrophosphate oxidoreductase
  • the formation of IPP as a general starting substrate for all plastidic isoprenoids was increased.
  • 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 activity of the enzyme geranylgeranyl pyrophosphate oxidoreductase is additionally increased in a further step of the invention by overexpression of a corresponding gene.
  • an increased formation of phytyl pyrophosphate is achieved by an increased conversion of geranylgeranyl pyrophosphate to phytyl pyrophosphate.
  • the GGPPOR gene from Arabidopsis thaliana (SEQ-ID No. 7) is increasingly expressed in transgenic plants.
  • the Arabidopsis GGPPOR is preceded by a transit signal sequence.
  • 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 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.
  • 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.
  • 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).
  • 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):
  • 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.
  • a chemically inducible promoter 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.
  • 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.
  • 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 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. 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.
  • 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.
  • DOXS l-deoxy-D-xylulose-5-phosphate synthase
  • HPPD p-hydroxyphenylpyruvate dioxygenase
  • GGPPOR geranylgeranylpyrophosphate oxidoreductase
  • the formation of IPP as a general starting substrate for all plastidic isoprenoids was increased.
  • 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 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.
  • an increased formation of phytyl pyrophosphate is achieved by an increased conversion of geranylgeranyl pyrophosphate to phytyl pyrophosphate.
  • the GGPPOR gene from Arabidopsis thaliana (SEQ-ID No. 7) is increasingly expressed in transgenic plants.
  • the Arabidopsis GGPPOR is preceded by a transit signal sequence.
  • 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.
  • HPPD p-hydroxyphenylpyruvate dioxygenase
  • cDNAs coding for this enzyme have been described from various organisms such as, for example, from microorganisms, from plants and from humans.
  • the cloning of the HPPD gene from Streptomyces avermitilis is described (Denoya et al., J. Bacteriol. 176 (1994), 5312-5319; SEQ ID No. 5).
  • the HPPD from Streptomyces is preceded by a transit signal sequence.
  • 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 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.
  • an expression cassette also 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 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.
  • 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).
  • 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.
  • 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 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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, 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 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).
  • 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.
  • 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.
  • 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)
  • 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).
  • the expression cassette can therefore, for example, be a seed-specific promoter (preferably the phaseolin
  • 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.
  • 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 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.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • 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.
  • 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.
  • a constitutive promoter preferably the CaMV 35 S promoter
  • the amino acid sequence KDEL lysine, aspartic acid, glutamic acid, leucine
  • KDEL lysine, aspartic acid, glutamic acid, leucine
  • 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.
  • 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.
  • 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).
  • 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 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 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.
  • 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, edited by SD Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • constitutive expression of the exogenous DOXS gene is advantageous.
  • 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.
  • 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
  • Plants in the sense of the invention are mono- and dicotyledonous plants or algae.
  • 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.
  • 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).
  • 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.
  • 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).
  • the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used.
  • 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).
  • 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).
  • the Arabisopsis thaliana DOXS gene was described in Mandel et al. (1996) described as a complete cDNA cloned into the vector pBluescript KS- (Stratagene).
  • 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.
  • 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
  • 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
  • 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.
  • DOXS Acc. Number AF035440
  • the oligonucleotide at the 5 'end comprises the sequence 5' -A1GGATCCATGAGTTTT-GATATTGCCAAATAC-3 '
  • 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
  • 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:
  • 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:
  • 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.
  • 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).
  • transgenic rape plants 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.
  • 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).
  • the cDNA of DOXS (SEQ-ID No. 1) was provided with a CaMV35S promoter and overexpressed in rapeseed using the 35S promoter.
  • 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 ⁇ -tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the ⁇ -tocopherol concentration was increased compared to the non-transformed plant.
  • 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 ⁇ g 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.
  • 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)
  • 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 /
  • 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
  • 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
  • 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.
  • 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.
  • 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:
  • 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).
  • 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 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.,
  • 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
  • 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.,
  • Fragment E contains the gene of the DOXS.
  • tobacco leaf disks were transformed with 45 sequences of the DOXS and the HPPD.
  • 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.
  • transgenic rapeseed plants which have a changed prenyl lipid content
  • 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.
  • 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
  • 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.
  • 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 ⁇ -tocopherol content of the whole plant or the seeds of the plant was then determined. In all In some cases, the ⁇ -tocopherol concentration was increased compared to the non-transformed plant.
  • RNA 20 ⁇ g of total RNA were first mixed with 3.3 ⁇ l of 3M sodium acetate solution and 2 ⁇ l of IM magnesium sulfate solution and made up to 100 ⁇ l of final volume with DEPC water. 1 ⁇ l 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 ⁇ l DEPC water. 2.5 ⁇ g RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco, Life Technologies).
  • a cDNA kit Gibco, Life Technologies
  • 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).
  • 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 ⁇ g RNA).
  • the PCR program was:
  • 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.
  • 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 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 is 5 '-ATTCTAGAC ⁇ TG- 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.
  • 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.
  • 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 -
  • 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.
  • 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 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' -ATGAATTCGGACAATC ⁇ GA- 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 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.
  • 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.
  • 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.
  • 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 ⁇ -tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the ⁇ -tocopherol concentration was increased compared to the non-transformed plant.
  • 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.
  • 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 ⁇ -tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the ⁇ -tocopherol concentration was increased compared to the non-transformed plant.

Abstract

Procédé d'obtention de plantes à capacité de biosynthèse de vitamine E accrue, par surexpression d'un gène végétal de 1-déoxy-D-xylulose-5-phosphate synthase d'Arabidopsis ou de E. coli.
PCT/EP1999/005467 1998-08-05 1999-07-30 Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes WO2000008169A1 (fr)

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EP99940083A EP1102852A1 (fr) 1998-08-05 1999-07-30 Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes
JP2000563793A JP2002525034A (ja) 1998-08-05 1999-07-30 １−デオキシ−ｄ−キシルロース−５−リン酸シンターゼをコードするｄｎａ配列および植物におけるその過剰産生
CA002339519A CA2339519A1 (fr) 1998-08-05 1999-07-30 Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes
AU54157/99A AU757440B2 (en) 1998-08-05 1999-07-30 DNA sequence coding for a 1-deoxy-d-xylulose-5-phosphate synthase and the overproduction thereof in plants

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DE1998135219 DE19835219A1 (de) 1998-08-05 1998-08-05 DNA-Sequenz codierend für eine 1-Deoxy-D-Xylulose-5-Phosphat Synthase und deren Überproduktion in Pflanzen
DE19835219.0 1998-08-05
DE19845224.1 1998-10-01
DE1998145224 DE19845224A1 (de) 1998-10-01 1998-10-01 DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Phosphat Synthase und eine Geranylgeranyl-Pyrosphat Oxidoreduktase und deren Überproduktion in Pflanzen
DE19845216.0 1998-10-01
DE19845231.4 1998-10-01
DE1998145216 DE19845216A1 (de) 1998-10-01 1998-10-01 DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Phosphat Synthase und eine Hydroxyphenylpyruvat Dioxygenase und deren Überproduktion in Pflanzen
DE1998145231 DE19845231A1 (de) 1998-10-01 1998-10-01 DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Pphosphat Synthase, eine Hydroxyphenylpyruvat Dioxygenase und eine Geranylgeranylpyrophosphat Oxidoreduktase und deren Überproduktion in Pflanzen

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US7208298B2 (en) 1998-04-14 2007-04-24 Kyowa Hakko Kogyo Co., Ltd. Process for producing isoprenoid compounds by microorganisms and a method for screening compounds with antibiotic or weeding activity
WO2000044912A1 (fr) * 1999-01-28 2000-08-03 Royal Holloway And Bedford New College Manipulation de l'expression d'un isoprenoide
WO2000044911A1 (fr) * 1999-01-29 2000-08-03 Basf Aktiengesellschaft Surexpression d'une sequence d'adn codant pour une transketolase dans des vegetaux
US7067647B2 (en) 1999-04-15 2006-06-27 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US7335815B2 (en) 1999-04-15 2008-02-26 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US7265207B2 (en) 1999-04-15 2007-09-04 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7141718B2 (en) 1999-04-15 2006-11-28 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
WO2001012827A3 (fr) * 1999-08-11 2001-08-23 Sungene Gmbh & Co Kgaa Homogentisate-dioxygenase
WO2001012827A2 (fr) * 1999-08-11 2001-02-22 Sungene Gmbh & Co. Kgaa Homogentisate-dioxygenase
WO2001073031A3 (fr) * 2000-03-28 2003-01-23 Corixa Corp Compositions et methodes destinees au traitement et au diagnostic du cancer de l'ovaire
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WO2000008169A8 (fr) 2000-05-04
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CA2339519A1 (fr) 2000-02-17
AU5415799A (en) 2000-02-28
AU757440B2 (en) 2003-02-20

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