EP1315808A2 - Deshydroquinate deshydratase/shikimate deshydrogenase utilisee comme cible d'herbicide - Google Patents

Deshydroquinate deshydratase/shikimate deshydrogenase utilisee comme cible d'herbicide

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Publication number
EP1315808A2
EP1315808A2 EP01969344A EP01969344A EP1315808A2 EP 1315808 A2 EP1315808 A2 EP 1315808A2 EP 01969344 A EP01969344 A EP 01969344A EP 01969344 A EP01969344 A EP 01969344A EP 1315808 A2 EP1315808 A2 EP 1315808A2
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
plant
shikimate dehydrogenase
dehydroquinate dehydratase
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
EP01969344A
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German (de)
English (en)
Inventor
Annette Freund
Uwe Sonnewald
Li Ding
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BASF SE
Original Assignee
BASF SE
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Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1315808A2 publication Critical patent/EP1315808A2/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.)
    • C12N9/0006—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
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/20—Herbicides, e.g. DDT
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to the identification of plant dehydroquinate dehydratase / shikimate dehydrogenase (DHD / SHD) as a new target for herbicidal active compounds.
  • the present invention further relates to a method for producing a test system based on the use of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3, functional equivalents of SEQ-ID No: 1 or SEQ ID NO: 3 or parts of SEQ-ID No: 1 or SEQ-ID No: 3 coding for a plant polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity for identifying Inhibitors of plant dehydroquinate dehydratase / shikimate dehydrogenase.
  • the invention further relates to substances identified using these methods or this test systems and their use as herbicides or the use of the polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity as a target for herbicide
  • the present invention also relates to a method for producing transgenic plants containing SEQ-ID No: 1 or SEQ-ID No: 3, functional equivalents of SEQ-ID No: 1 or SEQ ID NO: 3 or parts of SEQ-ID No : l or SEQ-ID No: 3, which have an increased dry matter and / or an increased content of aromatic amino acids compared to a non-transgenic plant of the same type.
  • the invention relates to methods for identifying nucleic acid sequences of dehydroquinate dehydratase / shikimate dehydrogenase variants which are resistant to the inhibitors of plant de-hydroquinate dehydratase / shikimate dehydrogenase identified by the methods according to the invention, and to transgenic plants in that they characterize the nucleic acid sequences said dehydroquinate dehydratase / shikimate dehydrogenase variants contain.
  • dehydroquinate dehydratase / shikimate dehydrogenase is involved in the biosynthesis of chorismate, the precursor of the aromatic amino acids phenylalanine, tyrosine and tryptophan, see Figure 1.
  • the precursors for the formation of aromatic amino acids are erythrosis-4-phosphate and phosphoenolpyruvate.
  • the two substances condense to split off the two phosphates 2-keto-3-deoxy-D-arabinoheptulosonate-7-phosphate, a C7 compound that cyclizes to dehydroquinate.
  • dehydroquinate dehydratase EC 4.2.1.10
  • shikimate dehydrogenase 5 EC 1.1.1.25
  • shikimate dehydrogenase EC 1.1.1.25
  • Dehydroquinate dehydratase / shikimate dehydrogenase is a bifunctional enzyme that catalyzes the third and fourth steps in chorismate bio-synthesis, see also Mitsuhashi, S., Davis, BD,
  • Inhibitors were also identified for the dehydroquinate dehydratase. Acetates, succinates, D- (+) tartrate and diethyl dicarbonates inhibit the dehydroquinate dehydratase in Escherichia coli (Chaudhuri, S., Lambert, JM, McColl, LA, Coggins, JR, Biochem. J., 239, 699-704
  • 35 acid biosynthesis are involved as a target protein (target) for herbicides. Active substances have been described which inhibit plant de novo amino acid biosynthesis.
  • glyphosate which inhibits amino acid biosynthesis in planta.
  • the shikimate pathway plays a role in a large number of other substances which are produced in large quantities by the plant, such as ubiquinone, folate, flavonoids, coumarins, lignin, alkaloids, cyanogenic glucosides, plastoquinone and tocopherols.
  • ubiquinone folate
  • flavonoids flavonoids
  • coumarins lignin
  • alkaloids cyanogenic glucosides
  • plastoquinone and tocopherols tocopherols.
  • the sum of all these substances can make up up to 50% of the dry substance of a plant.
  • the suitability of an enzyme as a target for herbicides can be demonstrated by reducing the enzyme activity, for example using antisense technology in transgenic plants. If reduced growth is brought about by introducing an antisense DNA for a specific gene into a plant, this indicates the suitability of the enzyme whose activity is reduced as a site of action for herbicidal active ingredients.
  • the antisense inhibition of acetolactate synthase (ALS) in transgenic potato plants leads to comparable phenotypes, such as the treatment of control plants with ALS-inhibiting herbicides (Höfgen et al., Plant Physiology 107, 469-477 (1995)).
  • Transgenic in the sense of the invention means that the nucleic acids used in the method are not in their natural place in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously.
  • Tansgen also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been changed.
  • Transgenic is preferably to be understood as meaning the expression of the nucleic acids at a non-natural location in the genome, that is to say there is homologous or preferably heterologous expression of the nucleic acids. The same applies to the nucleic acid construct according to the invention or the vector.
  • the object of the present invention was to demonstrate that dehydroquinate dehydratase / shikimate dehydrogenase is a suitable herbicidal target in plants, and to produce an efficient and simple dehydroquinate dehydratase / shikimate dehydrogenase test system for carrying out inhibitor-enzyme binding studies. Furthermore, the object was to identify dehydroquinate dehydratase / shikimate dehydrogenase variants which are resistant to the inhibitors found according to the invention.
  • the object was achieved by isolating DNA sequences which code for the plant enzyme dehydroquinate dehydratase / shikimate dehydrogenase, the production of antisense or co-suppression constructs of the plant dehydroquinate dehydrogenase. tase / Shikimate dehydrogenase and its expression in plants, as well as the functional expression of the vegetable dehydroquinate dehydratase / Shikimate dehydrogenase in prokaryotic or eukaryotic cells.
  • Tobacco (variety Samsun NN) was used as a model plant for the expression of the dehydroquinate dehydratase / shikimate dehydrogenase in sense and antisense orientation.
  • the dehydroquinate dehydratase / shikimate dehydrogenase was expressed heterologously in E. coli for the production of recombinant enzyme for carrying out enzyme assays.
  • a cDNA coding for plant dehydroquinate dehydratase / shikimate dehydrogenase was isolated from tobacco and sequenced, see example 1 or sequence listing SEQ-ID No. 1, SEQ ID No. 3 and Bonner, C. and Jensen, R. Biochem. J., 302, 11-14 (1994).
  • the gene can be functionally overexpressed in various heterologous systems such as in E. coli, yeast or baculovirus and in test systems for the identification of
  • Inhibitors are used. The evidence that dehydroquinate dehydratase / shikimate dehydrogenase is an essential gene for plants was first demonstrated with the help of antisense or cosuppression plants.
  • the plants show growth retardation to different degrees. Wild type and transgenic DHD / SHD plants are shown in side view ( Figure 2) and in top view ( Figures 3 and 4). There is a clear inhibition of growth in transgenic DHD / SHD plants compared to the wild type ( Figure 2, wild type on the far left).
  • the activity of the DHD / SHD enzyme was measured by the method as described in Example 5. It was found that the DHD / SHD enzyme activity is zero in cosuppression plants and an enzyme activity of 0.025 - 0.06 ⁇ M / min / g can be measured in wild type plants.
  • a nucleic acid sequence for identifying inhibitors of plant dehydroquinate dehydratase / shikimate dehydrogenase can be used to produce these test systems, the said nucleic acid sequence being, for example, the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 containing the coding region of a vegetable dehydroquinate dehydratase / shikimate dehydrogenase, or a nucleic acid sequence which is associated with the DNA sequence SEQ-ID No. 1 or SEQ-ID No.
  • 3 or parts or derivatives which are derived from these sequences by insertion, deletion or substitution, can be hybridized and encoded for a protein which has the biological activity of a plant dehydroquinate dehydratase / shikimate dehydrogenase.
  • Another object of the invention thus relates to methods for identifying new herbicides based on the use of a protein with dehydroquinate dehydratase / shikimate dehydrogenase activity encoded by a nucleic acid sequence, said nucleic acid sequence comprising the following sequence:
  • nucleic acid sequence which, on the basis of the degenerate genetic code, can be derived from the amino acid sequences represented by reverse 2 translation of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4; or c) functional analogues of those in SEQ ID NO: 1 or
  • SEQ ID NO: 3 nucleic acid sequences shown coding for a polypeptide with the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4; or
  • SEQ ID NO: 3 nucleic acid sequence shown coding for functional analogs of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4; or
  • homology between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the respective total sequence length, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group ( GCG), Madison, USA) using the following parameters:
  • Gap Weight 12 Length Weight: 4
  • Nucleotide sequence still have the desired functions. Functional equivalents thus encompass 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 an organism (see above), but also sequences that hybridize with the sequences according to the invention or parts of these sequences.
  • oligonucleotides for example the conserved or other regions, which can be determined by comparison with other related genes in a manner known to the person skilled in the art, are advantageously used.
  • longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • the hybridization conditions for DNA: DN hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately
  • the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between approximately 30 ° C. to 55 ° C., preferably between approximately 45 ° C. to 55 ° C.
  • These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid
  • a functional equivalent is understood to mean, in particular, natural or artificial mutations of an originally isolated one for a dehydroquinate dehydratase / shikimate dehydro- genes coding sequence, which still shows 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 this nucleotide sequence. The aim of such a modification can, for example, be to further narrow down the coding sequence contained therein or, for example, also to insert further restriction enzyme interfaces.
  • Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
  • nucleotide sequence according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components.
  • synthetic nucleotide sequences with codons are generated which are preferred by the respective host organism. These preferred codons can be determined from the highest protein abundance codons expressed in most interesting species.
  • nucleic acid sequences which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 1 or SEQ ID NO. 3 advantageously from 40 to 100%, preferably from 60 to 100% and particularly preferably from 70 to 100%, very particularly preferably 80-100%, or 85-100%, or 90-100%, or 95-100 %, or 96-100%, or 97-100%, or 98-100%, or 99-100%.
  • the method according to the invention can be carried out in individual, separate method approaches. However, the implementation in a high-throug put screening is preferred.
  • substances with herbicidal activity can be determined which reduce or block the transcription, expression, translation or the activity of a polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity.
  • These substances are potential herbicides, the effects of which can be further improved via classic chemical synthesis.
  • Test systems suitable for this are both in vitro and in vivo test systems.
  • proteins with dehydroquinate dehydratase / shikimate dehydrogenase activity can be used, the preferred
  • the enzyme quantities required for the in vitro test systems are preferably provided via the functional expression of plant dehydroquinate dehydratase / shikimate dehydrogenase, in particular dehydroquinate dehydratase / shikimate dehydrogenase from tobacco in suitable expression systems.
  • the enzyme isolated from plants, here preferably from tobacco can also be used instead of the recombinantly produced enzyme.
  • transgenic organisms are also preferred for in vivo test systems.
  • nucleic acid sequence such as the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 containing the coding region of a plant dehydroquinate dehydratase / shikimate dehydrogenase, or a nucleic acid sequence which is associated with the DNA sequence SEQ-ID No. 1 or SEQ-ID No.
  • Expression cassettes are therefore a further subject of the invention, the sequence of which for a dehydroquinate dehydratase / shikimate dehydrogenase from tobacco or its functional equivalent code for the production of a test system for finding compounds with herbicidal activity.
  • the nucleic acid sequence can a) be a nucleic acid sequence with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; or
  • SEQ ID NO: 2 or SEQ ID NO: 4 can be derived amino acid sequences
  • e) contain parts of the nucleic acid sequences a), b), c) or d); or
  • f) comprise at least 300 nucleotide building blocks of the nucleic acid sequences a), b), c) or d);
  • artificial DNA sequences are suitable as long as they impart the desired property of the expression of the dehydroquinate dehydratase / shikimate dehydrogenase gene, as described above, for example.
  • Such artificial DNA sequences can be determined, for example, by back-translation of proteins constructed by means of molecular modeling, which have dehydroquinate dehydratase / shikimate dehydrogenase activity, or by in vitro selection. Coding DNA sequences obtained by back-translating a polypeptide sequence according to the codon usage specific for the host organism are particularly suitable. A specific expert familiar with genetic methods can easily determine the specific codon usage by computer evaluations of other, known genes of the organism to be transformed. This methodology can also be used here for the expression of the target protein in bacteria, fungi, plants, insect cells and mammalian cells.
  • DNA fragments When preparing an expression cassette, various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • For the Connection of the DNA fragments to one another can be applied to the fragments adapters or linkers.
  • This methodology can be used in the expression of the target protein in bacteria, fungi, plants, insect cells and mammalian cells.
  • an expression cassette according to the invention comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a terminator and optionally a polyadenylation signal and optionally further regulatory elements which are operatively linked to the sequence coding for the polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity.
  • 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 in the expression of the coding sequence.
  • Such an expression cassette is produced by fusing a suitable promoter or a genetic control sequence with a suitable dehydroquinate dehydratase / shikimate dehydrogenase DNA sequence and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. 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).
  • Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties.
  • tissue-specific expression can also take place depending on certain stress factors.
  • Corresponding elements are, for example, for water stress, abseic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131 -17135) and heat stress (Schoffl F et al., Molecular & General Genetics 217 (2-3): 246-53, 1989).
  • Advantageous control sequences for the expression cassettes or vectors according to the invention are, for example, in promoters such as cos, tac, trp, tet, Ipp, lac, laclq, T7, T5, T3, gal, trc -, ara-, SP6 ⁇ , 1-PR or in the 1-PL promoter, which can be used to express the dehydroquinate dehydratase / shikimate dehydrogenase in gram-negative bacterial strains.
  • promoters such as cos, tac, trp, tet, Ipp, lac, laclq, T7, T5, T3, gal, trc -, ara-, SP6 ⁇ , 1-PR or in the 1-PL promoter, which can be used to express the dehydroquinate dehydratase / shikimate dehydrogenase in gram-negative bacterial strains.
  • control sequences are, for example, in the amy and SP02 promoters, which can be used to express the dehydroquinate dehydratase / shikimate dehydrogenase in gram-positive bacterial strains, and in the yeast or fungal promoter.
  • any promoter 20 that can control the expression of foreign genes in plants is suitable for expression in plants.
  • a plant promoter or a plant virus-derived promoter is preferably used.
  • the CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred, see Franck et al., Cell 21, 285-294 (1980). 25
  • 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, 2195-2202 (1989).
  • the expression cassette to be used for plants can also contain a chemically inducible promoter, by means of which the expression of the exogenous dehydroquinate dehydratase / shikimate dehydrogenase 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 dehydroquinate dehydratase / shikimate dehydrogenase gene in the plant can be controlled at a specific point in time.
  • promoters as e.g. the
  • promoters of the phosphoribosyl pyrophosphate amidotransferase from Glycine max see also Genbank Accession number U87999
  • a node-specific promoter as in EP 249676.
  • promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of amino acids or their 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, 2445-245 (1989)).
  • a foreign protein can be stably expressed up to a proportion of 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio / Technology 10, 1090-1094 (1995)).
  • promoters such as the Napingen promoter from rapeseed (US Pat. No. 5,608,152), the oleosin promoter from Arabidopsis (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US5, 504, 200), the Bce4 promoter from Brassica (WO91 / 13980) or the leguminous B4 promoter (LeB4, Baeumlein et al., Plant J., 2, 2, 1992: 233-239) or for Monocot-suitable promoters such as the promoters of the barley Ipt2 or Iptl gene promoters (WO95 / 15389 and WO95 / 23230) or the barley Hordein gene promoters, the glutelin rice gene, the orycin rice gene, or the prolamin rice Gene, the wheat gliadin gene, the wheat glutelin gene, the corn zein gene, the
  • the biosythesis site of amino acids is generally the leaf tissue, so that leaf-specific expression of the dehydroquinate dehydratase / shikimate dehydrogenase gene is useful.
  • the amino acid biosynthesis need not be limited to the leaf antlers, but also in all others Parts of the plant - for example in fatty seeds - can be tissue-specific.
  • Expression cassettes can also be constructed for expression in plants, the DNA sequence of which codes for a dehydroquinate dehydratase / shikimate dehydrogenase 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 enzymatically split off from the dehydroquinate dehydratase / Shikimate dehydrogenase part after translocation of the dehydroquinate dehydratase / shikimate dehydrogenase gene into the chloroplasts.
  • the transit peptide derived from the plastidic dehydroquinate dehydratase / shikimate dehydrogenase or a functional equivalent of this transit peptide e.g. the transit peptide of the Rubisco small subunit or the ferredoxin NADP oxidoreductase.
  • a plant expression cassette according to the invention can contain, for example, a constitutive promoter (preferably the CaMV 35 S promoter), the LeB4 signal peptide, the gene to be expressed and the ER retention signal.
  • the amino acid sequence KDEL lysine, aspartic acid, glutamic acid, leucine
  • the plant expression cassette can, for example, be built into the plant transformation vector pBinAR.
  • constitutive expression of the exogenous dehydroquinate dehydratase / shikimate dehydrogenase gene can be advantageous.
  • inducible expression may also appear desirable.
  • promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable expression in other plant tissues or in other organisms, such as E. coli bacteria.
  • all promoters described above can be used as plant promoters.
  • preferred polyadenylation signals are those which essentially contain T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid correspond to pTiACH5 (Gielen et al., EMBO J., 3, 835 (1984)) or functional equivalents.
  • the promoter and terminator regions in the transcription direction can optionally be provided 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, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter according to the invention can be both native or homologous and foreign or heterologous to the host plant.
  • the expression cassette according to the invention contains in the
  • the promoter according to the invention, any sequence and a region for the transcriptional termination. Different termination areas are interchangeable.
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as, for example, transitions and trans versions can be used, vi ro mutagenesis, "primer pair", restriction or ligation can be used. With suitable manipulations, such as, for example, restriction, "chewing-back” or filling in overhangs for "blunt ends”, complementary ends of the fragments can be made available for the ligation.
  • an expression cassette is inserted as an insertion into a vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally. Chromosomal replication is preferred.
  • the nucleic acid construct according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized plasmid or only of the nucleic acid construct as a vector or the nucleic acid sequences used.
  • nucleic acid sequences used in the method according to the invention can also be introduced into an organism alone.
  • nucleic acid sequences in addition to the nucleic acid sequences, further genes are to be introduced into the organism, all of them can be introduced into the organism together in a single vector or each individual gene can be introduced into the organism, the different vectors being able to be introduced simultaneously or successively.
  • the vector advantageously contains at least one copy of the nucleic acid sequences used and / or the nucleic acid construct according to the invention.
  • the expression cassettes according to the invention and the vectors derived from them, as already indicated above, can also contain other functional elements in addition to the promoters mentioned above. Examples include, but are not limited to:
  • Reporter genes code for easily quantifiable proteins. Via a growth, fluorescence, Che o, bioluminescence or resistance assay or via a photometric measurement (self- staining) or enzyme activity, these genes can be used to evaluate the transformation efficiency or the place or time of expression. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as the "green fluorescence protein" (GFP) (Gerdes HH and Kaether C, FEBS Lett 1996; 389 (1): 44-47; Chui WL et al., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques.
  • GFP green fluorescence protein
  • Selection markers that confer resistance to antibiotics include the npt gene which confers resistance to the aminoglyciside antibiotics neomycin (G 418), kanamycin and paromycin (Deshayes A et al., EMBO J. 4 (1985) 2731-2737), the hygro gene (Marsh JL et al., Gene. 1984; 32 (3): 481-485), the sul gene (Guerineau F et al., Plant Mol Biol. 1990; 15 (1): 127-136) and the she- ble gene that confers resistance to the bleomycin antibiotic zeocin.
  • selection marker genes are genes which confer resistance to 2-deoxyglucose-6-phosphate (WO 98/45456) or phosphinotricin etc. or those which confer resistance to antimetabolites, for example the dhfr gene ( Reiss, Plant Physiol. (Life Sei. Adv.) 13 (1994) 142-149). Also suitable are genes such as trpB or hisD (Hartman SC and Mulligan RC, Proc Natl Acad Sei U S A. 85 (1988) 8047-8051).
  • Mannose phosphate iso erase WO 94/20627
  • ODC ornithine decarboxylase
  • McConlogue 1987 in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed.
  • Deaminase from Aspergillus terreus Teamura K et al., Biosci Biotechnol Biochem. 59 (1995) 2336-2338).
  • affinity tags coding for a peptide or polypeptide, the nucleic acid sequence of which can be fused with the sequence coding for the target protein directly or by means of a linker using common cloning techniques.
  • the affinity tag is used to isolate the recombinant target
  • teins by means of affinity chromatography can also be used for the detection of the expressed fusion protein.
  • the above-mentioned linker can optionally contain a protease interface (eg for thrombin or factor Xa), as a result of which the affinity tag can be cleaved from the target protein if necessary.
  • affinity tags are the "His tag”, for example from Quiagen, Hilden, "Strep tag”, the “Myc tag”, the domain consisting of a chitin-binding domain and an integer from New England Biolab and that so-called CBD day from Novagen.
  • Examples of vectors for expression in E. coli are pGEX [Pharmacia Biotech Ine; Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein, or Protein A, the pTrc- Vectors (Amann et al., (1988) Gene 69: 301-315), the "pQE” vectors from Qiagen (Hilden), the "pKK233-2” from CLONTECH, Palo Alto, CA and the "pET” -, and the "pBAD” vector series from Strategagen, La Jolla as well as the Ml3mp series and pACYC184.
  • vectors for vectors for use in yeast are pYepSecl (Baldari, et al., (1987) E bo J. 6: 229-234), pMFa (Kur- jan and Herskowitz, (1982) Cell 30: 933-943) , pJRY88 . (Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives, pGAPZ derivatives, pPICZ derivatives and the vectors of the "Pichia Expression Kit” (all from Invitrogen Corporation, San Diego, CA).
  • insect cell expression vectors for example for expression in Sf 9 cells, are the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Luck-low and Summers (1989) Virology 170: 31-39).
  • plant expression vectors for expression in plant cells or algal cells can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, MW (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721.
  • Other suitable vectors are described, inter alia, in “Methods in Plant Molecular Biology and Biotechnology” (CRC Press, Chapter 6/7, 71-119).
  • Examples of expression vectors to be used in mammalian cells are pCDM8 and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBO J. 6: 187-195).
  • Promoters to be used are preferably of viral origin, e.g. Promoters of polyoma, adenovirus 2, cytomegalovirus or simian virus 40.
  • Further prokaryotic and eukaryotic expression systems are mentioned in chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Further advantageous vectors are described in Hellens et al. (Trends in plant science, 5, 2000).
  • the expression cassette and the vectors derived therefrom can also be used to transform bacteria, cyano-bacteria, yeasts, filamentous fungi and algae with the aim of increasing the content of ubiquinone, folate, flavonoids, cumarins, lignins, alkaloids, cyanogenic glucosides, Plastoquinones, tocopherols and aromatic amino acids can be used.
  • bacteria of the genus Escherichia (Escherichia coli), Erwinia, Flavobacterium, Alcaligenes or cyanobacteria, for example of the genus Synechocystis or Anabena, are preferred.
  • Bacteria of the genus Escherichia coli are particularly preferred here for economic reasons as well as for the many possibilities of genetic manipulation.
  • Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.
  • Preferred fungi are Aspergillus, Trichoderma, Ashbya, Mortierella, Saprolegnia, Pythium, Neurospora, Fusarium, Beauveria or others in Indian Chem Engr. Section B.
  • eukaryotic cell lines are e.g. common insect or mammalian cell lines known to those skilled in the art.
  • transgenic animals are also suitable as host organisms, for example C. elegans.
  • transgenic plants which have a functional or non-functional nucleic acid construct according to the invention or a functional or not according to the invention functional vector included.
  • Functional in the sense of the invention is to be understood that the nucleic acids used in the method are expressed alone or in the nucleic acid construct or in the vector and a biologically active gene product is produced.
  • non-functional means that the nucleic acids used in the method, alone or in the nucleic acid construct or in the vector, are not transcribed, are not expressed and / or a biologically inactive gene product is produced.
  • the so-called antisense RNAs are also nonfunctional nucleic acids or, when inserted into the nucleic acid construct or the vector, a nonfunctional nucleic acid construct or nonfunctional vector.
  • Both the nucleic acid construct according to the invention and the vector according to the invention can be used advantageously for the production of transgenic organisms, preferably plants.
  • Baculovirus expression systems “MaxBac 2.0 Kit” from Invitrogen, Calsbald or “Bac-PAK Baculovirus ExpressionsSystem” from CLONTECH, Palo Alto, CA Expression systems for yeasts such as the “Easy Select Pichia Expression Kit", the "Pichia Expression Kit” (all from Invitrogen, Calsbad
  • the vegetable dehydroquinate dehydratase / shikimate dehydrogenase protein expressed with the aid of an expression cassette is particularly suitable for the detection of inhibitors specific for dehydroquinate dehydratase / shikimate dehydrogenase in in vitro test systems.
  • the cDNA sequence of the dehydroquinate dehydratase / shikimate dehydrogenase or suitable fragments of the cDNA sequence of the dehydroquinate dehydratase / shikimate dehydrogenase from tobacco can be in one of the above-mentioned expression vectors such as, for example, the vector pQE is cloned and in one of the above-mentioned organisms or expression systems such as e.g. E. coli can be overexpressed because E. coli from the ones already mentioned! Reasons is particularly suitable for the expression of recombinant protein.
  • the method according to the invention for identifying inhibitors of a polypeptide with dehydroquinate, the hydratase / shikimate dehydrogenase activity, with herbicidal action is based on the transcription, expression, translation or the activity of the gene product selected from the group by a nucleic acid sequence:
  • nucleic acid sequence which can be derived on the basis of the degenerate genetic code from the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4, or
  • encoded amino acid sequence is influenced and selects those substances which reduce or block the transcription, expression, translation or the activity of the gene product.
  • the method is carried out in an organism, bacteria, yeasts, fungi or plants being used as the organism.
  • an organism can be used which is a conditional or natural mutant of the sequences SEQ ID NO: 1 or SEQ ID NO: 3.
  • a method is preferred in which the organism used is a transgenic organism.
  • a transgenic organism is an organism that has been transformed with an expression cassette according to the invention or a vector according to the invention.
  • transformation the transfer of foreign genes into the genome of an organism.
  • the described methods for transforming and regenerating plants can be used for the transformation of plants
  • Suitable methods are the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene cannon, the electroporation, the incubation of dry embryos in DNA-containing solution, the 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 S.D. Kung and R. Wu, Academic Press (1993), 128-143 and in Potrykus Annu.
  • An expression cassette according to the invention which binds to a dehydroquinate dehydratase / shikimate dehydrogenase gene, is preferably cloned into a vector, for example pBINAR, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res 12, 8711 (1984)). Agrobacts transformed with such a vector. Rien can then be used in a known manner to transform plants, in particular crop plants, such as tobacco plants, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then be cultivated in suitable media.
  • Transgenic plants can be regenerated in a known manner from the transformed cells of the wounded leaves or leaf pieces , which contain a gene integrated into the expression cassette for the expression of a dehydroquinate dehydratase / shikimate dehydrogenase gene.
  • Agrobacteria transformed with an expression cassette can also be used in a known manner to transform plants, in particular crop plants, such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, Salad and the various tree, nut and wine species and legumes can be used, for example by bathing wounded leaves or leaf pieces in an agrobacteria solution and then cultivating them in suitable media.
  • crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, Salad and the various tree, nut and wine species and legumes can be used, for example by bathing wounded leaves or leaf pieces in an agrobacteria solution and then cultivating them in suitable media.
  • Another object of the invention are in vitro methods for identifying substances having a herbicidal action which inhibit the activity of the vegetable dehydroquinate dehydratase / shikimate dehydrogenase.
  • the method according to the invention consists of the following steps:
  • a polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity is either expressed in one of the above-described embodiments of a transgenic organism in an enzymatically active form or an organism containing the protein according to the invention is cultured;
  • step b) the protein obtained in step a) is incubated in the growing or resting organism as a whole, in the cell disruption of the transgenic organism, in partially purified form or in a form purified to homogeneity with redox equivalents and with a chemical compound;
  • redox equivalents include, but are not limited to: NADPH / NADP + , NADH / NAD + and FAD / FADH.
  • a chemical compound is selected by step b) which inhibits a polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity in comparison to a sample not incubated with the chemical compound.
  • This method is particularly suitable for high-throughput screening.
  • the plant dehydroquinate dehydratase / shikimate dehydrogenase can be used, for example, in an enzyme test in which the activity of the dehydroquinate dehydratase / shikimate dehydrogenase 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 a high potency from a large number of substances, in order to subsequently carry out further in-depth tests known to the person skilled in the art with these substances.
  • inhibitors of the enzyme dehydroquinate dehydratase / shikimate dehydrogenase can be detected using techniques which show the interaction between protein and ligand.
  • three preferred embodiments are to be mentioned here, which in connection with the present invention are also suitable for high-throughput methods:
  • FCS fluorescence correlation spectroscopy
  • the average diffusion rate of a fluorescence molecule can be determined as a function of mass in a small sample volume.
  • FCS can be used to determine protein-ligand interactions.
  • the chemical compounds identified in this way, which bind to the dehydroquinate dehydratase / shikimate dehydrogenase, may be suitable as inhibitors.
  • the dehydroquinate dehydratase / shikimate dehydrogenase is then immobilized on a suitable carrier and incubated with the chemical compound to be investigated. After one or more suitable washing steps, the molecules of the chemical compound additionally bound to the protein can be detected using the above-mentioned methodology and thus possible inhibitors can be selected.
  • the chemical compounds identified in this way, which bind to the dehydroquinate dehydratase / shikimate dehydrogenase may be suitable as inhibitors.
  • Biacore is based on the change in the refractive index on a surface when a chemical compound binds to a protein immobilized on said surface. Since the change in the refractive index for a defined change in the mass concentration at the surface is virtually identical for all proteins and polypeptides, this method can in principle be applied to any protein (Lindberg et al. Sensor Actuators 4 (1983) 299-304; Malmquist Nature 361 (1993)
  • the chemical compound is injected into a reaction cell with a volume of 2-5ml, on the walls of which the protein has been immobilized.
  • the binding of the corresponding chemical compound to the protein and thus the identification of possible inhibitors can be done via Surface
  • Plasmon resonance can be made by picking up the laser light reflected from the surface.
  • the chemical compounds identified in this way which bind to the dehydroquinate dehydratase / shikimate dehydrogenase may be suitable as inhibitors.
  • the invention furthermore relates to in vivo methods for identifying substances having a herbicidal action which inhibit the dehydroquinate dehydratase / shikimate dehydrogenase activity in plants, consisting of
  • the following organisms or cell types are used to produce a transient organism: bacteria, in yeast, fungi, algae, plant cells, insect cells or mammalian cells.
  • Chemical compounds which reduce the biological activity, the growth or the vitality of the organisms are to be understood as compounds, the biological activity, the growth or the vitality of the organisms by at least 10%, advantageously by at least 30%, preferably by inhibit at least 50%, particularly preferably by at least 70%, very particularly preferably by at least 90%.
  • transgenic plants, plant cells, plant tissue or plant parts are used as transgenic organisms.
  • the invention further relates to compounds with herbicidal activity which can be identified using the test systems described above.
  • Another object of the invention is a method which consists in transferring the substances identified via the abovementioned methods to a plant in order to test their herbicidal activity and to select the substances which show herbicidal activity.
  • the identified substances can be chemically synthesized or microbiologically produced substances. be and e.g. in cell extracts of e.g. Plants, animals or microorganisms occur.
  • the reaction mixture may be a cell-free extract or comprise a cell or cell culture. Suitable methods are known to the person skilled in the art and are described, for example, generally in Alberts, Molecular Biology the cell, 3rd Edition (1994), e.g. Chapter 17.
  • the substances mentioned can be added to the reaction mixture or the culture medium, for example, or injected into the cells or sprayed onto a plant.
  • the sample can be divided into different groups, e.g. if it consists of a large number of different components, so as to reduce the number of different substances per sample and then to repeat the method according to the invention with such a "sub-sample" of the original sample.
  • the steps described above can be repeated several times, preferably until the sample identified according to the method according to the invention only comprises a small number of substances or only one substance.
  • the substance or derivative thereof identified according to the method according to the invention is further formulated so that it is suitable for use in plant breeding or plant row or tissue culture.
  • the substances which have been tested and identified in accordance with the method according to the invention can be: expression libraries, for example cDNA expression libraries, peptides, proteins, nucleic acids ren, antibodies, small organic substances, hormones, PNAs or the like (Milner, N ture Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein). These substances can also be functional derivatives or analogs of the known inhibitors or activators. Methods for the production of chemical derivatives or analogs are known to the person skilled in the art. The derivatives and analogs mentioned can be tested according to methods according to the prior art.
  • the cell or tissue which can be used for the method according to the invention is preferably a host cell according to the invention, a plant cell or a plant tissue, as described in the above-mentioned embodiments.
  • a further embodiment of the invention are substances which have been identified by the methods according to the invention described above and which are an antibody against the protein encoded by the sequences SEQ ID NO: 1 or SEQ ID NO: 3 or a functional equivalent of the protein encoded by the sequence SEQ ID NO: 1 or SEQ ID NO: 3.
  • Inhibitors of dehydroquinate dehydratase / shikimate dehydrogenase with herbicidal activity can be used as defoliants, desiccants, herbicides and in particular as weed killers. Weeds in the broadest sense are understood to mean all plants that grow up in places where they are undesirable. Whether the active ingredients found with the aid of the test system according to the invention act as total or selective herbicides depends, inter alia, on the amount used.
  • Inhibitors of dehydroquinate dehydratase / shikimate dehydrogenase with herbicidal activity can be used, for example, against the following weeds:
  • Echinochloa Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bro us, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbris yslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, Apera.
  • the substances identified or agents containing them in the process according to the invention can advantageously also be used in a further number of crop plants for eliminating undesired plants.
  • the following crops are considered, for example:
  • the substances found by the process according to the invention can advantageously also be used in crops which are tolerant to the action of herbicides by breeding, including genetic engineering methods.
  • the substances according to the invention or the herbicidal compositions comprising them can be sprayed, for example, in the form of directly sprayable aqueous solutions, powders, suspensions, and also high-strength aqueous, oily or other suspensions or dispersions, emulsions, oil dispersions, pastes, dusts, spreading agents or granules , Atomizing, dusting, scattering or pouring can be used.
  • the application forms depend on. the uses; in any case, they should ensure the finest possible distribution of the active compounds according to the invention.
  • Inert liquid and / or solid carriers include liquid additives such as mineral oil fractions from medium to high boiling points, such as kerosene or diesel oil, also coal tar oils and oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene, alkylated Naphthalenes or their derivatives, alkylated benzenes or their derivatives, alcohols such as methanol, ethanol, propanol, butanol, cyclohexanol, ketones such as cyclohexanone or strongly polar solvents, for example amines such as N-methylpyrrolidone or water.
  • liquid additives such as mineral oil fractions from medium to high boiling points, such as kerosene or diesel oil, also coal tar oils and oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene, alkylated Naphthalen
  • aqueous forms of use such as emulsion concentrates, suspensions, pastes, wettable powders or water-dispersible granules, which can be prepared, for example, by adding water.
  • emulsion concentrates such as emulsion concentrates, suspensions, pastes, wettable powders or water-dispersible granules
  • the substances and / or agents, the so-called substrates as such or dissolved in an oil or solvent can be homogenized in water by means of wetting agents, adhesives, dispersants or emulsifiers.
  • concentrates consisting of an active substance, wetting agent, tackifier, dispersant or emulsifier and possibly solvent or oil, which are suitable for dilution with water.
  • alkali, alkaline earth, ammonium salts of aromatic sulfonic acids e.g. Lignin, phenol, naphthalene and dibutylnaphthalenesulfonic acid, as well as of fatty acids, alkyl and alkylarylsulfonates, alkyl, lauryl ether and fatty alcohol sulfates, and salts of sulfated hexa-, hepta- and octadecanols as well as of fatty alcohol glycol ethers, condensation products of sulfonated naphthalene and its derivatives Formaldehyde, condensation products of naphthalene or naphthalenesulfonic acids with phenol and formaldehyde, polyoxyethylene octylphenol ether, ethoxylated isooctyl, octyl or nonylphenol, alkylphenyl, tributylphenyl poly
  • Powders, materials for broadcasting and dusts can advantageously be produced as solid carriers by mixing or grinding the active substances together with a solid carrier.
  • Granules for example coated granules, impregnated granules and homogeneous granules, can be prepared by binding the active ingredients to solid carriers.
  • Solid carriers are, for example, mineral such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, loess, clay, dolomite, diatomaceous earth, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas and vegetable products such as flour, tree bark, wood and nutshell flour, cellulose powder or other solid carriers.
  • the concentrations of the substances and / or agents according to the invention in the preparations ready for use can be varied within wide ranges.
  • the formulations generally contain 0.001 to 98% by weight, preferably 0.01 to 95% by weight, of at least one active ingredient.
  • the active ingredients are used in a purity of 90 ° s to 100%, preferably 95% to 100% (according to the NMR spectrum).
  • the herbicidal compositions or the substances can be applied pre- or post-emergence. If the active ingredients are less compatible with certain crop plants, application techniques can be used in which the herbicidal compositions or substances are sprayed with the aid of sprayers in such a way that the leaves of the sensitive crop plants are not struck wherever possible, while the active ingredients are applied to the leaves below them unwanted plants or the uncovered floor area (post-directed, lay-by).
  • the substances and / or agents according to the invention can be mixed with numerous representatives of other herbicidal or growth-regulating active compound groups and applied together.
  • active compound groups for example, 1, 2, 4-thiadiazoles, 1, 3, 4-thiadiazoles, amides, aminophosphoric acid and their derivatives, aminotriazoles, anilides, (het) -aryloxyalkanoic acid and their derivatives, benzoic acid and their derivatives, benzothiadiazinones , 2-aroyl-l, 3-cyclohexanediones, hetaryl aryl ketones, benzylisoxazolidinones, meta-CF3-phenyl derivatives, carbamates, quinolinic acid and their derivatives, chloroacetanilides, cyclohexane-1, 3-dione derivatives, diazines, dichloropropionic acid its derivatives, dihydrobenzofurans, dihydrofuran-3-ones
  • reesters phenylacetic acid and its derivatives, phenylpropionic acid and its derivatives, pyrazoles, phenylpyrazoles, pyridazines, pyridinecarboxylic acid and their derivatives, pyrimidyl ethers, sulfonamides, Sulfonylureas, triazines, triazinones, triazolinones, triazole carboxamides, uraciles into consideration.
  • the application rates of active ingredient are 0.001 to 3.0, preferably 0.01 to 1.0 kg / ha of active substance, depending on the control target, season, target plants and growth stage.
  • Another object of the invention is the use of a substance identified by one of the methods or agents according to the invention containing these substances as a herbicide or for regulating the growth of plants.
  • the invention also relates to transgenic organisms, preferably plants, transformed with an expression cassette containing the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or their functional equivalents, which are obtained by additional expression of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 or a functional equivalent of one of these sequences have become tolerant towards inhibitors of dehydroquinate dehydratase / shikimate dehydrogenase, as well as transgenic cells, tissues, parts and propagation material of such transgenic organisms, preferably plants.
  • Transgenic crop plants such as e.g. Barley, wheat, rye, corn, soybean, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato,
  • Another object of the invention is thus the use of an expression cassette containing DNA sequences SEQ-ID No. 1, SEQ ID No. 3 or with these hybridizing DNA sequences for the transformation of plants, cells, tissues or parts of plants.
  • the aim of the use is preferably the production of plants with herbicide-resistant forms of the dehydroquinate dehydratease / shikimate dehydrogenase.
  • the gene coding for a polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity can impart resistance to inhibitors.
  • the expression of such a gene leads to a herbicide-resistant plant, as could be shown for another enzyme from chorismate biosynthesis, the enolpyruvylshikimate-3-phosphate synthase.
  • the provision of the herbicidal target further enables a method for identifying a dehydroquinate dehydratase / shikimate dehydrogenase which is not inhibited by the inhibitors according to the invention.
  • an enzyme which differs from the dehydroquinate dehydratase / shikimate dehydrogenase according to the invention is referred to as the dehydroquinate dehydratase / shikimate dehydrogenase variant.
  • the above method is also the subject of the present invention.
  • the above-mentioned method for generating variants of the nucleic acid sequences SEQ ID NO: 1 or SEQ ID NO: 3 consists of the following steps:
  • a further subject of the invention is therefore an organism produced by this method, preferably the organism is a plant. Then whole plants are regenerated and the resistance to the herbicide in intact plants is checked.
  • Modified proteins and / or nucleic acids which can impart resistance to herbicides in plants can also be produced from the sequences SEQ ID NO: 1 or SEQ ID NO: 3 by means of the so-called "site directed mutagenesis" Stability and / or enzymatic activity of enzymes or the properties such as binding of the above-mentioned inhibitors according to the invention can be very specifically improved or changed.
  • Zhu et al. (Nature Biotech., Vol. 18, May 5, 2000: 555-558) describes a "site directed mutagenisis" method in plants which can be used advantageously.
  • EP-A-0 909 821 describes a method for changing proteins using the microorganism E. coli 5 XL-1 Red. During replication, this microorganism generates mutations in the introduced nucleic acids and thus leads to a change in the genetic information. Isolation of the modified nucleic acids or the modified proteins and testing for resistance can easily identify advantageous nucleic acids and the proteins encoded by them. After introduction into plants, these can then express resistance there and thus lead to resistance to the herbicides.
  • mutagenesis and selection are, for example, methods such as the in vivo mutagenesis of seeds or pollen and selection of resistant alleles in the presence of the invention Inhibitors, followed by genetic and molecular identification of the altered, resistant alley. Furthermore, the mutagenesis and selection of resistances in cell culture by multiplying the culture in the presence of successively increasing concentrations of the inhibitors according to the invention. The increase in the spontaneous mutation rate can be exploited by chemical / physical mutagenic treatment. As described above, modified genes can also be isolated with microorganisms which have an endogenous or recombinant activity of the proteins coded by the nucleic acids used in the Q method according to the invention and which are sensitive to the inhibitors identified according to the invention. The cultivation of the microorganisms on media with an increasing concentration of inhibitors according to the invention allows the selection and evolution of 5 resistant variants of the targets according to the invention. The frequency of the mutations can in turn be increased by mutagenic treatments.
  • a further subject of the invention is therefore a method for creating nucleotide sequences which code for gene products which have a changed biological activity, the biological activity being changed in contrast to the fact that there is increased activity.
  • Increased activity is to be understood as an activity which is at least 10%, preferably at least 30%, particularly preferably at least 50%, very particularly preferably at least 100% higher than that of the starting organism or of the starting gene product.
  • the biological activity may have been changed so that the substances and / or agents according to the invention no longer or no longer correctly target the nucleic acid sequences. and / or bind the gene products encoded by them.
  • not more or no longer correct means that the substances have changed by at least 30%, preferably at least 50%, particularly preferably by at least 70%, very particularly preferably by at least 80% or not at all Bind nucleic acids and / or gene products in comparison to the starting gene product or the starting nucleic acids.
  • Yet another aspect of the invention therefore relates to a transgenic plant genetically modified by the method according to the invention described above.
  • transgenic plants which are resistant to the substances and / or agents comprising these substances found by the method according to the invention can also be produced by overexpression of the nucleic acids SEQ ID NO: 1 or SEQ ID NO: 3 used in the methods according to the invention.
  • a further subject of the invention is therefore a method for producing transgenic plants which are resistant to substances found by a method according to the invention, characterized in that nucleic acids with the sequences SEQ ID NO: 1 or SEQ ID NO: 3 overexpressed.
  • a similar method is described by way of example in Lermantova et al. Plant Physiol., 122, 2000: 75-83.
  • the present invention therefore furthermore includes the use of plants which have genes which have been hit by the T-DNA insertion and have the nucleic acid sequences SEQ ID NO: 1 or SEQ ID NO: 3 for the development of new herbicides.
  • Alternative methods for identifying the homologous nucleic acids for example in other plants with similar sequences, such as using transposons, are known to those skilled in the art.
  • This invention therefore also relates to the use of alternative insertion utagenesis methods for inserting foreign nucleic acid into the nucleic acid sequences SEQ ID NO: 1 or SEQ ID NO: 3, in sequences derived from these sequences on the basis of the genetic code and / or their derivatives in others Plants.
  • Another method variant for identifying polypeptides resistant to the inhibitors according to the invention with dehydroquinate dehydratase / shikimate dehydrogenase activity is based on the fact that the dehydroquinate dehydratase / shikimate dehydrogenase pathway occurs not only in plants but also in bacteria and fungi. Some of these microorganisms may now contain dehydroquinate dehydratase / shikimate dehydrogenase variants.
  • the method according to the invention for the targeted detection of said dehydroquinate dehydratase / shikimate dehydrogenase variants is based on the fact that an organism is incubated with an inhibitor identified using the method according to the invention. If there is no or only partial inhibition of growth, the dehydroquinate dehydratase / shikimate dehydrogenase is isolated from the said organism and characterized with regard to its nucleic acid sequence. Partial inhibition of growth is understood here to mean that the growth is reduced by only 50%, preferably 45%, particularly preferably 20%, compared to an organism which has not been incubated. If necessary, the existing resistance is reinforced by further mutations.
  • the methods for mutagenesis described above can be used here. Any organism that has enzymes of the Shikimate pathway can be used here. Bacteria, plants and fungi are particularly preferred.
  • the present invention furthermore relates to transgenic organisms, preferably plants, their reproductive material and their plant cells, tissue or parts, transformed with an expression cassette containing the sequence of a dehydroquinate dehydratase / shikimate dehydrogenase variant which is not inhibited by the inhibitors according to the invention.
  • the expression cassette is identical to the above-described embodiments of an expression cassette for the expression of the dehydroquinate dehydratase / shikimate dehydrogenase, except for the fact that instead of the nucleic acid sequence of the dehydroquinate dehydratase / shikimate dehydrogenase, said dehydroquinate dehydratase / shikimate drogenase is present.
  • transgenic plants are produced using one of the above-described embodiments of the expression cassette according to the invention using the common transformation methods also described above.
  • the effectiveness of the expression of the transgenically expressed dehydroquinate dehydratase / shikimate dehydrogenase gene can be determined, for example, in vitro by increasing the number of shoots or by a germination test.
  • a change in the type and level of expression of the dehydroquinate dehydratase / shikimate dehydrogenase gene and its effect on the resistance to inhibitors of dehydroquinate dehydratase / shikimate dehydrogenase on test plants can be tested in greenhouse experiments.
  • Another object of the invention relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or parts of plants.
  • the aim of the use is preferably to increase the dehydroquinate dehydratase / shikimate dehydrogenase content or the content of a polypeptide with dehydroquinate dehydratase / shikimate dehydrogenase activity in the plant.
  • the transgenic plants are produced as described above by transforming a plant with at least one expression cassette according to the invention or at least one vector according to the invention. However, increased expression can also be achieved by targeted mutagenesis of the promoter region of the respective natural dehydroquinate dehydratase / shikimate dehydrogenase gene.
  • the following further embodiments of the invention are also based on overexpression of the dehydroquinate dehydratase / shikimate dehydrogenase.
  • the overexpression of dehydroquinate dehydratase / shikimate dehydrogenase can be mediated by means of an expression cassette according to the invention or a vector according to the invention, each containing one of the nucleic acid sequences described above coding for a polypeptide with increased dehydroquinate dehydratase / shikimate dehydrogenase activity.
  • increased activity means that at least 10%, preferably by at least 30%, particularly preferably by at least 50%, very particularly preferably by at least one, compared to the dehydroquinate dehydratase / shikimate dehydrogenase encoded by SEQ ID NO: 1 or SEQ ID NO: 2 Understand 100% higher activity.
  • the dry matter of a plant can also be increased by increasing the chorismate and the aromatic amino acids. This leads to an increase in dry matter and increases the overall yield of the plants.
  • biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan in plants can be increased by overexpression of the dehydroquinate dehydratase / shikimate dehydrogenase.
  • Plants to be used preferably are crop plants, such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and and wine species as well as legumes.
  • crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and and wine species as well as legumes.
  • transgenic plants Depending on the choice of the promoter, the expression can take place specifically in the leaves, in the seeds or in other parts of the plant.
  • Such transgenic plants, their reproductive material and their plant cells, tissues or parts are a further object of the present invention.
  • the invention is illustrated by the following examples, but is not limited to these.
  • Cloning methods such as: restriction cleavage, DNA isolation, 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, recombinant sequence analysis DNA was according to Sambrook et al. , Cold Spring Harbor Laboratory Press (1989); ISBN 0-87969-309-6.
  • the transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Willmitzer (Nucl. Acid Res. 16, 9877 (1988)).
  • Agrobacteria were grown in YEB medium (Vervliet et al., Gen. Virol. 26, 33 (1975)).
  • the bacterial strains used below (E. coli, XL-I Blue) were obtained from Stratagene or Qiagen.
  • the Agrobacterium strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) was developed by Deblaere et al. in nucl. Acids Res. 13, 4777 (1985).
  • the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used.
  • the vectors pUC19 (Yanish-Perron, Gene 33, 103-119 (1985)) pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12,
  • the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
  • H0 Water system water treatment plant (Millipore, Eschborn).
  • DNA-modifying enzymes and molecular biological kits were developed by the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Röche (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach / Taunus), Novagen ( Madison, Wisconsin, USA), Perkin-Elmer (further adt), Pharmacia (Freiburg) Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise stated, they were used according to the manufacturer's instructions.
  • the dehydroquinate dehydratase / shikimate dehydrogenase was cloned from tobacco flowers using the RT-PCR method. A sequencing analysis confirmed that the dehydroquinate dehydratase / shikimate dehydrogenase is from tobacco. The following primers were used for this procedure:
  • the PCR product is 1088 base pairs in size and was used for the antisense and co-suppression inhibition of the dehydroquinate dehydratase / shikimate dehydrogenase gene.
  • the full-length clone was amplified using the PCR method from tobacco flower DNA.
  • This 1668 base pair long cDNA fragment contains an open reading frame of 1668 bases and codes for a protein with 556 amino acids.
  • the transit peptide belonging to the pre-protein was not cloned by this procedure.
  • Analyzes of the polypeptide using the GCG program (Oxford Molecular) showed a value of 100% identity at the nucleic acid and amino acid level with a protein from Nicotiana tabacum described in the database (Acc number: L 32794).
  • the 1088 base pair fragment of the Nicotiana tabacum dehydroquinate dehydratase / shikimate dehydrogenase was cloned in the sense orientation and in the antisense orientation into the binary vector pBINAR under the control of the 35S promoter, see Figure 6.
  • For cloning the dehydroquinate dehydratase / Shikimate dehydrogenase in the binary vector could use the BamHI interfaces specified by the primers.
  • the PCR product was purified using the Gene Clean Kit (Dianova GmbH, Hilden) and digested with BamHI.
  • the vector pBinl9AR was also cut with BamHI for ligation.
  • This construct was transformed into tobacco by Agrobacterium-mediated transformation. Regenerated plants were examined for dehydroquinate dehydratase / shikimate dehydrogenase mRNA amounts. All investigated antisense and sense plants with reduced dehydroquinate dehydratase / Shiki at dehydrogenase mRNA levels showed a clear phenotype. A strict correlation between the phenotype and the reduced amount of mRNA was found. Plants with reduced dehydroquinate dehydrogenase / Shikimate dehydrogenase mRNA showed mosaic leaves, reduced size - see Figures 2 to 4 - and died during plant development.
  • transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) tobacco leaf disks were transformed with sequences of the dehydroquinate dehydratase / shikimate dehydrogenase.
  • tobacco plants 10 ml of an overnight culture of Agrobacterium tumefaciens grown under selection were centrifuged off, 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.
  • RNA from plant tissues was determined as in Logemann et al., Anal. Biochem. 163, 21 (1987) isolated. For the analysis, 20 ⁇ g of RNA were separated in a 1.5% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond, Amersham). The detection of specific transcripts was carried out as described for Amasino (Anal. Biochem. 152,
  • the DNA fragments used as a probe were radioactively labeled with a random primed DNA labeling kit (Boehringer, Mannheim) and hybridized according to standard methods (see Hybond user instructions, Amersham). Hyridization signals were visualized by autoradiography using X-OMAT AR films from Kodak.
  • Figure 5 shows a Northern analysis of five tobacco plants (19-1, 19-4, 19-5, 83-2, 83-5), which contain a pBinAR antisense construct from DHD / SDH were transformed. As a control, the RNA from two wild-type plants is applied. The expression of DHD / SDH is reduced in the transgenic tobacco plants.
  • Wild type and transgenic DHD / SDH plants are shown in side view ( Figure 2) and in top view ( Figures 3 and 4). There is a clear inhibition of growth compared to the wild type ( Figure 2, wild type on the left). The reduced growth correlates with a decrease in DHD / SDH gene expression ( Figure 5 A and 5 B).
  • Figure 5 A shows Northern analyzes of transgenic DHD / SHD plants from the TI generation with strong phenotypic changes. The analysis shows that DHD / SHD gene expression is inhibited in plants with a strong phenotypic change.
  • Figure 5 B shows Northern analyzes of transgenic DHD / SHD plants of the T1 generation with a normal phenotype.
  • the formation of NADPH can be measured at an OD of 334 nm over 10 minutes.
  • the reaction is started by adding 1 microliter of the extracted crude protein.
  • the reaction buffer contains:
  • Another enzyme assay of dehydroquinate dehydratase / shikimate dehydrogenase is carried out by measuring both enzymes in a coupled back reaction:
  • Expression vectors are suitable for the expression of recombinant proteins in E. coli, but also baculovirus vectors for the expression of dehydroquinate dehydratase / shikimate dehydrogenase in insect cells (Gibco BRL).
  • Bacterial expression vectors are e.g. derived from pBR322 and carry a bacteriophage T7 promoter for expression.
  • the plasmid is propagated in an E. coli strain which carries an inducible gene for the T7 polymerase (e.g. JM109 (DE3); Promega).
  • the expression of the recombinant protein is activated via the induction of the T7 polymerase by IPTG.
  • IPTG-inducible systems from Quiagen (pQE vectors) or Novagen (pET vectors) are suitable. Depending on the interfaces available, there are vectors with different reading frames.
  • the full-length dehydroquinate dehydratase / shikimate dehydrogenase gene was cloned into the pQE vector ( Figure 7) and transformed into E. coli.
  • a single colony of this E. coli strain was transferred to "2xYT" (1 l: Bacto-trypton 16 g, yeast extract 10 g, 5 g NaCl, 5 mg, 50 mg / 1 ampicillin and 50 mg / 1 kanamycin per 1 liter) Incubated overnight at 37 ° C. The next day, 50 ml of 2 * YT were inoculated with 0.5 ml of the overnight culture and grown at 25 ° C. to an ODgoo of 0-6.
  • Figure 8 shows the expressed DHD / SDH protein with a size of approx. 60 kD in SDS-Page gel electrophoresis.
  • Lane 1 (left to right): protein markers, molecular weights from top to bottom: 97.4 KD; 66 KD; 46 KD; 30 KD; 21.5 KD and 14.3 KD
  • Lane 2 induced DHD / SHD protein (crude extract, denatured) in the presence of 2 mM IPTG, 37 ° C. molecular weight DHD / SHD: approx. 60 KD
  • Lane 4 induced DHD / SHD protein (crude extract, native) in the presence of 0.05 mM IPTG, 25 ° C
  • Lane 5 induced DHD / SHD protein (purified on Ni-NTA material, native)
  • Lemna inor is grown under non-sterile conditions in plastic trays in 17 mMOl / 1 MES buffer pH 5.5 + 1.5 mmol / 1 CaCl — 2 + 1 g / 1 Hakaphos special.
  • the Lemna cultures are washed and separated in 0.5 ml fresh nutrient solution in 48-well microtiter plates.
  • the active ingredients are dissolved in 5 mmol / 1 in DMSO and diluted 1: 5 in water. 2501 of this solution are used for the test.
  • the fluorescence of chlorophyll is measured during the treatment.
  • a herbicidal action can be detected by comparison with an untreated control and is abbreviated to H in Table 1.

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Abstract

L'invention concerne l'utilisation de séquences d'acides nucléiques, codant un polypeptide présentant l'activité de la déshydroquinate déshydratase/shikimate déshydrogénase, pour produire un système de test servant à trouver des inhibiteurs de la déshydroquinate déshydratase/shikimate déshydrogénase. Pour la première fois, on a montré au moyen de la technologie antisens que la déshydroquinate déshydratase/shikimate déshydrogénase constitue une cible d'herbicide. L'invention concerne en outre un procédé de production de plantes transgéniques qui contiennent une séquence d'acides nucléiques codant un polypeptide présentant l'activité de la déshydroquinate déshydratase/shikimate déshydrogénase (E.C. 4.2.1.10/E.C. 1.1.1.25) et qui sont caractérisées par une production de biomasse accrue et/ou une teneur plus élevée en acides aminés aromatiques, par rapport à une plante non transgénique.
EP01969344A 2000-07-07 2001-07-06 Deshydroquinate deshydratase/shikimate deshydrogenase utilisee comme cible d'herbicide Withdrawn EP1315808A2 (fr)

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US7319013B2 (en) * 2002-03-20 2008-01-15 Basf Aktiengesellschaft Serine hydroxymethyltransferase as a target for herbicides
WO2005068625A1 (fr) * 2004-01-05 2005-07-28 The Regents Of The University Of California Vegetaux modifies pour avoir des teneurs elevees en acide gallique, et procedes pour produire ces vegetaux
CA2891956A1 (fr) * 2012-11-20 2014-05-30 J.R. Simplot Company Insertion d'adn de transfert a mediation par tal
CN113174395B (zh) * 2021-04-30 2022-06-24 中国烟草总公司郑州烟草研究院 与烟气苯酚释放量相关的突变基因dhq-sdh1

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WO2000005386A2 (fr) * 1998-07-21 2000-02-03 E.I. Du Pont De Nemours And Company Enzymes impliquees dans la biosynthese du chorismate
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