EP1525304A1 - Cytochrome b5 reductase nadh-dependante utilisee comme cible pour herbicides - Google Patents

Cytochrome b5 reductase nadh-dependante utilisee comme cible pour herbicides

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Publication number
EP1525304A1
EP1525304A1 EP03764982A EP03764982A EP1525304A1 EP 1525304 A1 EP1525304 A1 EP 1525304A1 EP 03764982 A EP03764982 A EP 03764982A EP 03764982 A EP03764982 A EP 03764982A EP 1525304 A1 EP1525304 A1 EP 1525304A1
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European Patent Office
Prior art keywords
acid sequence
nucleic acid
seq
nadh
reductase
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German (de)
English (en)
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Andreas Reindl
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BASF SE
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BASF SE
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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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/004—Cytochrome-b5 reductase (1.6.2.2)
    • 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

Definitions

  • the present invention relates to the use of a polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase (EG 1.6.2.2), which causes growth retardation and chlorotic leaves in the absence of presence, and by the nucleic acid sequence SEQ ID NO: 1 or functional equivalents of the above Nucleic acid sequence is encoded as a target for herbicides.
  • SEQ ID NO: 1 nucleic acid sequence SEQ ID NO: 1 or functional equivalents of the above Nucleic acid sequence is encoded as a target for herbicides.
  • functional equivalents of SEQ ID NO: 1 are provided.
  • the present invention comprises the use of the polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase in a method for identifying compounds with herbicidal activity which inhibit NADH-dependent cytochrome b5 reductase.
  • the invention relates to the use of these compounds identified via the method as herbicides.
  • the object of the present invention is therefore to identify new targets which are essential for the growth of plants or whose inhibition for the plant lead to reduced growth, and to provide methods which are suitable for identifying compounds with herbicidal activity.
  • the object was achieved by using a polypeptide with the biological activity of an NADH-dependent cytochrome b5 reductase encoded by a nucleic acid sequence consisting of
  • nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 2 on the basis of the degenerate genetic code by back-translation;
  • nucleic acid sequence which can be derived on the basis of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 39%;
  • affinity tag denotes a peptide or polypeptide, the coding nucleic acid sequence of which can be fused with the nucleic acid sequence according to the invention directly or by means of a linker using common cloning techniques.
  • the affinity tag is used for the isolation, enrichment and / or targeted purification of the recombinant target protein by means of affinity chromatography from whole cell extracts.
  • the linker mentioned above can advantageously contain a protease interface (for example for thrombin or factor Xa), as a result of which the affinity tag can be split off from the target protein if necessary.
  • affinity tags examples include the "His tag” from Quiagen, Hilden, “Strep tag”, the “Myc tag” (Invitrogen, Carlsberg), the chitin-binding domain and an Intein tag from New England Biolabs, the maltose-binding protein (pMal) from New England Biolabs and the so-called CBD tag from Novagen.
  • the affinity tag can be at the 5 'or 3' end of the coding nucleic acid sequence with the sequence coding for the target protein.
  • An expression cassette contains a nucleic acid sequence according to the invention functionally linked with at least one genetic control element, such as a promoter, and advantageously with a further control element, such as a terminator.
  • the nucleic acid sequence of the expression cassette can be, for example, a genomic or a complementary DNA sequence or an RNA sequence as well as semisynthetic or fully synthetic analogues thereof. These sequences can be in linear or circular form, extra-chromosomal or integrated into the genome.
  • the corresponding nucleic acid sequences can be produced synthetically or obtained naturally, or contain a mixture of synthetic and natural DNA components, and consist of different heterologous gene segments from different organisms.
  • Artificial nucleic acid sequences are also suitable here as long as they enable the expression of a polypeptide encoded by a nucleic acid sequence according to the invention with the biological activity of a NADH-dependent cytochrome b5 reductase in a cell or an organism.
  • synthetic nucleotide sequences can be generated which have been optimized with regard to the codon usage of the organisms to be transformed.
  • nucleotide sequences mentioned above can be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleotide building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • various DNA fragments can be manipulated so that a nucleotide sequence with the correct reading direction and correct reading frame is obtained.
  • the nucleic acid fragments are connected to one another using general cloning techniques, as described, for example, in T.
  • a functional or operative linkage means the sequential arrangement of regulatory sequences or genetic control elements in such a way that each of the regulatory sequences or each of the genetic control elements can perform its function as intended when expressing the coding sequence.
  • “Functional equivalents” here describe nucleic acid sequences which hybridize under standard conditions with the nucleic acid sequence SEQ ID NO: 1 or parts of SEQ ID NO: 1 and are capable of expressing a polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase in a cell or an organism.
  • oligonucleotides For hybridization, it is advantageous to use short oligonucleotides with a length of about 10-50 bp, preferably 15-40 bp, for example of the conserved or other areas, which can be determined by comparison with other related genes in a manner known to the person skilled in the art.
  • longer fragments of the nucleic acids according to the invention with a length of 100-500 bp or the complete sequences for the hybridization can also be used.
  • the length of the fragment or the complete sequence or depending on the type of nucleic acid, i.e. DNA or RNA used for hybridization vary these standard conditions. For example, the melting temperatures for DNA: DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.
  • the hybridization conditions for DNA: DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 65 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between approximately 30 ° C.
  • These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for DNA hybridization are described in relevant textbooks on genetics, such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, for example, and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content. The specialist can obtain further information on hybridization from the following textbooks: Ausubel et al.
  • a functional equivalent of SEQ ID NO: 1 also means nucleic acid sequences which are homologous or identical to SEQ ID NO: 1 up to a defined percentage and furthermore in particular also natural or artificial mutations of the above-mentioned nucleic acid sequences which are necessary for a polypeptide encode with the biological activity of a NADH-dependent cytochrome b5 reductase.
  • the present invention also encompasses those nucleotide sequences which are obtained by modifying the abovementioned nucleic acid sequences.
  • modifications can be exemplified by techniques familiar to the person skilled in the art, such as “site directed mutagenesis”, “error prone PCR", “DNA shuffling” (Nature 370, 1994, pp. 389-391) or “staggered extension process” (Nature Biotechnol. 16, 1998, pp.258-261).
  • the aim of such a modification can e.g. the insertion of further restriction enzyme interfaces, the removal of DNA to shorten the sequence, the exchange of nucleotides for codon optimization or the addition of further sequences. Proteins that are encoded via modified nucleic acid sequences must still have the desired functions despite a different nucleic acid sequence.
  • the term functional equivalent can also refer to the amino acid sequence encoded by the corresponding nucleic acid sequence.
  • the term functional equivalent describes a protein whose amino acid sequence with SEQ ID NO: 2 is identical or homologous to a defined percentage.
  • Functional equivalents thus include naturally occurring variants of the sequences described here, as well as artificial nucleic acid sequences, for example those obtained by chemical synthesis and adapted to codon use, or the amino acid sequences derived therefrom.
  • Genetic control sequence describes sequences which have an influence on the transcription and, if appropriate, translation of the nucleic acids according to the invention in prokaryotic or eukaryotic organisms. Examples of this are promoters, terminators or so-called “enhancer” sequences.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified such that the natural regulation has been switched off and the expression of the target gene has been modified, that is to say increased or decreased.
  • control sequence is selected depending on the host organism or starting organism. Genetic control sequences also include the 5 'untranslated region, introns, or the 3' non-coding region of genes. Control sequences are also to be understood as those which enable homologous recombination or insertion into the genome of a host organism or which allow removal from the genome. Genetic control sequences also include further promoters, promoter elements or minimal promoters, and sequences influencing the chromatin structure (for example matrix attachment regions (MAR's)) which can modify the expression-controlling properties. Genetic control sequences can, for example, also result in tissue-specific expression depending on certain stress factors. Corresponding elements are, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991;
  • 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 length of the shorter of the two sequences, which by comparison using the program algorithm GAP (Wisconsin Package Version 10.2 Genetics Computer Group (GCG), Madison, Wisconsin, USA) setting the following parameters for polypeptides
  • Gap Weight 8 Length Weight: 2
  • Gap Weight 50 Length Weight: 3
  • Natural genetic environment means the natural chromosomal locus in the organism of origin.
  • the natural genetic environment of the nucleic acid sequence is preferably at least partially preserved.
  • the environment flanks the nucleic acid sequence at least on the 5 'or 3' side and has a sequence length of at least 50 bp, preferably at least 100 bp, particularly preferably at least 500 bp, very particularly preferably at least 1000 bp, most preferably at least 5000 bp.
  • Plants in the sense of the invention are plant cells, tissues, organs or whole plants such as seeds, bulbs, flowers, pollen, fruits, seedlings, roots, leaves, stems or other parts of plants. Plants are also understood to mean propagation material such as seeds, fruits, seedlings, cuttings, tubers, cuts or rhizomes.
  • Response time means the time it takes to complete an activity test until a significant statement about an activity is obtained and depends both on the specific activity of the protein used in the test and on the method used and the sensitivity of the devices used. The determination of the reaction times is known to the person skilled in the art. In methods based on photometric methods for identifying compounds with a herbicidal action, the reaction times are, for example, generally between> 0 to 120 minutes.
  • Recombinant DNA describes a combination of DNA sequences that can be produced by recombinant DNA technology.
  • Recombinant DNA technology generally known techniques for fusing DNA sequences (described, for example, in Sambrook et al., 1989, Cold Spring Habour, NY, Cold Spring Habour Laboratory Press).
  • Reporter genes code for easily quantifiable proteins. These genes can be used to carry out an evaluation of the transformation efficiency or of the expression site or time by means of growth, fluorescence, chemo, bioluminescence or resistance assay or by means of a photometric measurement (intrinsic color) or enzyme activity. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as "green fluorescence protein” (GFP) (Gerdes HH and Kaether C, FEBS Lett 1996; 389 (1): 44-47; Chui WL et al., Gurr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques.
  • GFP green fluorescence protein
  • Selection markers confer resistance to antibiotics or other toxic compounds: Examples include the neomycin phosphotransferase gene, which is resistant to the aminoglycoside antibiotics neomycin (G 418), kamanyne, paromycin (Deshayes A et al ., EMBO J. 4 (1985) 2731-2737), the sul gene coding for a mutated dihydropteroate synthase (Guerineau F et al., Plant Mol Biol. 1990; 15 (1): 127-136), the hygromycin B phosphotransferase Gene (Gen Bank Accession NO: K 01193) and the shble resistance gene, which is resistant to bleomycin antibiotics such as. Zeocin gives.
  • selection marker genes are genes which confer resistance to 2-deoxyglucose-6-phosphate (WO 98/45456) or phosphinotricin etc. or those which confer antimetabolite resistance, 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 US A. 85 (1988) 8047-8051).
  • Mannose phosphate isomerase (WO 94/20627), the ODC (ornithine decarboxylase) gene (McConlogue, 1987 in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed.) Or the deaminase from Aspergillus are also suitable terreus (Tamura K et al., Biosci Biotechnol Biochem. 59 (1995) 2336-2338).
  • Transformation describes a process for introducing heterologous DNA into a pro- or eukaryotic cell.
  • a transformed cell describes not only the product of the transformation process itself, but also all transgenic progeny of the transgenic organism produced by the transformation
  • Target / Target Protein a polypeptide encodes the nucleic acid sequence according to the invention, which can be an enzyme in the classical sense or e.g. a structural protein, a protein relevant for development processes, regulatory proteins such as transcription factors, kinases, phosphatases, receptors, subunits of channels, transport proteins, regulatory subunits which give an enzyme complex substrate or activity regulation. What all targets or sites of action have in common is that their functional presence is essential for survival or normal development and growth.
  • Transgene Relating to a nucleic acid sequence, an expression cassette or a vector containing a nucleic acid sequence according to the invention or an organism transformed with the abovementioned nucleic acid sequence, expression cassette or vector, the expression transgenic describes all such constructions which have been produced by genetic engineering methods and in which either the nucleic acid sequence of the target protein or a genetic control sequence functionally linked to the nucleic acid sequence of the target protein or a combination of the abovementioned possibilities are not in their natural genetic environment or have been modified by genetic engineering methods. The modification can be achieved here, for example, by mutating one or more nucleotide residues of the corresponding nucleic acid sequence.
  • the nucleic acid sequence SEQ ID NO: 1 codes for a cytochrome b5 reductase that is specifically dependent on NADH (E.G. 1.6.2.2).
  • Characteristic of higher eukaryotes is the electron transfer system located on the membrane of the endoplasmic reticulum and consists of a NADH-dependent cytochrome b5 reductase and cytochrome b5 (cytb ⁇ ).
  • the NADH-dependent cytochrome b5 reductase uses an FAD as a prosthetic group to transfer electrons from NADH to Cytb5, a protein containing heme.
  • Polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase thus denotes an enzyme which is able to transfer electrons from NADH to Cytb5, a protein containing heme, using a FAD as a prosthetic group.
  • the enzymatic activity of an enzyme with the biological activity of an NADH dependent cytochrome b5 reductase can be determined by means of suitable activity tests, as are described below by way of example (see also example 5).
  • Cytb ⁇ has been described as a component of electron transport in the modification of fatty acids (Kearns et al, 1991; Spektrum Akademischer Verlag Heidelberg, Berlin, Oxford, page 751). The electrons are then likely to be transferred from cytochrome b5 to desaturases or P450 monooxygenases (Fukuchi-Mizutani, Plant Physiology, 119; 353-361; 1999). Plant-based NADH-dependent cytochrome b5 reductases are found in almost all cell types, especially in immature seeds (Fukuchi-Mizutani, Plant Physiology, 119; 353-361; 1999).
  • NADH-dependent cytochrome b5 reductase was isolated and characterized for the first time from human erythrocytes (Yubisui T, Takeshita M., J Biol Chem., 1980; 255 (6): 2454-1456) and since then also from many other organisms.
  • Nucleic acid sequences of plant-dependent NADH-dependent cytochrome b5 reductases are known, e.g. from Arabidopsis (Gen Bank Acc. No. AB007799; Mizutani and Fukuchi-Mizutani, Plant Physiol.
  • NADH-dependent cytochrome b5 reductase from human erythrocytes can be inhibited by millimolar concentrations of inositol hexaphosphate (Palmieri et al, Archives of Biochemistry and Biophysics, 1990, 280 (1), 224-228).
  • Thenoyltrifluoroacetone shows a 50% inhibition of the NADH-dependent cytochrome b5 reductase from rat liver microsomes at 0.5 mM (Golf et al., Biol. Chem. Hoppe- Seyler 1985, 366, 647-653).
  • NADH-dependent cytochrome b5 reductase Other substances which can inhibit NADH-dependent cytochrome b5 reductase from various organisms are Amytal, Mepacrin, Dicoumarol (Golf et al; 1985, Biol. Chem. Hoppe-Seyler, 366, pp. 647-653), or N-ethylmaleimides and Atebrin (Tamura et al; 1983, J. Biochem. 94, pp. 1547-1555). However, inhibitors for plant-based NADH-dependent cytochrome b5 reductases have not yet been described.
  • the present invention relates to the use of a polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase encoded by a nucleic acid sequence consisting of
  • nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 2 on the basis of the degenerate genetic code by back-translation;
  • nucleic acid sequence which can be derived on the basis of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 39%;
  • the functional equivalents according to c) are characterized by essentially the same functionality, i.e. they have the physiological function of an NADH-dependent cytochrome b5 reductase.
  • SEQ ID NO: 1 The functional equivalents of SEQ ID NO: 1 according to the invention have a homology with SEQ ID No: 1 of at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, preferably at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76% particularly preferred at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • SEQ ID NO: 2 The functional equivalents of SEQ ID NO: 2 according to the invention have a homology with SEQ ID No: 2 of at least 39%, 40%, 41%, 42%, " 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% preferably at least 60%, 61%, 62%, 63%, 64 %, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% , 81% o, 82%, 83%, 84%, 85%, 86% particularly preferably at least 87%, 88%, 89%, 90%, 91%, 92%, 93% very particularly preferably at least 94%, 95 %, 96%, 97%, 98%), 99%.
  • Examples of functional equivalents are the plant nucleic acid sequences already encoding NADH-dependent cytochrome b5 reductase or amino acid sequences of a NADH-dependent cytochrome b5 reductase from Medicago truncatula (Gen Bank Acc. No. AA660929; Covitz, PA et al. Plant Physiol. 117 (4), 1325-1332 (1998)), Oryza sativa (Gen Bank Acc. No. BE039960), Solanum tuberosum (Gen Bank Acc. No. BE340917), Beta vulgaris (Gen Bank Acc. No. BI096337) and Pumpkin (Cucurbita maxima; Gen Bank Acc. No. AF274589).
  • nucleic acid sequences which code for a polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase, comprising a partial area, are claimed within the scope of the present invention.
  • nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 4 on the basis of the degenerate genetic code by back-translation;
  • nucleic acid sequence which can be derived on the basis of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 87%.
  • polypeptides encoded by the aforementioned nucleic acid sequences are also claimed.
  • the functional equivalents are characterized by a essentially the same functionality, ie they have the physiological function of a NADH-dependent cytochrome b5 reductase.
  • nucleic acid sequences or “comprising” based on nucleic acid sequences means that the nucleic acid sequence according to the invention can contain additional nucleic acid sequences at the 3 'or at the 5' end, the length of the additional nucleic acid sequences 75bp at the 5 'and 50bp 3' end of the nucleic acid sequences according to the invention, preferably does not exceed 50bp at the 5 'and 10bp at the 3' end.
  • SEQ ID NO: 3 The functional equivalents of SEQ ID NO: 3 according to the invention have a homology with SEQ ID No: 3 of at least 77%, 78%, 79%, 80%, preferably at least 81%), 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90% particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • SEQ ID NO: 4 The functional equivalents of SEQ ID NO: 4 according to the invention have a homology with SEQ ID No: 4 of at least 87%), preferably at least 88%), 89%), 89%> preferably at least 90%, 91%, 92%, 93 % particularly preferably at least 94%>, 95%, 96% very particularly preferably at least 97%, 98%, 99%.
  • nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 2 on the basis of the degenerate genetic code by back-translation;
  • nucleic acid sequence which can be derived on the basis of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 39%;
  • Nucleic acid sequences coding for a polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase containing a partial region a) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 3; or
  • Codes can be derived by back-translation from the amino acid sequence shown in SEQ ID NO: 4; or
  • nucleic acid sequence which can be derived on the basis of the degenerated genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 87%;
  • nucleic acid sequences according to the invention are referred to below as “nucleic acid sequences according to the invention”.
  • the polypeptides encoded by a nucleic acid sequence according to the invention with the biological activity of an NADH-dependent cytochrome b5 reductase are referred to below as "NCR" for the sake of simplicity.
  • NCR cause growth retardation in reduced amounts as well as necrotic leaves in plants.
  • a reduction in the polypeptide means that the amount of the polypeptide is reduced using genetic engineering methods.
  • a plant modified in this way is compared with a plant which has no genetic modifications with respect to this polypeptide, but is otherwise identical to the genotype of the genetically manipulated plant under identical growth conditions.
  • the gene products of the nucleic acids according to the invention represent new targets for herbicides which make it possible to provide new herbicides for controlling unwanted plants.
  • undesirable plants are understood to mean all plants that grow up in places where they are undesirable, for example:
  • SEQ ID NO: 1 or SEQ ID NO: 3 or parts of the above-mentioned nucleic acid sequences can be used for the production of hybridization probes, via which e.g. the corresponding full length genes and / or functional equivalents of SEQ ID NO: 1 or SEQ ID NO: 3 can be isolated.
  • the manufacture of these probes and the conduct of the experiments are known. This can be done, for example, by the targeted production of radioactive or non-radioactive probes by means of PCR and the use of appropriately labeled oligonucleotides with subsequent hybridization experiments. The technologies required for this are described, for example, in T. Maniatis, E.F. Fritsch and J.
  • the corresponding probes can also be modified using standard technologies (Lit. SDM or random mutagenesis) so that they can be used for other purposes, e.g. as a probe that hybridizes specifically to mRNA and the corresponding coding sequences for the purpose of analyzing the corresponding sequences in other organisms.
  • the above-mentioned probes can be used for the detection and isolation of functional equivalents of SEQ ID NO: 1 or SEQ ID NO: 3 from other plant species on the basis of sequence identities.
  • part or all of the sequence of the corresponding SEQ ID NO: 1 or SEQ ID NO: 3 is used as a probe for screening in a genomic or cDNA bank of the corresponding plant species or in a computer search for sequences of functional equivalents in electronic databases ,
  • Preferred plant species are the undesired plants already mentioned at the beginning.
  • the invention furthermore relates to expression cassettes containing
  • a nucleic acid sequence which, owing to the degenerate genetic code, is converted back from the amino acid sequence shown in SEQ ID NO: 4. lets lead; or functional equivalents of the nucleic acid sequence SEQ ID NO: 3 with an identity of at least 86% to SEQ ID NO: 3; a nucleic acid sequence which can be derived from the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 87%;
  • an expression cassette according to the invention comprises a promoter at the 5 'end of the coding sequence and a transcription termination signal at the 3' end and optionally further genetic control sequences which are functionally linked to the nucleic acid sequence according to the invention in between.
  • the expression cassettes according to the invention are also to be understood as analogs which can come about, for example, from a combination of the individual nucleic acid sequences on a polynucleotide (multiple constructs), on several polynucleotides in a cell (co-transformation) or through sequential transformation.
  • Advantageous genetic control sequences according to point a) for the expression cassettes according to the invention or for vectors containing expression cassettes according to the invention are, for example, promoters such as cos, tac, trp, tet, Ipp, lac, laclq, T7, T5, T3 , gal, trc, ara, SP6, ⁇ -PR or in the ⁇ -PL promoter, which can be used to express the NCR in gram-negative bacterial strains.
  • promoters such as cos, tac, trp, tet, Ipp, lac, laclq, T7, T5, T3 , gal, trc, ara, SP6, ⁇ -PR or in the ⁇ -PL promoter, which can be used to express the NCR in gram-negative bacterial strains.
  • Further advantageous genetic control sequences are, for example, in the amy and SPO2 promoters, which can be used to express the NCR in gram-positive bacterial strains, and in the yeast or fungal promoters AUG1, GPD-1, PX6, TEF, CUP1, PGK, GAP1, TPI, PHO5, AOX1, GAL10 / CYC1, CYC1, OliC, ADH, TDH, Kex2, MFa or NMT or combinations of the above promoters (Degryse et al., Yeast 1995 Jun 15; 11 (7): 629-40; Romanos et al. Yeast 1992 Jun; 8 (6): 423-88; Benito et al. Eur. J. Plant Pathol.
  • Suitable genetic control sequences for expression in insect cells are the polyhedrin promoter and the p10 promoter (Luckow, V.A. and Summers, M.D. (1988) Bio / Techn. 6, 47-55).
  • Advantageous genetic control sequences for the expression of the NCR in cell culture are in addition to polyadenylation sequences such as e.g. from Simian Virus 40 eukaryotic promoters of viral origin such as e.g. Promoters of polyoma, adenovirus 2, cytomegalovirus or simian virus 40.
  • Simian Virus 40 eukaryotic promoters of viral origin such as e.g. Promoters of polyoma, adenovirus 2, cytomegalovirus or simian virus 40.
  • Promoters of viral origin are particularly preferred, such as the promoter of the 35S transcript of the cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294; Odell et al., Nature 313 (1985), 810-812).
  • Further preferred constitutive promoters are, for example, the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al., Plant Mol Biol 1995, 29: 637- 649), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991).
  • the expression cassettes can also contain a chemically inducible promoter as a genetic control sequence, by means of which the expression of the exogenous gene in the plant can be controlled at a specific point in time.
  • a chemically inducible promoter such as, for example, the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), one that can be induced by salicylic acid (WO 95/19443), one that can be induced by benzenesulfonamide (EP-A-0388186), one which is inducible by tetracycline (Gatz et al., (1992) Plant J. 2, 397404), one which is inducible by abscisic acid (EP-A 335528) or one by ethanol or cyclohexanone inducible (WO 93/21334) promoter can also be used.
  • promoters which express tissue or organ-specific expression e.g. mediate in anthers, ovaries, flowers and flower organs, leaves, guard cells, trichomes, stems, lead tissues, roots and seeds.
  • tissue or organ-specific expression e.g. mediate in anthers, ovaries, flowers and flower organs, leaves, guard cells, trichomes, stems, lead tissues, roots and seeds.
  • those promoters which ensure leaf-specific expression are also suitable.
  • the promoter of the cytosolic FBPase from potato WO 97/05900
  • the SSU promoter small subunit
  • the Rubisco ribulose-1, 5-bisphosphate carboxylase
  • ST-LSI promoter from potato
  • Seed-specific promoters are, for example, the promoter of phaseolin (US 5,504,200, Bustos MM et al., Plant Cell. 1989; 1 (9): 839-53), of the 2S albuming gene (Joseffson LG et al., J Biol Chem 1987 , 262: 12196-12201), leguminum (Shirat A et al., Mol Gen Genet. 1989; 215 (2): 326-331), USP (unknown seed protein; Bäumlein H et al., Molecular & General Genetics 1991, 225 (3): 459-67) of the Napin gene (Stalberg K, et al., L.
  • phaseolin US 5,504,200, Bustos MM et al., Plant Cell. 1989; 1 (9): 839-53
  • the 2S albuming gene Joseffson LG et al., J Biol Chem 1987 , 262: 12196-12201
  • leguminum Shirat
  • sucrose binding protein WO 00/26388
  • LeB4 promoter Baumlein H et al., Mol Gen Genet 1991, 225: 121-128; Fiedler, U. et al., Biotechnology (NY) (1995), 13 (10) 1090.
  • promoters suitable as genetic control sequences are, for example, specific promoters for tubers, storage roots or roots, such as, for example, the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato, the promoter of the starch synthase (GBSS1) or the sporamine promoter, fruit-specific promoters, such as the fruit-specific promoter from tomato (EP-A 409625), fruit-ripening-specific promoters, such as the fruit-ripening-specific promoter from tomato (WO 94/21794), flower-specific promoters such as the phytoene synthase promoter (WO 92 / 16635) or the promoter of the P-rr gene (WO 98/22593) or specific plastid or chromoplast promoters, such as the RNA polymerase promoter (WO 97/06250) or the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max (see also
  • Additional functional elements b) are to be understood as examples, but not by way of limitation, of reporter genes, origins of replication, selection markers and so-called affinity tags, fused with the NCR directly or optionally containing a protease interface by means of a linker.
  • Other suitable additional functional elements are sequences which ensure targeting in the apoplasts, plastids, the vacuole, the mitochondrium, the peroxisome, the endoplasmic reticulum (ER) or, due to the lack of corresponding operative sequences, a retention in the compartment of formation, the cytosol (Kermode, Grit. Rev. Plant Sei. 15, 4 (1996), 285-423).
  • Vectors according to the invention also contain at least one copy of the nucleic acid sequences according to the invention and / or the expression cassettes according to the invention.
  • 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 can be 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.
  • the expression cassette according to the invention and vectors derived therefrom can be used for the transformation of bacteria, cyanobacteria, yeast, filamentous fungi and algae and eukaryotic, non-human cells (for example insect cells) with the aim of recombinantly producing the NCR, the production of which a suitable expression cassette according to the organism in which the gene is to be expressed.
  • 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, they can all 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.
  • nucleic acid (s) according to the invention, the expression cassette or the vector can be introduced into the corresponding organisms (transformation) by all methods known to the person skilled in the art.
  • Suitable methods are the biolistic method or by protoplast transformation (cf. e.g. Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New Y-ork-Basel-Cambridge), electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the gene transfer mediated by Agrobacterium. The methods mentioned are published, for example, in B.
  • the transformation using agrobacteria and the vectors to be used for the transformation are known to the person skilled in the art and are described in detail in the literature (Bevan et al., Nucl. Aeids Res. 12 (1984) 8711.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E.
  • coli and agrobacteria contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (; Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187), EP A 0 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V; Fraley et al., Grit. Rev. Plant. Sci., 4: 1-46 and An et al. EMBO J. 4 (1985) 277-287).
  • Agrobacteria transformed with a vector according to the invention can also be used in a known manner to transform plants such as test plants such as Arabidopsis or crops such as cereals, corn, oats, rye, barley, wheat, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes, carrots, peppers, rapeseed, tapioca, cassava, arrowroot, tagetes , Alfaifa, lettuce and the various tree, nut and wine species can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • test plants such as Arabidopsis or crops such as cereals, corn, oats, rye, barley, wheat, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes, carrots, peppers, rapeseed, tapioca, cassava, arrowroot, tage
  • the genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by S.D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer can be found.
  • transgenic organisms produced by transformation with one of the above-described embodiments of an expression cassette containing a nucleic acid sequence according to the invention or a vector containing the abovementioned expression cassette and the recombinant NCR obtainable by expression from the transgenic organism are the subject of the present invention.
  • the present invention also relates to the use of transgenic organisms containing an expression cassette according to the invention, e.g. for the provision of recombinant protein and / or the use of these organisms in in vivo test systems.
  • yeasts, mosses, algae and fungi preferred organisms for recombinant expression are also eukaryotic cell lines.
  • mosses are Physcomitrella patens or other mosses described in Kryptogamen, Vol. 2, Moose, Farne, 1991, Springer Verlag (ISBN 3540536515).
  • bacteria of the genus Escherichia, Erwinia, Flavobacterium, Alcaligenes or Cyanobacteria, for example of the genus Synechocystis or Anabena are preferred.
  • yeasts are Candida, Saccharomyces, Schizosaccheromyces, Hansenula or Pichia.
  • Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria, Mortierella, Saprolegnia, Pythium, or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995).
  • Preferred plants are selected in particular from monocotyledonous crop plants, such as, for example, cereals such as wheat, barley, millet, rye, ritical, maize, rice or oats, and sugar cane.
  • the transgenic plants according to the invention are selected in particular from dicotyledonous crop plants, such as, for example, Brassicacae such as rape, cress, Arabidopsis, Kohiart or Canola; Leguminosae such as soybean, alfalfa, pea, bean family or peanut solanaceae such as potato, tobacco, tomato, eggplant or paprika; Asteraceae such as sunflower, tagetes, lettuce or calendula; Cucurbitaceae such as melon, pumpkin or zucchini, or flax, cotton, hemp, flax, red pepper, carrot, carrot, sugar beet or various tree, nut and wine species.
  • Brassicacae such as rape, cress, Arabidopsis, Kohiart or Canola
  • Leguminosae such as soybean, alfalfa, pea, bean family or peanut solanaceae such as potato, tobacco, tomato, eggplant or paprika
  • Asteraceae such as sunflower,
  • transgenic animals such as C. elegans are also suitable as host organisms.
  • the typical advantageous, commercially available fusion and expression vectors 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 "pKK233-2" from CLONTECH, Palo Alto, CA and the "pET” and the "pBAD” vector series from Stratagene, La To call Jolla.
  • vectors for use in yeast are pYepSed (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan 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” (Invitrogen Corporation, San Diego, CA).
  • Vectors for use in filamentous fungi are described in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge.
  • insect cell expression vectors can also be used advantageously, for example for expression in Sf9, Sf21 or Hi5 cells, which are infected via recombinant baculoviruses.
  • These are, for example, the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • the Baculovirus expression systems "MaxBac 2.0 Kit” and "Insect Select System” from Invitrogen, Calsbald or “BacPAK Baculovirus Ex- pression system "from CLONTECH, Palo Aito, CA.
  • Insect cells are particularly suitable for overexpression of eukaryotic proteins, since they carry out post-translational modifications of the proteins which are not possible in bacteria and yeasts.
  • the handling of insect cells in cell culture and their infection for the expression of Proteins are known to the person skilled in the art and can be carried out analogously to known methods (Luckow and Summers, Bio / Tech. 6, 1988, pp.47-55; Glover and Harnes (eds) in DNA Cloning 2, A practical Approach, Expression Systems, Second Edition, Oxford University Press, 1995, 205-244).
  • plant cells or algal cells can advantageously be used for gene expression.
  • plant expression vectors 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, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721.
  • nucleic acid sequences according to the invention can be expressed in mammalian cells.
  • Examples of corresponding expression vectors are pCDM ⁇ and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EM-BO 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.
  • 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).
  • transgenic organism according to the invention The organisms transformed with an expression cassette according to the invention are referred to under the term “transgenic organism according to the invention”.
  • Another object of the present invention is the use of NCR in a method for identifying compounds with herbicidal activity.
  • the method according to the invention for identifying compounds having a herbicidal action preferably comprises the following steps:
  • the detection according to step (ii) of the above method can be carried out using techniques which show the interaction between protein and ligand.
  • Either the test compound or the enzyme may contain a detectable label, e.g. a fluorescent, radioisotope, chemiluminescent or enzymatic label.
  • a detectable label e.g. a fluorescent, radioisotope, chemiluminescent or enzymatic label.
  • enzymatic labels are horseradish peroxidase, alkaline phosphatase or lucifierase.
  • the subsequent detection depends on the marking and is known to the person skilled in the art.
  • FCS fluorescence correlation spectroscopy
  • Test connections is displaced ("displacement assay").
  • the compounds identified in this way can be suitable as inhibitors.
  • the fluorescence polarization uses the property of a quiescent fluorophore excited with polarized light also to emit polarized light again. However, if the fluorophore can rotate during the excited state, the polarization of the emitted fluorescent light is more or less lost. Under otherwise identical conditions (e.g. temperature, viscosity, solvent), the rotation is a function of the molecular size, which means that the measurement signal provides information about the size of the residue bound to the fluorophore. (Methods in Enzymology 246 (1995), pp. 283-300).
  • a method according to the invention can be set up directly for measuring the binding of a test compound marked by a fluorescent molecule to the NCR. Alternatively, the method according to the invention can also be designed in the form of the "displacement assay" described under 1. The compounds identified in this way can be suitable as inhibitors.
  • Fluorescence Resonance Energy Transfer is based on the radiationless energy transfer between two spatially adjacent fluorescence molecules under suitable conditions. A prerequisite is the overlap of the
  • the fluorescence labeling of the NCR and the bonded tet compound can be measured using FRET (Cytometry 34, 1998, pp. 159-179).
  • FRET Fluorescence labeling 34, 1998, pp. 159-179
  • the method according to the invention can also be designed in the form of the "displacement assay” described under 1.
  • a particularly suitable embodiment of the FRET technology is the "Homogeneous Time Resolved Fluorescence" (HTRF), as sold by Packard BioScience. The compounds identified in this way can be suitable as inhibitors.
  • the measurement of surface plasmon resonance is based on the change in the refractive index on a surface when a test 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) 186-187). For example, the measurement can be carried out in one throughput with the aid of the automated analyzers based on surface pasmon resonance sold by Biacore (Freiburg) of currently up to 384 samples per day.
  • Biacore Biacore
  • a method according to the invention can be set up directly for measuring the binding of a test compound to the NCR.
  • the method according to the invention can also be designed in the form of the "displacement assay" described under 1.
  • the compounds identified in this way can be suitable as inhibitors.
  • a preferred embodiment of the method according to the invention which is based on steps i) and ii), consists in that
  • an NCR is expressed in a transgenic organism according to the invention or an organism which naturally contains an NCR is cultivated;
  • the NCR from step i) is brought into contact with a test compound in the cell disruption of the transgenic or non-transgenic organism, in partially purified form or in a form purified to homogeneity;
  • a compound is selected which reduces or blocks the activity of the NCR, the activity of the NCR incubated with the test compound being determined with the activity of an NCR not incubated with a test compound.
  • the NCR-containing solution can consist of the lysate of the original organism or of the transgenic organism transformed with an expression cassette according to the invention. If necessary, the NCR can be partially or completely cleaned using common methods.
  • common methods for protein purification can be found, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley- Interscience (1994); ISBN 0-87969-309-6.
  • the protein fused to an affinity tag can be purified using affinity chromatography known to those skilled in the art.
  • the purification of the NCR from human tissue can, for example, by the method of Jollie et al. (Plant Physiol. 85, pp. 457-462, 1987).
  • the NCR required for in vitro methods can thus be isolated either by heterologous expression from a transgenic organism according to the invention or from an organism which contains an NCR activity, preferably from an undesired plant, the term “undesired plant” being used at the beginning mentioned species are to be understood.
  • the NCR is now incubated with a test compound. After a reaction time, the activity of the NCR incubated with the test compound is determined with the activity of an NCR not incubated with a test compound.
  • NCR is inhibited, a significant decrease in activity compared to the activity of the non-inhibited polypeptide according to the invention is observed, a decrease of at least 10%, advantageously at least 20%, preferably at least 30%, particularly preferably by at least 50% up to a 100 % Reduction (blocking) is achieved.
  • at least 50% inhibition at concentrations of the test compound of 10 ⁇ M, preferably at 10 ⁇ M, particularly preferably of 10 ⁇ M based on enzyme concentration in the micromolar range.
  • the determination of the enzymatic activity of the NCR can be carried out, for example, using an activity test in which the increase in the product, the decrease in the substrate (or starting material) or the decrease in a specific cofactor, or in a combination of at least two of the aforementioned parameters depending on a defined one Period of time can be determined.
  • suitable substrates are, for example, ferricytochrome b5, potassium ferricyanide, 2,6-dichlorophenolindophenol, methemerythrin, p-benzoquinone or 5-hydroxy-1, 4-naphthoquinone, preferably ferricytochrome b5, potassium ferricyanide, 2,6-dichlorophenolindophenol, particularly preferably ferricytochrome b5, potassium ferricyanide very particularly preferably potassium ferricyanide and, for suitable cofactors, NADH.
  • derivatives of the abovementioned compounds which contain a detectable label such as, for example, a fluorescent, radioisotopic or chemiluminescent label, can also be used.
  • the amounts of substrate to be used for the activity test are between 0.5-10 mM and amounts of NADH between 0.1-5 mM based on 1-10 ⁇ jt / g / ml enzyme.
  • the conversion of a substrate is monitored photometrically, based on a method described by Mihara and Sato (Methods Enzymol., 52, 1978, pp. 102-108), which is based on the reduction of potassium ferrcyanide and the photometric measurement based at 420 nm.
  • a preferred embodiment of the method according to the invention which is based on steps i) and iii), consists of the following steps:
  • test compounds that cause a reduced growth or limited survivability of the non-transgenic organism compared to the growth of the transgenic organism.
  • the polypeptide with the biological activity of an NCR is overexpressed in the transgenic organism according to i).
  • the transgenic organism thus has an increased NCR activity compared to a non-transgenic organism, wherein with increased NCR activity of the transgenic organism one compared to the non-transgenic organism of the same genus by at least 10%, preferably by at least 25%, particularly preferably by at least 40%, particularly preferred to understand at least 50% higher activity.
  • the growth difference in step iv) for selecting an inhibitor with a herbicidal action is at least 10%, preferably 20%, preferably 30%, particularly preferably 40% and very particularly preferably 50%.
  • the transgenic organism here is a bacterium, a yeast, a fungus, a plant or a eukaryotic cell line (from insects or mammals such as, for example, mouse), preferably plants, bacteria or yeasts, which can be easily transformed using common techniques, such as Arabidopsis thaliana, Solanum tuberosum, Nicotiana Tabacum or Saccharomyces cerevisiae in which the poly- peptide coding sequence was incorporated via transformation. These transgenic organisms therefore have an increased tolerance to compounds which inhibit the polypeptide according to the invention.
  • Saccharomyces cerevisiae is particularly useful here because its genome is completely sequenced and it can easily be used to produce "knock-out” mutants and the analog NCR gene present in this organism can be specifically switched off (eg Methods in Yeast Genetics, Kaiser, Michaelis, Mitchell (eds.) CSHL Press, Cold Spring Harbor Laboratory Press, 1994: 73-85).
  • the above-mentioned embodiment of the method according to the invention can, however, also be used to identify compounds with a growth regulatory effect.
  • a plant is used as a transgenic organism.
  • the method for identifying compounds with growth regulatory activity thus comprises the following steps:
  • the polypeptide with the biological activity of an NCR is overexpressed in the transgenic plant according to i).
  • the transgenic plant thus has an increased NCR activity compared to a non-transgenic plant of the same genus, with an increased NCR activity of the transgenic plant compared to a non-transgenic plant of the same genus by at least 10%, preferably by at least 25%. , particularly preferably by at least 40%, very particularly preferably by at least 50% higher activity.
  • test compounds are selected which bring about a change in the growth of the non-transgenic organism compared to the growth of the transgenic organism.
  • Modified growth is to be understood as an inhibition of the vegetative growth of the plants, which can manifest itself in particular in a reduction in the growth in length.
  • the treated Plants accordingly have a compact stature; a darker leaf coloration can also be observed.
  • test compounds can also be used in one inventive method. If the target is influenced by a group of test compounds, then it is either possible to directly isolate the individual test compounds or to divide the group of test compounds into different subgroups, e.g. if it consists of a large number of different components, so as to reduce the number of different test compounds in the method according to the invention.
  • the method according to the invention is then repeated with the individual test compound or the corresponding subgroup of test compounds.
  • the steps described above can be repeated several times, preferably until the subgroup identified according to the method according to the invention comprises only a small number of test compounds or only one test compound.
  • All of the compounds or substances identified using the method according to the invention can then be checked for their herbicidal activity in vivo.
  • One way of testing the compounds for herbicidal activity is to use the Lemna minor duckweed in microtiter plates. Changes in chlorophyll content and photosynthesis performance can be measured as parameters. It is also possible to apply the compound directly to undesired plants, the herbicidal action e.g. about limited growth can be determined.
  • the method according to the invention can also advantageously be carried out in high-throughput methods, so-called HTS, which enables parallel testing of a large number of different connections.
  • HTS high-throughput methods
  • the use of carriers which carry one or more of the nucleic acid molecules according to the invention, one or more vectors containing the nucleic acid sequence according to the invention, one or more transgenic organisms which contain at least one of the nucleic acid sequences according to the invention or one or more (poly ) contain peptides encoded via the nucleic acid sequences according to the invention.
  • the carrier used can be solid or liquid, is preferably solid, particularly preferably a microtiter plate.
  • the above-mentioned carriers are also the subject of the present invention.
  • microtiter plates According to the most widespread technique, 96-well, 384-well and 1536-well microtiter plates are used, which can usually contain volumes of 200 // l.
  • the other components of a HTS system are commercially available to match the corresponding microtiter plates, such as many instruments, materials, automatic pipetting devices, robots, automated plate readers and plate washers.
  • the invention further relates to compounds with herbicidal activity identified by the processes according to the invention.
  • These compounds are referred to below as "selected compounds". They have a molecular weight of less than 1000 g / mol, advantageously less than 500 g / mol, preferably less than 400 g / mol, particularly preferably less than 300 g / mol.
  • Compounds with herbicidal activity have a Ki value of less than 1 mM, preferably less than 1 ⁇ M, particularly preferably less than 0.1 ⁇ M, very particularly preferably less than 0.01 ⁇ U.
  • the selected compounds can of course also be present in the form of their agriculturally useful salts.
  • Agriculturally useful salts include, in particular, the salts of those cations or the acid addition salts of those acids whose cations or anions do not adversely affect the herbicidal activity of the selected compounds.
  • the selected compounds contain asymmetrically substituted ⁇ -carbon atoms, either as racemates, mixtures of enantiomers, pure Enantiomers or, if they have chiral substituents, also exist as mixtures of diastereomers.
  • the selected compounds can be chemically synthesized or microbiologically produced substances and can occur, for example, in cell extracts from, for example, plants, animals or microorganisms.
  • the reaction mixture can be a cell-free extract or can comprise a cell or cell culture. Suitable methods are known in the art and are generally described in Alberts, Molecular Biology the cell, 3rd Edition (1994), for example chapter 17.
  • the selected compounds from comprehensive substance libraries come for example.
  • test compounds can be expression libraries such as cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs , Cell. 79 (1994), 193-198 and references cited therein).
  • the selected compounds can be used to control undesired plant growth, under certain circumstances also for defoliation, for example of potatoes, or desiccation, for example of cotton, and as growth regulators.
  • Herbicidal compositions containing the selected compounds control plant growth very well on non-cultivated areas. In crops such as wheat, rice, maize, soybeans and cotton, they act against weeds and grass weeds without significantly damaging the crop plants. This effect occurs especially at low application rates.
  • the selected compounds can be used to control the harmful plants already mentioned above.
  • selected compounds or herbicidal compositions containing them can advantageously also be used in a further number of crop plants for eliminating unwanted plants.
  • the following crops are considered, for example:
  • the selected compounds can also be used in crops which are tolerant to the action of herbicides by breeding, including genetic engineering methods. The preparation of these cultures is described below.
  • the invention further relates to a process for the preparation of the herbicidal composition already mentioned above, characterized in that selected compounds are formulated with suitable auxiliaries to give crop protection agents.
  • the selected compounds can e.g. be formulated in the form of directly sprayable aqueous solutions, powders, suspensions, including high-proof aqueous, oily or other suspensions or suspoemulsions or dispersions, emulsifiable concentrates, emulsions, oil dispersions, pastes, dusts, sprinkling agents or granules, and by spraying, atomizing, dusting, Scattering or pouring can be applied.
  • the application forms depend on the intended use and the nature of the selected compounds and should in any case ensure the finest possible distribution of the selected compounds.
  • the herbicidal composition contains a herbicidally effective amount of at least one selected compound and auxiliaries customary for the formulation of herbicidal compositions.
  • emulsions, pastes or aqueous or oil-containing formulations and dispersible concentrates the selected compounds can be dissolved or dispersed in an oil or solvent, it being possible to add further formulation auxiliaries for homogenization.
  • liquid or solid concentrates which are suitable for dilution with water can also be prepared from selected compound, optionally solvents or oil and optionally further auxiliaries.
  • EC, EW emulsifiable concentrates
  • SC suspensions
  • SL soluble concentrates
  • DC dispersible concentrates
  • pastes, pastilles, wettable powders or granules emulsifiable concentrates
  • Corresponding powders or granules or Tablets can also be provided with a solid coating which prevents abrasion or premature release of the active ingredient ("coating").
  • auxiliary means the following classes of compounds: anti-foaming agents, thickeners, wetting agents, adhesives, dispersants, emulsifiers, bactericides and / or thixotrophic agents.
  • anti-foaming agents thickeners, wetting agents, adhesives, dispersants, emulsifiers, bactericides and / or thixotrophic agents.
  • SLs, EWs and ECs can be produced by simply mixing the corresponding ingredients, powder by mixing or grinding in special mill types (e.g. hammer mills).
  • DC, SCs and SEs are usually produced by wet milling, it being possible to produce an SE from a SC by adding an organic phase which may contain further auxiliaries or selected compounds.
  • the manufacture is known.
  • Powders, materials for broadcasting and dusts can advantageously be prepared by mixing or grinding the active compounds together with a solid carrier.
  • Granules, e.g. Coating, impregnation and homogeneous granules can be produced by binding the selected compounds to solid carriers. Further details of the production are known to the person skilled in the art, and e.g.
  • liquid additives such as medium to high boiling point mineral oil fractions such as kerosene or diesel oil, also coal tar oils as well as oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, e.g.
  • Solid carriers are, for example, mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, loess, clay, dolomite, diatomaceous earth. de, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas and vegetable products such as cereal flour, tree bark, wood and nutshell flour, cellulose powder or other solid carriers.
  • mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, loess, clay, dolomite, diatomaceous earth. de, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas and vegetable products such as cereal flour, tree
  • surfactants suitable for the formulations according to the invention are known to the person skilled in the art, such as, for example. Alkali, alkaline earth, ammonium salts of aromatic sulfonic acids, e.g.
  • the herbicidal compositions or the selected compounds can be applied pre- or post-emergence. If the selected compounds are less compatible with certain crop plants, application techniques can be used in which the selected compounds are sprayed with the aid of sprayers in such a way that the leaves of the sensitive crop plants are not hit, if possible, while the selected compounds are applied to the leaves underneath growing unwanted plants or the uncovered floor area (post-directed, lay-by).
  • the application rates of selected compounds are 0.001 to 3.0, preferably 0.01 to 1.0 kg / ha, depending on the control target, season, target plants and growth stage.
  • the provision of the herbicidal target further enables a method for identifying a protein with the biological activity of an NCR which is not or only to a limited extent inhibited by a herbicide which has the NCR as the site of action, for example the herbicidally active selected compounds.
  • a protein which differs from the NCR is referred to as an NCR variant, which is encoded by a nucleic acid sequence which i) encodes a polypeptide with the biological activity of a NADH-dependent cytochrome b5 reductase which is not inhibited by substances having a herbicidal action which inhibit NCR and have been determined by the abovementioned methods; and
  • the above-mentioned method for generating nucleic acid sequences encoding NCR variants of nucleic acid sequences comprises the following steps:
  • SEQ ID NO: 1 The functional equivalents of SEQ ID NO: 1 according to the invention have a homology with SEQ ID No: 1 of at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, preferably at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76% particularly preferred at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%), 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%., 98%, 99%.
  • SEQ ID NO: 2 The functional equivalents of SEQ ID NO: 2 according to the invention have a homology with SEQ ID No: 2 of at least 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48 %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% preferably at least 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%), 80% , 81%, 82%, 83%, 84%, 85%, 86% particularly preferably at least 87%, 88%, 89%, 90%), 91%>, 92%, 93% very particularly preferably at least 94%, 95%, 96%, 97%, 98%, 99%.
  • the sequences selected according to the method described above are advantageously introduced into an organism.
  • Another object of the invention is therefore an organism produced by this method.
  • the organism is preferably a plant, particularly preferably one of the crop plants defined above.
  • Modified proteins and / or nucleic acids which can impart resistance to the selected compounds in plants, can also be produced from the nucleic acid sequences mentioned above by means of the so-called "site directed mutagenesis".
  • This mutagenesis can, for example, improve the stability and / or activity of the Target proteins or the properties such as binding and action of the above-mentioned inhibitors according to the invention can be specifically improved or changed.
  • Zhu et al. (Nature Biotech., Vol. 18, May 2000: 555-558) describes a "site directed mutagenesis" method in plants which can be used advantageously.
  • this microorganism creates mutations in the introduced nucleic acids and thus leads to a change in the genetic information.
  • isolating the modified nucleic acids or the modified proteins and testing for resistance it is easy to identify advantageous nucleic acids and the proteins encoded by them. These can then express resistance there after introduction into plants 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 inhibitors according to the invention, followed by genetic and molecular identification of the modified, resistant alleis. Furthermore, 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 using microorganisms which have an endogenous or recombinant activity of the proteins coded by the nucleic acids used in the 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 resistant variants of the targets according to the invention. The frequency of the mutations can in turn be increased by mutagenic treatments.
  • Another object of the invention is therefore a method for producing nucleic acid 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 means that at least 10% of the parent organism or the parent gene product is preferably by at least 30%, particularly preferably by at least 50%, very particularly preferably by at least 100% higher activity.
  • the biological activity may have been changed so that the substances and / or agents according to the invention no longer or no longer bind correctly to the nucleic acid sequences and / or the gene products encoded by them.
  • no longer or no longer correctly 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 which has been transformed with a nucleic acid sequence which codes for a gene product which has an altered biological activity or with a nucleic acid sequence which codes for an NCR variant.
  • Methods for transformation are known to the person skilled in the art and are exemplified above.
  • transgenic plants which are resistant to the substances and / or agents containing these substances found by the method according to the invention can also be produced by transformation followed by overexpression of a nucleic acid sequence according to the invention.
  • a further subject of the invention is therefore a method for producing transgenic plants which are resistant to substances which have been found by a method according to the invention, characterized in that nucleic acids coding for an NCR variant are overexpressed in these plants.
  • a similar method is described by way of example in Lermantova et al., Plant Physiol., 122, 2000: 75-83.
  • Protein or nucleic acid such as RNA or DNA is changed so that the changed structure no longer has a herbicidal effect, that is to say the interaction of the herbicide with the target site can no longer take place.
  • transgenic plants are produced using one of the above-described embodiments of the expression cassette according to the invention using common transformation methods also described above.
  • the effectiveness of the expression of the transgenically expressed NCR can be determined, for example, in vitro by proliferation or by a germination test.
  • a change in the type and level of expression of the NCR gene and its effect on the resistance to inhibitors of the NCR on test plants can be tested in greenhouse experiments.
  • the invention is further illustrated by the following examples, which should not be construed as limiting.
  • Cloning methods such as RestrictionNC switching, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of Escherichia coli cells, cultivation of bacteria 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) and Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6.
  • the bacterial strains used below (E. coli DH5 ⁇ , XL-1 blue, XL10 Gold, BL21DE (3), JM 109) were obtained from Stratagene, BRL Gibco or Invitrogen, Carlsberg, CA.
  • the primer pair Rei 156 / Rei 157 became a cDNA sequence coding for NCR from Arabidopsis thaliana (accession number AB007799; Mizutani.M. And Fukuchi-Mizutani.M., 1997)
  • Rei 156 5'-TATACCCGGGATGGATACCGAGTTTCTCCGAA-3 '(SEQ ID NO: 5) and
  • Rei 157 5 - TATACCCGGGGAACTGGAATTGCATCTCCGGA-3 '(SEQ ID NO: 6).
  • the primers Rei 156/157 were then used in a PCR reaction to amplify a cDNA fragment from an Arabidopsis thaliana cDNA library (Stratagene). The PCR was carried out according to the conditions listed in Table 1. Table 1
  • the vector pBinAR (Höfgen and Willmitzer 1990, Plant Science 66, 221-230) was cleaved with Smal and ligated with the NCR fragment isolated from the vector pPCRScript-NCR via Smal (pBinAR-NCR).
  • the ligation mixture was transformed into XL10 Gold E. coli cells and clones containing pBinAR-NCR were identified using a digoxigenin-labeled NCR probe (Röche) with the aid of anti-digoxigenin antibodies.
  • the detection of the antisense orientation of the NCR cDNA in pBinAR-NCR was carried out by sequencing and by PCR reactions with 2 different primer pairs (Rei 143 and Rei 196, or Rei 144 and Rei 195) according to the conditions listed in Table 2.
  • the antisense orientation of the NCR could match the primers
  • the construct pBinAR-NCR was transformed into agrobacterial strain pGV 2260.
  • a positively transformed agrobacterial colony was used to transform Arabidopsis plants.
  • the detection of the presence of the construct pBinAR-NCR in an agrobacterial colony was carried out via PCR with the primers Rei 156 and Rei 157 via PCR according to the conditions listed in Table 2. An amount of an agrobacterial colony taken directly from the agar plate served as the DNA template.
  • a 4 ml LB medium culture (LB medium: 10 g / l peptone, 5 g / l yeast extract, 10 g / l NaCl; pH 7.0; 80 mg / l kanamycin) was taken from a single colony of positively transformed agrobacteria on a plate / I and 25 mg / l rifampicin), incubated overnight at 28 ° C.
  • a 400 ml LB medium culture (LB medium with 80 mg kanamycin / ml and 25 mg / ml rifampicin) was subsequently inoculated with this culture. After 12 hours of incubation at 28 ° C.
  • Plants grown on the selection plates were placed on soil after 3-4 weeks and incubated for 4-8 weeks in long-term conditions in climatic chambers (22-24 ° C during the day, 19 ° C at night; 65% relative humidity). The seeds were harvested after 6 weeks.
  • the integration of the antisense NCR gene into the genome of the transgenic plants was verified by PCR according to the conditions listed in Table 3.
  • Genomic DNA (isolation using the "DNeasy Plant Mini” kit from QIAGEN according to the manufacturer's instructions), which was prepared from leaf material of the corresponding transgenic lines, served as a template.
  • the NCR antisense pBinAR construct used for the transformation served as a positive control.
  • the intrinsic genomic gene which contains an intron and is therefore longer than the antisense NCR cDNA, as well as the antisense NCR cDNA itself could be detected by targeted selection of the primers.
  • the expected fragment length for the genomic NCR was approximately 1800 bp when using the primers
  • Transgenic plants which contained the construct pBinAR-NCR Antisense, showed high anthocyanin accumulations, i.e. heavily stressed leaves and veins, chlorotic leaves and a drastic growth delay: Transgenic plants (TO generation) only had 1-10% of the fresh mass of wild type plants 6 weeks of cultivation on earth in long-term conditions in climatic chambers (daytime 22-24 ° C, night 19 ° C; 65% relative humidity. The seeds that developed in the pods of the plants of the transformed TO generation were stunted and germinated in 100 % of all cases not.
  • NCR is suitable as a herbicide target.
  • an Arabidopsis cDNA coding for an NCR (Genbank Accession Number: AB007799) was overexpressed in E. coli bacteria.
  • the nucleic acid sequence coding for NCR was determined according to standard conditions via PCR (for example according to Sambrook, J. et al. (1989) "Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press; 34 cycles; annealing temperature 60 ° C; polymerization time 2 min) with pBinAR-NCR as template and the primers
  • NCR The activity of the NCR was compared with NCR which was recombinantly expressed according to Example 4 (Fukuchi-Mizutani et al., Plant Physiol. 119, pp. 353-361, 1999) or according to the method described by Jollie et al. (Plant Physiol. 85, pp. 457-462, 1987) was isolated, determined by the method of Mihara and Sato (Methods Enzymol., 52, 1978, pp. 102-108).
  • the reduction of potassium ferricyanide at 420 nm and 25 ° C. over a period of 5 to 15 minutes is measured photometrically.
  • Example 6 Identification of a functional analogue from tobacco
  • cDNA bank a cDNA library (hereinafter referred to as "binary cDNA bank") in a vector which can be used directly for the transformation of plants
  • mRNA was isolated from various plant tissues and analyzed using the TimeSaver cDNA synthesis kit (Amersham Pharmacia Biotech, Freiburg) rewritten into double-stranded cDNA.
  • the first strand cDNA synthesis was performed with T 2 . 18 oligonucleotides carried out according to the manufacturer's instructions.
  • the method according to Kohci et al, 1995, Plant Journal 8, 771-776 was used, the cDNA being amplified by PCR with the oligonucleotide N1 under the conditions listed in Table 4.
  • the PCR product obtained was bound to the column matrix of the PCR purification kit (Qiagen, Hilden) and eluted with 300 mM NaP buffer, pH 7.0, 0.5 mM EDTA, 0.04% SDS.
  • the DNA was denatured in a boiling water bath for 5 minutes and then renatured at 60 ° C. for 24 hours. 50 l of the DNA were applied to a hydroxyl pathite column and this was washed 3 times with 1 ml of 10 mM NaP buffer, pH 6.8.
  • the bound single-stranded DNA was eluted with 130 mM NaP buffer, pH 6.8, precipitated with ethanol and dissolved in 40 // I water. Of these, 20 / I were used for a further PCR amplification as described above. After further enrichment of ssDNA, a third PCR amplification was carried out as described above.
  • the plant transformation vector for recording the cDNA population prepared as described above was produced by restriction enzyme digestion of the vector pUC18 with Sbfl and BamHI, purification of the vector fragment followed by filling in the overhangs with Pfu DNA polymerase and religation with T4 DNA ligase (Stratagene).
  • the construct thus produced is referred to below as pUC18Sbfl-.
  • the vector pBinAR was first cleaved with Notl, religated after filling in the ends, cleaved with Sbfl, religated after filling in the ends and then cleaved with EcoRI and HindIII.
  • the resulting fragment was ligated into a derivative of the binary plant transformation vector pPZP (Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) Plant Mol Biol 25: 989-994), which enables plants to be transformed using Agrobacterium and mediated kanamycin resistance in transgenic plants.
  • the construct generated here is referred to below as pSun12 / 35S.
  • pUC18Sbfl- was used as a template for a polymerase chain reaction (PCR) with the oligonucleotides V1 and V2 (see Table 3) and Pfu DNA polymerase.
  • PCR polymerase chain reaction
  • the resulting fragment was ligated into the Smal digested pSun12 / 35S, generating pSunblues2. After cleavage with Notl, dephosphorylation with
  • pSunblues2 was ligated with the normalized and also NotL-cleaved cDNA population. After transformation in E.coli XMblue (Stratagene), the clones generated in this way were placed in microtiter plates.
  • the binary cDNA bank contains cDNAs in “sense” and in “antisense” orientation under the control of the flower Kohlmosaikvirus 35s Promotors, which can lead to "Cosuppressions” and "Antisene” effects after the transformation in tobacco plants.
  • NCR A sequence coding for NCR was identified using a digoxygenin-labeled probe produced using the DIG DNA Labeling Mix (Röche, Mannheim) according to the manufacturer's instructions, the plasmid pMAL-c2x-NCR under standard conditions using PCR with the primers
  • Rei 111 S'-ATGGATACCGAGTTTCTCCGAA-S '(SEQ ID NO: 21) and
  • the probe thus produced was used to screen the Nicotiana tabacum cDNA bank.
  • the cDNA bank was plated out with a titer of 2.5 ⁇ 10 5 plaque-forming units and analyzed using the plaque screening method (T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1989). 12 phage populations were isolated, with which a second screening was carried out, whereby genetically uniform phage populations could be isolated, which were used for in vivo excision. Restriction analysis showed no differences between the cDNA clones and four clones with the largest insertions were selected for sequencing. The sequence data of these clones gave SEQ ID NO: 3, which is 77% identical to SEQ ID NO: 1.

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Abstract

La présente invention concerne l'utilisation comme cible pour herbicides d'un polypeptide présentant l'activité biologique d'une cytochrome b5 réductase NADH-dépendante (E.C. 1.6.2.2), dont l'absence entraîne des retards de croissance ainsi que l'apparition de feuilles chlorotiques, et qui est codée par la séquence nucléotidique correspondant à SEQ ID NO:1 ou par des équivalents fonctionnels de cette séquence. Dans ce contexte, les équivalents fonctionnels de la séquence correspondant à SEQ ID NO:1 sont donnés. L'invention concerne également l'utilisation de ce polypeptide présentant l'activité biologique d'une cytochrome b5 réductase NADH-dépendante dans un procédé d'identification de composés ayant un effet herbicide qui inhibe la cytochrome b5 réductase NADH-dépendante. L'invention concerne en outre l'utilisation des composés ayant été identifiés, selon ledit procédé, comme herbicides.
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US8895596B2 (en) 2010-02-25 2014-11-25 Merck Sharp & Dohme Corp Cyclic benzimidazole derivatives useful as anti-diabetic agents
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RU2015140066A (ru) 2013-02-22 2017-03-30 Мерк Шарп И Доум Корп. Противодиабетические бициклические соединения
WO2014139388A1 (fr) 2013-03-14 2014-09-18 Merck Sharp & Dohme Corp. Nouveaux dérivés d'indole utiles en tant qu'agents antidiabétiques
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US11072602B2 (en) 2016-12-06 2021-07-27 Merck Sharp & Dohme Corp. Antidiabetic heterocyclic compounds
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