EP1587920A1

EP1587920A1 - Malate dehydrogenase as a target for herbicides

Info

Publication number: EP1587920A1
Application number: EP03789308A
Authority: EP
Inventors: Thomas Ehrhardt; Andreas Reindl; Annette Freund; Ralf-Michael Schmidt; Kirsten Deist; Uwe Sonnewald; Marc Stitt Nigel; Wolfgang Lein; Frederik BÖRNKE
Original assignee: BASF SE
Current assignee: BOERNKE, FREDERIK; DEIST, KIRSTEN; Ehrhardt Thomas; Freund Annette; Lein Wolfgang; REINDL, ANDREAS; Schmidt Ralf-Michael; SONNEWALD, UWE; Stitt Nigel Marc; BASF SE
Priority date: 2002-12-20
Filing date: 2003-12-17
Publication date: 2005-10-26
Also published as: US20060058190A1; AU2003293906A1; CA2510561A1; WO2004058956A1; JP2006512069A

Abstract

The invention relates to the use of malate dehydrogenase, the absence of which causes growth retardation and chlorotic leaves and which is coded by the nucleic acid sequence SEQ ID NO:2 or a functional equivalent thereof, as a target for herbicides. Novel nucleic acid sequences comprising SEQ ID NO:2 and functional equivalents of SEQ ID NO:2 are provided in said framework. The invention also relates to the use of said malate dehydrogenase and the functional equivalents thereof in a method for identifying compounds having a herbicidal or growth-regulating effect and the use of the compounds identified via said method as herbicides or growth regulators.

Description

Malate dehydrogenase as a target for herbicides

description

The present invention relates to the use of malate dehydrogenase, which causes growth retardation and chlorotic leaves in the absence of presence and is encoded by the nucleic acid sequence SEQ ID NO: 2 or a functional equivalent of this nucleic acid sequence as a target for herbicides. In this context, new nucleic acid sequences comprising SEQ ID NO: 2 and functional equivalents of SEQ ID NO: 2 are provided. Furthermore, the present invention relates to the use of the abovementioned malate dehydrogenase and its functional equivalents in a method for identifying compounds having a herbicidal or growth-regulating action, and to the use of these compounds identified via the method as herbicides or growth regulators.

The basic principle of identifying herbicides by inhibiting a defined target is known (for example US 5,187,071, WO 98/33925, WO 00/77185). In general, there is a great need to detect enzymes that could be new targets for herbicides. The reasons for this are resistance problems of herbicidal active substances which act on known targets and the constant effort to identify new herbicidal active substances which are distinguished by the broadest possible range of action, ecological and toxicological compatibility and / or low application rates.

The detection of new targets is associated with great difficulties in practice because the inhibition of an enzyme, which is part of a metabolic pathway, often no longer influences the growth of the plant. This may be due to the fact that the plant switches to alternative metabolic pathways, the existence of which is not known, or that the inhibited enzyme is not limiting for the pathway. Furthermore, plant genomes are characterized by a large functional redundancy. In the genome of Arabidopsis thaliana, functionally equivalent enzymes are more common in gene families compared to insects or mammals (Nature, 2000, 408 (6814): 796-815). This assumption is confirmed experimentally by the fact that large gene knock-out programs by T-DNA or transposon insertion in Arabidopsis have so far produced less pronounced phenotypes than expected (Curr. Op. Plant Biol. 4, 2001, pp.111- 117).

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 used to identify compounds with herbicidal and / or growth regulators - toric effect are suitable.

The object was achieved by using malate dehydrogenase in a method for identifying herbicides.

At this point, further terms used in the description are now defined.

"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 to isolate, enrich and / or purify the recombinant target protein by means of affinity chromatography from whole cell extracts. The linker mentioned above can advantageously contain a protease interface (e.g. for thrombin or factor Xa), as a result of which the affinity tag can be cleaved from the target protein if necessary. Examples of common affinity tags are the "His tag" e.g. by 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 attached at the 5 'or 3' end of the coding nucleic acid sequence with the sequence coding for the target protein.

"Biological activity of a glyoxysomal malate dehydrogenase": In the context of the present invention, this term describes that the presence of the glyoxysomal malate dehydrogenase imparts growth and survivability. If the activity of a protein with the biological activity of a glyoxysomal malate dehydrogenase is inhibited, this leads to reduced growth, a growth arrest or a death of the plant.

"Enzymatic activity of a glyoxysomal malate dehydrogenase": The term enzymatic activity describes the ability of an enzyme to convert a substrate into a product. The enzymatic activity can be determined in a so-called activity test via the increase in the product, the decrease in the substrate (or starting material) or the decrease in a specific cofactor or via a combination of at least two of the above-mentioned parameters depending on a defined period of time. "Enzymatic activity of a glyoxysomal malate dehydrogenase" here means the ability of an enzyme to also catalyze a reaction catalyzed by the glyoxysomal malate dehydrogenase. "Expression cassette": 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 enzymatic activity of a glyoxysomal malate dehydrogenase, preferably the biological activity of a glyoxysomal malate dehydrogenase in a cell or an organism. For example, synthetic nucleotide sequences can be generated which have been optimized with regard to the codon usage of the organisms to be transformed.

All of the 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). When preparing an expression cassette, 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. 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 (1994).

"Functional link": A functional or operative link is understood to mean 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 has its function in the expression of the coding sequence can fulfill as intended.

"Functional equivalents" here describe nucleic acid sequences which hybridize under standard conditions with the nucleic acid sequence SEQ ID NO: 2 or parts of SEQ ID NO: 2 and are capable of expressing a polypeptide with the enzymatic, preferably biological activity of a glyoxysomal malate dehydrogenase in a cell or an organism.

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 those skilled in the art. However, 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. Depending on the nucleic acid / oligonucleotide 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.

Under standard hybridization conditions, for example, depending on the nucleic acid, temperatures between 42 and 58 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide such as 42 ° C in 5 x SSC, 50% formamide. The hybridization conditions for DNA: DNA hybrids are advantageously at 0.1 × SSC and temperatures between approximately 20 ° C. to 65 ° C., preferably between approximately 30 ° C. to 45 ° C. For DNA: RNA hybrids, the hybridization conditions are advantageously 0.1 × SSC and temperatures between approximately 30 ° C. to 65 ° 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 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, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be made 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 person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), 1985, "Current Protocols in Molecular Biology", John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Ap- proach, IRL Press at Oxford University Press, Oxford.

A functional equivalent of SEQ ID NO: 2 also means nucleic acid sequences which are homologous or identical to SEQ ID NO: 2 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 enzymatic, preferably biological activity of a glyoxysomal malate dehydrogenase.

The present invention also encompasses, for example, nucleotide sequences which are obtained by modification of the nucleic acid sequences mentioned above. Such 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. In this case, the term functional equivalent describes a protein whose amino acid sequence with SEQ ID NO: 3 is identical or homologous to a defined percentage.

Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. nucleic acid sequences obtained by chemical synthesis and adapted to the use of codons 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. In addition to these control sequences or instead of these 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. The 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;

266 (26): 17131-17135), cold and dry stress (Plant Cell 1994, (6): 251-264) and. Heat stress (Molecular & General Genetics, 1989, 217 (2-3): 246-53).

"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 is determined by comparison using the BESTFIT program algorithm (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA setting the following parameters for polypeptides

Gap Weight: 8 Length Weight: 2

Average Match: 2,912 Average Mismatch: -2 _. 003

and the following parameter for nucleic acids

Gap Weight: 50 Length Weight: 3

Average Match: 10,000 Average Mismatch: -9,000

is calculated.

Instead of the term "homologous" or "homology", the term identity is also used synonymously below.

“Mutations” of nucleic acid or amino acid sequences include substitutions (= replacements), additions (additions), deletions (deletions), inversions (changes) or insertions (insertions) of one or more nucleotide residues, which also means that the corresponding amino acid sequence of the target protein can be determined Substitution, insertion or deletion of one or more amino acids can change, but overall the functional properties of the target protein are essentially retained. "Natural genetic environment" means the natural chromosomal locus in the organism of origin. In the case of a genomic library, 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 for a test to determine the enzymatic activity to obtain significant information about an enzymatic activity 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 (e.g. described in Sambrook et al., 1989, Cold Spring Habour, NY, Cold Spring Habour Laboratory Press).

"Replication origins" ensure the multiplication of the expression cassettes or vectors according to the invention in microorganisms and yeasts, e.g. the pBR322 ori or the P15A ori in E. coli (Sambrook et al .: "Molecular Cloning. A Laboratory Manual", 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and the ARS1 ori in yeast ( Nucleic Acids Research, 2000, 28 (10): 2060-2068).

"Reporter genes" code for easily quantifiable proteins. These genes can be used to evaluate the transformation efficiency or the location or time of expression by means of growth, fluorescence, chemo-, bioluminescence or resistance assay or via photometric measurement (intrinsic color) or enzyme activity. 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. 23 (5): 912-8, 1997), chloramphenicol acetyl transferase, a luciferase (Giacomin, Plant Sei 1996, 116: 59-72; Scikantha, J Bact 1996, 178: 121; Millar et al., Plant Mol Biol Rep 1992 10: 324-414), and lziferase genes, in general the β-galactosidase or the β-glucuronidase (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or the Ura3 gene.

"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), kanamycin, 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. Further examples of 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 U S A. 85 (1988) 8047-8051). The gene of 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 are also suitable from Aspergillus 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 classic sense or, for example, 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 that give an enzyme complex a 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": In relation 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 produced by genetic engineering methods 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.

Malate dehydrogenase catalyzes the reversible, pyridine nucleotide-dependent conversion of oxaloacetate and malate. Due to the implementation of these important metabolites of primary metabolism, the reaction catalyzed by malate dehydrogenase is of importance for various processes (review in Faske et al. 1997, Plant Physiology 115, 705-715). NAD ⁺ -dependent malate dehydrogenases can be found in mitochondria, micro-bodies such as peroxysomes or glyoxysomes and the cytosol, as well as in root nodules, while chloroplasts contain an NADP ⁺ -dependent malate dehydrogenase that is regulated depending on the light. Malate dehydrogenases are encoded in Arabidopsis by a gene family. The individual isoforms contain specific N-terminal transit sequences for transport into the chloroplasts, mitochondria and microbodies. There are also sequences for malate dehydrogenases that presumably remain in the cytosol due to the lack of transit peptides. Sequences coding for malate dehydrogenases were isolated from various C3 and C4 plants (see Miller et al. 1998 Plant Journal 15, 173-184). Nucleic acid sequences coding for glyoxysomal malate dehydrogenase and protein sequences from watermelon (P19446, Swissprot; identity to SEQ ID NO: 2 = 77.5%; identity to Seq ID NO: 3 = 85.4%), cucumber, Arabidopsis, soya and Rice.

The antisense inhibition of a cytosolic malate dehydrogenase in potatoes up to 40% residual activity did not lead to pronounced growth phenotypes and no reduced tuber yield (Jenner et al. 2001, Plant Physiology 126, 1139-1149). According to Faske et al. (1997, Plant Physiology 115, 705-715), the reduction in the expression of a plastid malate dehydrogenase from tobacco by antisense and cosuppression in transgenic tobacco plants does not affect the vitality of the transgenic plants, even with a greatly reduced malate dehydrogenase expression (<20% residual activity). Only a slight change in the growth parameters (fresh weight, dry weight) was observed. The attempted overexpression of a plastid malate dehydrogenase from sorghum in the C4 plant Flaveria led to a drastic inhibition of malate dehydrogenase activity to 5-50% by cosuppression. Residual activity (Trevanion et al. 1997, Plant Physiology 113, 1153-1165) without significant effects on the vitality of the plants.

In the context of the present invention, it was surprisingly found that plants in which glyoxysomal malate dehydrogenase was specifically reduced had phenotypes which are comparable with phenotypes produced by herbicide application. Drastic growth retardations and damage such as chlorosis and necrosis were observed.

Although eytosolic and plastid malate dehydrogenase is not essential for plants, these enzymes can in principle also be used to determine inhibitors of glyoxysomal malate dehydrogenase, since the reaction catalyzed by eytosolic and plastid malate dehydrogenase is the same as that catalyzed by glyoxysomal malate dehydrogenase ,

The present invention relates to the use of malate dehydrogenase in a method for identifying herbicides, preferably vegetable malate dehydrogenase, particularly preferably vegetable malate dehydrogenase encoded by a nucleic acid sequence comprising

a) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 1; or

b) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 2; or

c) a nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 3 on the basis of the degenerate genetic code by back-translation; or

d) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 63%,

whereby the functional equivalents according to c) and d) are characterized by the same functionality, i.e. they have the enzymatic activity of a glyoxsysomal malate dehydrogenase.

The term “comprising” or “comprising” based on nucleic acid sequences means that the nucleic acid sequence according to the invention has additional ends at the 3 'or 5' May contain nucleic acid sequences, the length of the additional nucleic acid sequences not exceeding 500 bp at the 5 'and 500 bp 3' end of the nucleic acid sequences according to the invention, preferably 250 bp at the 5 'and 250 bp at the 3' end, particularly preferably 100 bp at the 5 'and 100 bp at the 3 'end.

Examples of functional equivalents according to d) are also the plant nucleic acid sequences coding for malate dehydrogenase from cucumber (Gen Bank Accession Number: L31900) Arabidopsis (Gen Bank Accession Number: AC005671) Soya (Gen Bank Accession Number: L01628) and rice (Gen Bank Accession Number : D85763) and the nucleic acid sequence which is encoded by the amino acid sequence of a malate dehydrogenase from watermelon (Accession Number: P19446, Swissprot).

All of the above-mentioned sequences are also the subject of the present invention.

The functional equivalent of SEQ ID NO: 3 according to d) according to the invention has a homology with SEQ ID No: 3 of at least 63%, 64%, 65%, 66%, preferably at least 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% preferably at least 75%, 76%, 77%, 78%, 79%, 80% preferably at least 81%, 82%, 83%, .84%, 85%, 86% , 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93% particularly preferably at least 94%, 95%, 96%, 97%, 98%, 99%.

The use of glyoxysomal malate dehydrogenase in a method for identifying herbicides encoded by a nucleic acid sequence is particularly preferred

d) a nucleic acid sequence, which is due to the degenerate genetic code by back-translating the amino acid sequence of a functional Equivalents of SEQ ID NO: 3, which have an identity with SEQ ID NO: 3 of at least 66%, can be derived.

The functional equivalents according to d) are characterized by the same functionality, i.e. they have the biological function of a glyoxysomal malate dehydrogenase.

The nucleic acid sequences mentioned above are preferably from a plant.

The functional equivalent of SEQ ID NO: 3 according to d) according to the invention has a homology with SEQ ID No: 3 of at least 66%, 67%, 68%, preferably at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% preferably at least 81%, 82%, 83% preferably at least 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, particularly preferably at least 94%, 95%,. 96%, 97%, 98%, 99%.

Furthermore, nucleic acid sequences encoding a plant glyoxysomal malate dehydrogenase are claimed in the context of the present invention comprising:

d) functional equivalents of the nucleic acid sequence SEQ ID NO: 2 with an identity of at least 79% to SEQ ID NO: 2; or

e) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 87%.

The polypeptides encoded by the aforementioned nucleic acid sequences are also claimed. Draw the functional equivalents according to c) and d) have the same functionality, ie they have the enzymatic, preferably biological activity of a glyoxysomal malate dehydrogenase.

The functional equivalents of SEQ ID NO: 2 according to the invention have an identity with SEQ ID No: 2 of at least 79%, 80%, 81%, 82%, 83%, preferably at least 84%, 85%, 86%, 88 %, 89%, preferably at least 90%, 91%, 92%, 93%, 94%, 95% particularly preferably at least 96%, 97%, 98%, 99%.

The functional equivalents of SEQ ID NO: 3 according to the invention have an identity with SEQ ID No: 3 of at least 87%, 88%, 89%, 89%, preferably at least 90%, 91%, 92%, 93%, preferably at least 94%, 95%, 96% particularly preferably at least 97%, 98%, 99%.

The term “nucleic acid sequences according to the invention” used below stands for nucleic acid sequences encoding a malate dehydrogenase, preferably encoding nucleic acid sequences encoding a plant malate dehydrogenase, particularly preferably encoding nucleic acid sequences encoding a plant malate dehydrogenase

very particularly preferably comprising nucleic acid sequences encoding a plant glyoxysomal malate dehydrogenase.

a) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 1; or b) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 2; or

d) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 66%.

The polypeptides encoded by a nucleic acid sequence according to the invention with the enzymatic, preferably biological activity of a glyoxsysomal malate De- _. For the sake of simplicity, hydrogenase are referred to below as "MDH". The term "malate dehydrogenase" refers to any protein / enzyme with the enzymatic activity of a malate dehydrogenase.

Glyoxysomal MDHs cause a reduced amount of growth retardation as well as necrotic and chlorotic leaves in plants.

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. Furthermore, the gene products of the nucleic acids according to the invention represent new targets for growth regulators which make it possible to provide new growth regulators for regulating the growth of plants.

In the broadest sense, undesirable plants are understood to mean all plants that grow up in places where they are undesirable, for example:

Dicotyledon weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Mat caria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirschus, Carduus Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, Taraxacum.

Monocotyledonous weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittariapus, Eleocharis Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, Apera.

SEQ ID NO: 2 or parts of the above-mentioned nucleic acid sequences can be used for the production of hybridization probes. 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. Sambröok, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). 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 probes can be used for the detection and isolation of functional equivalents of SEQ ID NO: 2 from other plant species based on sequence identities. Here, part or all of the sequence of the corresponding SEQ ID NO: 2 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) comprising genetic control sequences in functional linkage with a nucleic acid sequence

i. a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 2; or

ii. a nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 3 on the basis of the degenerate genetic code by back-translation; or

iii. functional equivalents of the nucleic acid sequence SEQ ID NO: 2 with an identity of at least 79% to SEQ ID NO: 2; iv. a 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: 3, which has an identity with SEQ ID NO: 3 of at least 92%.

b) additional functional elements; or

c) a combination of a) and b);

and containing the use of expression cassettes

a) genetic control sequences in functional. Linking to a nucleic acid sequence according to the invention;

b) additional functional elements; or

c) a combination of a) and b);

for the expression of a malate dehydrogenase, preferably MDH, which can be used in vitro test systems. Both embodiments of those described above

Expression cassettes are referred to below as the expression cassette according to the invention.

According to a preferred embodiment, 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, if appropriate, further genetic control sequences which are functionally linked to the nucleic acid sequence according to the invention located therebetween.

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, λ-PR or in the λ-PL promoter, which can be used to express a malate dehydrogenase, preferably MDH, 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 malate dehydrogenase, preferably MDH, 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. 104, 207-220 (1998); Cregg et al. Biotechnology (NY) 1993 Aug; 11 (8): 905-10; Luo X., Gene 1995 Sep 22; 163 (i): 127-31; Nacken et al., Gene 1996 Oct 10; 175 (1-2): 253-60; Turgeon et al., Mol Cell Biol 1987 Sep; 7 (9): 3297-305) or the transcription terminators NMT, Gcy1, TrpC, AOX1, nos, PGK or CYC1 (Degryse et al., Yeast 1995 Jun 15; 11 (7 ): 629-40; Brunelli et al. Yeast 1993 Dec9 (12): 1309-18; Frisch et al., Plant Mol. Biol. 27 (2) , 405-409 (1995); Scorer et al., Biotechnology (NY) 12 (2), 181-184 (1994), Genbank acc. number Z46232; Zhao et al. Genbank acc number: AF049064; Punt et al., (1987) Gene 56 (1), 117-124), which can be used to express malate dehydrogenase, preferably MDH, in yeast strains.

Examples of 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 malate dehydrogenase, preferably MDH 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.

Further advantageous genetic control sequences for the expression of malate dehydrogenase, preferably MDH in plants, are found in the plant promoters CaMV / 35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, LEB4, USP, STLS1, B33, NOS; Contain FBPaseP (WO 98/18940) or in the ubiquitin or phaseolin promoter, preferably a plant promoter or a promoter which is derived from a plant virus is preferably used. Particularly preferred are promoters of viral origin 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. Such promoters, such as the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible (WO 95/19443), a benzenesulfonamide-inducible (EP -A-0388186), one that can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397404), one that can be induced by abscisic acid (EP-A 335528) or one that can be induced by ethanol or cyclohexanone ( WO 93/21334) promoters can also be used.

Also suitable are 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. In addition to the above-mentioned constitutive promoters, especially those promoters which ensure leaf-specific expression are also suitable. Worth mentioning are the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit), the Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989), 2445-245). Promoters which control expression in seeds and plant embryos are also preferred. 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. Planta 1996, 199: 515-519), the sucrose binding protein (WO. 00/26388) or the LeB4 promoter (Baumlein H et al., Mol Gen Genet 1991, 225: 121-128; Fiedler, U. et al., Biotechnology (NY) (1995), 13 (10) 1090).

Further 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, for example, the RNA polymerase promoter (WO 97/06250) or the promoter of the phosphoribosyl pyrophosphate amidotransferase from glycine max (see also Genbank Accession No. U87999) or another no- diene-specific promoters as in EP-A 249676 can advantageously be used.

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 malate dehydrogenase, preferably MDH, directly or by means of a linker optionally containing a protease interface. Further suitable additional functional elements are sequences which target in the apoptlasts, plastids, the vacuole, the mitochondrium, the peroxisome, the endoplasmic reticulum (ER) or, due to the lack of corresponding operative sequences, remain in the compartment of the development, the cytosol (Kermode, Crit. 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.

In addition to plasmids, 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.

In a further embodiment of the vector, 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.

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 according to the invention and vectors derived therefrom can be used to transform bacteria, cyanobacteria (for example the genus Synechocystes, Anabaena, Calothrix, Scytonema, Oscillatoria, Plectonema and Nostoc), proteobacteria such as Magnetococcus sp. MC1, yeast, filamentous fungi and algae and eukaryotic, non-human cells (eg insect cells) with the aim of recombinant production of malate dehydrogenase, preferably MDH, are used, whereby the production of a suitable expression cassette depends on the organism in which the gene is to be expressed.

Containing vectors

a) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 2; or

b) a nucleic acid sequence which can be derived from the amino acid sequence shown in SEQ ID NO: 3 on the basis of the degenerate genetic code by back-translation; or

c) functional equivalents of the nucleic acid sequence SEQ ID NO: 2 with an identity of at least 79% to SEQ ID NO: 2;

d) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 87%.

are the subject of the present invention.

In a further advantageous embodiment, the nucleic acid sequences used in the method according to the invention can also be introduced into an organism alone.

If, 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.

In principle, the 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.

For microorganisms, the person skilled in the art can use the corresponding textbooks from Sambrook, J. et al. (1989) "Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press, by FM Ausubel et al. (1994) "Current protocols in molecular biology", John Wiley and Sons, by DM Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or Guthrie et al. "Guide to Yeast Genetics and Molecular Biology ", Methods in Enzymology, 1994, Academic Press. For the transformation of filamentous fungi, the production of protoplasts and transformation using PEG (Wiebe et al. (1997) Mycol. Res. 101 (7) : 971-877; Proctor et al. (1997) Microbiol. 143, 2538-2591), on the other hand the transformation with the aid of Agrobacterium tumefaciens (de Groot et al. (1998) Nat. Biotech. 16, 839-842 ) on.

For dicotyledonous plants, the described methods for transforming and regenerating plants from plant tissues or plant cells for transient or stable transformation can be used. Suitable methods are the biolistic method or by protoplast transformation (see, for example, 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 York-Basel-Cambridge), electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the gene transfer mediated by Agrobacterium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225).

The transformation by means of 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. Acids Res. 12 (1984) 8711. The intermediate vectors can be homologous due to sequences Sequences in the T-DNA are integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination, which also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria The intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation) by helper plasmids. Binary vectors can replicate both in E. coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker which is linked from the right and left T- DNA border region can be framed. 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 B.V., Alblasserdam (1985), Chapter V; Fraley et al., Grit. Rev. Plant. Sci., 4: 1-46 and An et al. EMBO J. 4 (1985) 277-287).

The transformation of monocotyledonous plants using Agrobacterium-based vectors has also been described (Chan et al, Plant Mol. Biol. 22 (1993), 491-506; Hiei et al, Plant J. 6 (1994) 271-282; Deng et al; Science in China 33 (1990), 28-34; Wilmink et al, Plant Cell Reports 11, (1992) 76-80; May et al; Biotechnology 13 (1995) 486-492; Conner and Domisse; Int. J. Plant Sei. 153 (1992) 550-555; Ritchie et al; Transgenic Res. (1993) 252-265). Alternative systems for the transformation of monocotyledonous plants are transformation using the biolistic approach (Wan and Lemaux; Plant Physiol. 104 (1994), 37-48; Vasil et ai; Biotechnology 11 (1992), 667-674; Ritala et al, Plant Mol. Biol 24, (1994) 317-325; Spencer et al, Theor. Appl. Genet. 79 (1990), 625-631) the protoplast transformation, the electroporation of partially permeabilized cells; the introduction of DNA using glass fibers. In particular, the transformation of maize has been described several times in the literature (cf., for example, WO 95/06128; EP 0513849 A1; EP 0465875 A1; EP 0292435 A1; Fromm et al, Biotechnology 8 (1990), 833-844; Gordon-Kamm et al, Plant Cell 2 (1990), 603-618; Koziel et al, Biotechnology 11 (1993) 194-200; Moroc et al, Theor Applied Genetics 80 (190) 721-726).

The successful transformation of other cereals has also been described, e.g. for barley (Wan and Lemaux, see above; Ritala et al, see above; wheat (Nehra et al, Plant J. 5 (1994) 285-297).

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 crop plants such as cereals, maize, oats, rye, barley, wheat, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, Potato, tobacco, tomato, carrot, paprika, rapeseed, tapioca, cassava, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species can be used, eg by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.

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.

The 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 malate dehydrogenase, preferably MDH, obtainable by expression from the transgenic organism are the subject of the present invention. The present invention also relates to

Use of transgenic organisms containing an expression cassette according to the invention, for example for the provision of recombinant protein and / or the use of these organisms in in vivo test systems. In addition to bacteria, yeasts, mosses, algae and fungi, preferred organisms for recombinant expression are also eukaryotic cell lines.

Preferred mosses are Physcomitrella patens or other mosses described in Kryptogamen, Vol. 2, Moose, Farne, 1991, Springer Verlag (ISBN 3540536515).

Within the bacteria, bacteria of the genus Escherichia, Erwinia, Flavobacterium, Alcaligenes or Cyanobacteria, for example of the genus Synechocystes, Anäbaena, Calothrix, Scytonema, Oscillatoria, Plectonema and Nostoc, are particularly preferred. prefers Synechocystis or Anabena.

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, triticale, maize, rice or oats, and sugar cane. Furthermore, the transgenic plants according to the invention are selected in particular from dicotyledonous crop plants, such as, for example, Brassicacae such as rapeseed, cress, Arabidopsis, cabbages or canola; Leguminosae such as soy, alfalfa, pea, bean crops 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.

In principle, transgenic animals such as C. elegans are also suitable as host organisms.

It is also preferred to use expression systems and vectors that are publicly available or commercially available.

For use in E. coli bacteria, the typical advantageous, commercially available fusion and expression vectors pGEX [Pharmacia Biotech Ine; Smith, DB and Johnson, KS (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 to call "pET" - and the "pBAD" vector series from Stratagene, La Jolla.

Further advantageous 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.MJ.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.

Alternatively, insect cell expression vectors can also be used advantageously, e.g. for expression in Sf9, Sf21 or Hi5 cells, which are infected via recombinant baculoviruses. These are e.g. 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 Expression System" from CLONTECH, Palo Alto, CA. Insect cells are particularly suitable for overexpressing eukaryotic proteins because they carry out post-translational modifications of the proteins that 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).

Furthermore, plant cells or algal cells can advantageously be used for gene expression. Examples of 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.

Furthermore, the nucleic acid sequences according to the invention can be expressed in mammalian cells. Examples of corresponding expression vectors are pCDMδ and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBO J. 6: 187-195). In this case, promoters to be used are of viral origin, such as 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 transgenic organisms which contain a nucleic acid sequence are claimed in the context of the present invention.

All of the above-described embodiments of the transgenic organisms are summarized under the term “transgenic organism according to the invention”.

Another object of the present invention is the use of malate dehydrogenase, preferably MDH, in a method for identifying test compounds with a herbicidal action.

The method according to the invention for identifying compounds having a herbicidal action preferably comprises the following steps:

i. Bringing malate dehydrogenase, preferably MDH, into contact with one or more test compounds under conditions which allow the test compound (s) to bind to the malate dehydrogenase, preferably MDH; and

ii. Evidence of whether the test compound binds to the malate dehydrogenase, preferably to the MDH from i); or

iii. Evidence as to whether the test compound reduces or blocks the enzymatic or biological activity of the malate dehydrogenase, preferably the MDH from i); or

iv. Detection of whether the test compound reduces or blocks the transcription, translation or expression of a malate dehydrogenase, preferably MDH.

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. Examples of 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.

In this context, five preferred embodiments are to be mentioned in particular, which in connection with the present invention are also suitable for high-throughput methods (HTS): 1. Using fluorescence correlation spectroscopy (FCS) (Proc. Natl. Acad. Sei. USA (1994) 11753-11575), the average diffusion rate of a fluorescence molecule as a function of mass can be determined in a small sample volume. By measuring the change in mass or the resulting change in the diffusion rate of a test compound when binding to the malate dehydrogenase, preferably the MDH, FCS can be used to determine protein-ligand interactions. A method according to the invention can be set up directly for measuring the binding of a test compound marked by a fluorescence molecule. Alternatively, the method according to the invention can be designed in such a way that a chemical reference compound marked by a fluorescence molecule is displaced by further test compounds ("displacement assay").

2. The fluorescence polarization uses the property of a resting fluorophore excited with polarized light to also 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 (eg temperature, viscosity, solvent) the rotation is a function of the molecular size, with which one can make a statement about the size of the residue bound to the fluorophore via the measurement signal (Methods in Enzymology 246 (1995), pp. 283-300). A method according to the invention can be established directly for measuring the binding of a test compound marked by a fluorescent molecule to the malate dehydrogenase, preferably the MDH. Alternatively, the method according to the invention. can also be designed in the form of the "displacement assay" described under 1.

3. Fluorescence Resonance Energy Transfer "(FRET) is based on the radiationless energy transfer between two spatially adjacent fluorescence molecules under suitable conditions. A prerequisite is the overlap of the emission spectrum of the donor molecule with the excitation spectrum of the acceptor molecule. By fluorescence labeling of the malate dehydrogenase, preferably the MDH and Binding to the Tets connection can be measured using FRET (Cytometry 34, 1998, pp. 159-179). Alternatively, 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 "Homogeneous Time Resolved Fluorescence" (HTRF), as distributed by Packard BioScience.

4. Surface enhanced laser desorption / ionization (SELDI) in combination with a "Time of Flight" mass spectrometer (MALDI-TOF) enables fast

Analysis of molecules on a support and can be used to analyze protein Ligand interactions can be used (Worral et al., (1998) Anal. Biochem. 70: 750-756). In a preferred embodiment, the malate dehydrogenase, preferably the MDH, is then immobilized on a suitable carrier and incubated with the test compound. After one or more suitable washing steps, the malate dehydrogenase, preferably MDH, can be added

^' Detect additionally bound molecules of the test compound by means of the method mentioned above and thus select test compounds bound to the malate dehydrogenase, preferably to the MDH.

5. 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). The measurement can be carried out, for example, with the aid of the automated analyzers based on surface plasmon resonance sold by Biacore (Freiburg) in a throughput of currently up to 384 samples per day. A method according to the invention can be set up directly for measuring the binding of a test compound to the malate dehydrogenase, preferably to the MDH. Alternatively, the method according to the invention can also be designed in the form of the "displacement assay" described under 1.

The compounds identified via the above-mentioned methods 1 to 5 can be suitable as inhibitors. All of the substances identified by the abovementioned methods can then be checked for their herbicidal action in another embodiment of the method according to the invention.

There is also the possibility, by elucidating the three-dimensional structure of the malate dehydrogenase, preferably the MDH, using X-ray structure analysis to detect further potential herbicidal active ingredients by means of "molecular modeling". The person skilled in the art is familiar with the production and preparation of protein crystals required for the X-ray structure analysis and the corresponding measurements and subsequent evaluations of these measurements, the detection of a binding site in the protein and the prediction of possible inhibitor structures. In principle, "Molecular Modeling" can also be used to optimize the compounds identified using the above-mentioned methods.

A preferred embodiment of the method according to the invention, which is based on steps i) and ii), consists in selecting a test compound which before the enzymatic activity of a malate dehydrogenase, preferably MDH, is reduced or blocked.

A particularly preferred embodiment of the method according to the invention, which is based on steps i) and ii), consists in selecting a test compound which reduces or blocks the enzymatic activity of a malate dehydrogenase, preferably MDH, the activity of those incubated with the test compound Malate dehydrogenase, preferably MDH, is compared with the activity of a malate dehydrogenase, preferably MDH, not incubated with a test compound.

A preferred embodiment of the method based on steps i) and ii) is that

i. a malate dehydrogenase, preferably an MDH, is expressed in a transgenic organism according to the invention or an organism which naturally contains a malate dehydrogenase, preferably MDH, is cultured;

ii. the malate dehydrogenase, preferably the MDH 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; and

iii. a compound is selected which reduces or blocks the activity of the malate dehydrogenase, preferably the MDH, the activity of the malate dehydrogenase incubated with the test compound.

A preferred embodiment of the method based on steps i) and ii) is that

iii. a compound is selected which reduces or blocks the activity of the malate dehydrogenase, preferably the MDH, the activity of the malate dehydrogenase, preferably MDH, incubated with the test compound with the activity a malate dehydrogenase, preferably MDH, not incubated with a test compound.

The solution containing malate dehydrogenase, preferably MDH, 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 malate dehydrogenase, preferably MDH, can be partially or completely purified using standard methods. A general overview of common techniques for protein purification can be found, for example, in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6. In the case of recombinant representation, the protein fused to an affinity tag can be purified using affinity chromatography, which is known to the person skilled in the art.

The malate dehydrogenase, preferably MDH, 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 enzyme with the enzymatic activity, preferably biological, of a malate dehydrogenase, for example from an undesired plant , whereby the term undesired plant is to be understood as the species mentioned at the outset.

To identify herbicidal compounds, the malate dehydrogenase, preferably MDH, is then incubated with a test compound. After a reaction time, the enzymatic activity of the malate dehydrogenase, preferably MDH incubated with the test compound, is determined with the enzymatic activity of a malate dehydrogenase, preferably MDH not incubated with a test compound. When malate dehydrogenase, preferably MDH, is inhibited, a significant decrease in activity is observed in comparison to the activity of the non-inhibited polypeptide according to the invention, a decrease of at least 10%, advantageously at least 20%, preferably at least 30%, particularly preferably by at least 50% to towards a 100% reduction (blocking) is achieved. Preferably at least 50% inhibition at concentrations of the test compound of 10 ^"4 M, preferably at 10 ^{" 5} M, particularly preferably of 10 ^"6 M, based on the enzyme concentration in the micromolar range.

The enzymatic activity of malate dehydrogenase, preferably MDH, can be determined, for example, using an activity test in which the increase in the product, the decrease in the substrate (or starting material) or the name of the cofactor or by a combination of at least two of the The aforementioned parameters can be determined as a function of a defined period of time. Examples of suitable substrates are, for example, malate (-) - tartrate (S, S), (S) -malate, 2-hydroxybutyrate, 2-hydroxyglutarate, 2-hydroxymalonate, 2-oxobutyrate, 2-oxoglutarate, ketomalonate, L-malate, meso -Tartrate (S, R), oxaloacetate and for suitable cofactors NAD +, NADH, APAD, APADH. Depending on the malate dehydrogenase used, NADPH can also be used as a cosubstrate. If appropriate, 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 can be between 0.5-100 mM and amounts of cofactor between 0.1-5 mM based on 1-100 μg / ml enzyme.

The activity can be determined, for example, in analogy to the method described by Gietl et al (1996, BBA 1274, 48-58).

Furthermore, the determination of the activity in step iii) of the above-mentioned method

a) photometrically via the conversion from NADH to NAD +; and or

b) is carried out photometrically via the conversion from NAD + to NADH; or

c) photometrically via the conversion from APAD to APADH; or

respectively.

A preferred embodiment of the method according to the invention, which is based on steps i) and iii), consists of the following steps:

i. Production of a transgenic organism comprising a nucleic acid sequence encoding a glyoxsomal malate dehydrogenase comprising

b) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID N0: 2; or

c) a nucleic acid sequence which, owing to the degenerate genetic code, is converted back from the sequence shown in SEQ ID NO: 3. deduces th amino acid sequence; or

d) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 66%, preferably 81%;

ii. Applying a test compound to the transgenic organism according to i) and to a non-transgenic organism of the same genotype;

iii. Determining the growth or survivability of the transgenic and non-transgenic organisms after application of the test compound; and

iv. Selection of test compounds that cause reduced growth or limited survivability of the non-transgenic organism compared to the growth of the transgenic organism.

In the above-mentioned method, malate dehydrogenase is overexpressed in the transgenic organism.

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 is preferably a plant, algae, a cyanobacterium e.g. the genus Synechocystes or a proteobacterium such as Magnetococcus sp. MC1, preferably plants that can be transformed using common techniques, such as Arabidopsis thaliana, Solanum tuberosum, Nicotiana Tabacum, cyano-bacteria that can be easily transformed, such as Synechocystis, in which the sequence coding for a polypeptide according to the invention was incorporated via transformation. These transgenic organisms therefore have an increased tolerance to compounds which inhibit the polypeptide according to the invention. "Knock-out" mutants can also be used here in which the analog gene for malate dehydrogenase, preferably MDH, which is naturally present in this organism, has been specifically switched off.

However, the above-mentioned embodiment of the method according to the invention can also be used to identify substances with a growth regulatory effect. A plant is used as a transgenic organism. The procedure for the identification of substances with growth regulatory effects kung thus comprises the following steps:

i. Production of a transgenic plant comprising a nucleic acid sequence encoding a glyoxsomal malate dehydrogenase comprising

ii. Applying a test substance to the transgenic plant according to i) and to a non-transgenic plant of the same variety;

iii. Determining the growth or viability of the transgenic and non-transgenic plants after application of the test substance; and

iv. Selection of test substances that cause an altered growth of the non-transgenic plant compared to the growth of the transgenic plant.

In step iv), test compounds are selected which bring about a changed 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 color can also be observed. Furthermore, under changing growth, there is also a temporal change in the course of maturity, haymaking or propagation laterally. rather branching of the plants, a shortening or lengthening of the development stages, an increase in stability, the growth of larger quantities of buds, flowers, leaves, fruits, seeds, roots and tubers, an increase in the sugar content in plants such as sugar beet, sugar cane and citrus fruits ten, the protein content in plants such as cereals or soybeans, or a stimulation of the latex flow in rubber trees. The detection of this changed growth is known to the person skilled in the art.

According to the inventive method, several 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 isolate the individual test compounds directly 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. Depending on the complexity of the sample, 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 methods described above for the identification of inhibitors with herbicidal or growth-regulating activity are referred to below as “methods according to the invention”.

All of the compounds identified by the method according to the invention can then be checked for their herbicidal or growth-regulating action 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.

In the HTS, the use of carriers which contain 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, is suitable for practical implementation or several transgenic organisms which contain at least one of the nucleic acid sequences according to the invention or contain one or more (poly) 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. According to the most widespread technology, 96-well, 384-well and 1536-well microtiter plates are used, which can usually contain volumes of 200μl. In addition to the microtiter plates, 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.

In addition to the HTS methods based on microtiter plates, so-called "free format assays" or test systems that have no physical barriers between the samples can also be used, e.g. in Jayaickreme et al., Proc. Natl. Acad. Be U.S.A. 19 (1994) 161418; Chelsky, "Strategies for Screening Combinatorial Libaries, First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 710, 1995); Salmon et al., Molecular Diversity 2 (1996), 5763 and US 5,976,813.

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 μM, particularly preferably less than 0.1 μM, very particularly preferably less than 0.01 μM.

The invention furthermore relates to compounds having a growth regulator activity, identified by the processes according to the invention. These compounds are also called "selected compounds" in the following.

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 herbicidally active compounds identified by the process of the invention.

Furthermore, the selected compounds, if they contain asymmetrically substituted α-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 e.g. in cell extracts of e.g. Plants, animals or microorganisms occur. The reaction mixture can be a cell-free extract or can comprise a cell or cell culture. Suitable methods are known to the person skilled in the art and are e.g. generally described in Alberts, Molecular Biology the cell, 3rd Edition (1994), e.g. Chapter 17. The selected compounds can also come from extensive substance libraries.

Possible 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 undesirable plant growth and / or as growth regulators. Herbicidal compositions containing the selected compounds fight plant growth on non-cultivated areas very well. 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.

Depending on the particular application method, selected compounds or herbicidal compositions containing them can advantageously also be used in a further number of crop plants for eliminating undesired plants. The following crops are considered, for example:

Allium cepa, Ananas comosus, Arachis hypogaea, Asparagus officinalis, Beta vulgaris spec. altissima, Beta vulgaris spec. rapa, Brassica napus var. napus, Brassica napus var. napobrassica, Brassica rapa var. silvestris, Camellia sinensis, Carthamus tinctori- us, Carya illinoinensis, Citrus limon, Citrus sinensis, Coffea arabica (Coffea canephora, Coffea libericaus), Cucumis Cynodon dactylon, Daucus carota, Elaeis guineen- sis, Fragaria vesca, Glycine max, Gossypium hirsutum, (Gossypium arboreum, Gossypium herbaceum, Gossypium vitifolium), Helianthus annuus, Hevea brasiliensis, Hordeum batupatus, Humulus lupusatus, Humulus lupus, Iulus Juglans regia, Lens culinaris, Linum usitatissimum, Lycopersicon lycopersicum, Malus spec, Manihot esculenta, Medicago sativa, Musa spec, Nicotiana tabacum (N.rustica), Olea europaea, Oryza sativa, Pha- seolus lunatus, Phaseolus vulgaris, Picea abies, Pinus spec, Pisum sativum, Prunus avium, Prunus persica, Pyrus communis, Ribes sylestre, Ricinus communis, Saccharum officinarum, Seeale cereale, Solanum tuberosum, Sorghum bicolor (The vulgaris) cacao, Trifolium pratense, Triticum aestivum, Triticum durum, Vicia faba, Vitis vinifera, Zea mays.

In addition, 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 or growth-regulating 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. in the form of directly sprayable aqueous solutions, powders, suspensions, also high-proof aqueous, oily or other suspensions or suspoemulsions or dispersions, emulsifiable concentrates, emulsions, oldispersions, pastes, dusts, sprinkling agents or granules and formulated by spraying, atomizing, Dusting, scattering or pouring can be used. 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.

To produce emulsions, pastes or aqueous or oil-containing formulations and dispersible concentrates (DC), the selected compounds can be dissolved or dispersed in an oil or solvent, it being possible to add further formulation auxiliaries for homogenization. However, 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. Emulsifiable substances are mentioned here

Concentrates (EC, EW), suspensions (SC), soluble concentrates (SL), dispersible concentrates (DC), pastes, pastilles, wettable powders or granules, where the solid formulations are either soluble in water or soluble (water table) could be. Furthermore, appropriate powders or granules or tablets can still have a fixed, abrasion or premature release of the active ingredient preventing coating ("coating") are provided.

In principle, the term “auxiliary” means the following classes of compounds: anti-foaming agents, thickeners, wetting agents, adhesives, dispersants, emulsifiers, bactericides and / or thixotrophic agents. The meaning of the above-mentioned agents is known to the person skilled in the art.

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 jointly grinding the active substances with a solid carrier. Granules, for example coated granules, impregnated granules and homogeneous granules, can be prepared 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. listed in the following documents: US 3,060,084, EP-A 707445 (for liquid concentrates), Browning, "Agglomeration", Chemical Engineering ^" , Dec. 4, 1967, 147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw -Hill, New York, 1963, pages 8-57 and ff. WO 91/13546, US 4,172,714, US 4,144,050, US 3,920,442, US 5,180,587, US 5,232,701, US 5,208,030, GB 2,095,558, US 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al., Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology , Wiley VCH Verlag GmbH, Weinheim (Federal Republic of Germany), 2001.

A large number of inert liquid and / or solid carriers suitable for the formulations according to the invention are known to the person skilled in the art, 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. 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, e.g. Amines such as N-methylpyrrolidone or water.

Solid carriers are, for example, mineral earths 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 cereal flour, tree bark, wood and nutshell flour, cellulose powder or other solid carriers.

A large number of surface-active substances (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. 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 with formaldehyde, condensation products of naphthalene or naphthalenesulfonic acids with phenol and formaldehyde, polyoxyethylene octylphenol ether, ethoxylated isooctyl, octyl or nonylphenol, alkylphenyl, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl ether, polyethylenalkylene oil, ethoxylated ethoxylated alcohol, fatty alcohol ethoxylate, ethoxylated ethoxylated ethoxylated alcohol, fatty alcohol ethoxylate, ethoxylated ethoxylated alcohol, fatty acid ethoxylated alcohol, fatty acid ethoxylated alcohol, fatty alcohol ethoxylated alcohol, fatty acids , Lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin sulfite waste liquor or methyl cellulose.

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 on the Leaves of unwanted plants growing underneath 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 malate dehydrogenase, preferably MDH, which is not or only to a limited extent by a herbicide which has the malate dehydrogenase, preferably MDH, as the site of action, e.g. the herbicidal selected compounds is inhibited. In the following, a protein that differs from malate dehydrogenase, preferably MDH, is referred to as the MDH variant, which is encoded by a nucleic acid sequence which

i) encodes a polypeptide with the biological activity of a malate dehydrogenase, which is determined by substances determined according to the above-mentioned methods with herbicidal activity, which inhibit malate dehydrogenase, preferably MDH, is not inhibited;

ii) are encoded by a functional equivalent of the nucleic acid sequence SEQ ID NO: 3 with an identity of at least 63% to SEQ ID NO: 3.

In a preferred embodiment, the above-mentioned method for generating nucleic acid sequences coding for MDH variants of nucleic acid sequences comprises the following steps:

a) expression of the proteins encoded by the above-mentioned nucleic acids in a heterologous system or in a cell-free system;

b) Randomized or directed mutagenesis of the protein by modification of the nucleic acid;

c) measuring the interaction of the modified gene product with the herbicide;

d) identification of derivatives of the protein which have less interaction;

e) testing the biological activity of the protein after application of the herbicide;

f) Selection of the nucleic acid sequences which have an altered biological activity towards the herbicide.

The functional equivalent of SEQ ID NO: 3 according to ii) has a homology with SEQ ID No: 3 of at least 63%, 64%, 65%, 66%, preferably at least 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74% preferably at least 75%, 76%, 77%, 78%, 79%, 80% preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 89%, 90%, 91%, 92%, 93% 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. Then whole plants are regenerated and the resistance to the selected compound in intact plants is checked.

Modified proteins and / or nucleic acids which can impart resistance to the selected compounds in plants can also be produced from the above-mentioned nucleic acid sequences by means of the so-called "site directed mutagenesis", for example the stability and / or activity of the target protein by this mutagenesis or the properties such as binding and action of the above-mentioned inhibitors according to the invention can be specifically improved or changed.

For example, Zhu et al. (Nature Biotech., Vol. 18, May 2000: 555-558) describes a "site directed mutagenesis" method in plants which can be used advantageously.

Furthermore, changes can be made via the method described by Spee et al. (Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-78), the PCR method described can be achieved using dITP for random mutagenesis or by the method described by Rellos et al. (Protein Expr. Purif., 5, 1994: 270-277) further improved method.

Another possibility for producing these modified proteins and / or nucleic acids is one of Stemmer et al. (Proc. Natl. Acad. Sci. USA, Vol. 91, 1994: 10747-10751) described "in vitro" recombination technique for molecular evolution or that described by Moore et al. (Nature Biotechnology Vol. 14, 1996: 458-467) described combination of the PCR and recombination method.

Another route to mutagenesis of proteins is described by Greener et al. in Methods in Molecular Biology (Vol. 57, 1996: 375-385). EP-A-0 909 821 describes a method for changing proteins using the microorganism E. coli XL-1 Red. During replication, this microorganism creates mutations in the introduced nucleic acids and thus leads to a change in the genetic information. By 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.

Further methods of 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 alley. Mutagenesis and Selection of resistances in cell culture by increasing 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.

In addition, methods for the targeted modification of nucleic acids are available (Zhu et al. Proc. Natl. Acad. Sei. USA, Vol. 96, 8768-8773 and Beethem et al., Proc. Natl. Acad. Sei. USA, Vol 96, 8774-8778). These methods make it possible to replace those amino acids in the proteins which are important for the binding of inhibitors with functionally analogous amino acids which, however, prevent the inhibitor from binding.

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 is understood to mean 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. Furthermore, 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. For the purposes of the invention, no longer or no longer correctly means that the substances have at least 30%, preferably at least 50%, particularly preferably at least 70%, very particularly preferably at least 80% or not at all of the modified nucleic acids and / or bind 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 MDH variant. Methods of transformation are known to those skilled in the art known and exemplified above.

Genetically modified 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 process for the production of transgenic plants which are resistant to substances found by a process according to the invention, characterized in that nucleic acids coding for a malate dehydrogenase, preferably MDH, are overexpressed in these plants. A similar method is described by way of example in Lermantova et al., Plant Physiol., 122, 2000: 75-83.

The methods according to the invention for producing resistant plants described above enable the development of new herbicides which have the most comprehensive possible plant species independent action (so-called total herbicides), in combination with the development of crop plants resistant to the total herbicide. Useful plants resistant to total herbicides have already been described in various ways. Several principles can be distinguished to achieve resistance:

a) Generation of resistance in a plant via mutation or genetic engineering processes, in that the protein serving as the target for the herbicide is clearly overproduced and because of the large excess of the protein serving as the target for the herbicide, which is produced by this protein in the cell. exercised function is maintained even after application of the herbicide.

b) Modification of the plant in such a way that a modified version of the protein which acts as the target of the herbicide is introduced and that the function of the newly introduced modified protein is not impaired by the herbicide.

c) Modification of the plant in such a way that a new protein / a new RNA is introduced which is characterized in that the chemical structure of the herbicidal action of the low molecular weight substance

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. d) that the function of the target is replaced by a new gene introduced into the plant, thus creating a so-called "alternative pathway".

e) That the function of the target is taken over by another gene present in the plant or its gene product.

Alternative methods for identifying the homologous nucleic acids, for example in other plants with similar sequences such as, for example, using transposons, are known to the person skilled in the art. This invention therefore also relates to the use of alternative insertion mutagenesis methods for inserting foreign nucleic acids into the nucleic acid sequence SEQ ID NO: 3 in sequences derived from these sequences on the basis of the genetic code and / or their derivatives in other plants.

The 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 MDH variant can be determined, for example, in vitro by multiplication of the shoot meristem or by a germination test.

In addition, a change in the type and level of expression of the malate dehydrogenase, preferably MDH gene, and its effect on the resistance to inhibitors of malate dehydrogenase, preferably MDH, can be tested on test plants in greenhouse experiments.

The invention is further illustrated by the following examples, which should not be construed as limiting.

Example 1: Generation of a cDNA library in the plant transformation vector

To generate 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 carried out with T12-18 oligonucleotides according to the manufacturer's instructions. After size fractionation and ligation of EcoRI-Notl adapters according to the manufacturer's instructions and filling in the overhangs with Pfu DNA polymerase (Stratagene), the cDNA population was normalized. This was done the method according to Kohci et al, 1995, Plant Journal 8, 771-776, the cDNA being amplified by PCR with the oligonucleotide N1 under the conditions listed in Table 1.

Table 1

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 μl of the DNA were applied to a hydroxylapathite 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 μl water. Of these, 20 μl 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 a transformation of plants by means of Agrobacterium and a kanamycin resistance mediated in transgenic plants telt, ligated. 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 2) and Pfu DNA polymerase. The resulting fragment was ligated into the Smal digested pSun12 / 35S to generate pSunblues2. After cleavage with Notl, dephosphorylation with shrimp alkaline phosphatase (Röche Diagnostics, Mannheim) and purification of the vector fragment, pSunblues2 was ligated to the normalized and also NotL-cleaved cDNA population. After transformation in E.coli XMblue (Stratagene), the clones produced in this way were placed in microtiter plates. The binary cDNA library contains cDNAs in "sense" and in "antisense" orientation under the control of the cauliflower mosaic virus 35s promoter, which can accordingly lead to "cosuppression" and "antisene" effects after transformation in tobacco plants.

Table 2: Oligonucleotides used

Example 2: Transformation and analysis of tobacco plants

Selected clones of the binary cDNA library were transformed into Agrobacterium tumefaciens C58C1: pGV2260 (Deblaere et al., Nucl. Acids. Res. 13 (1984), 4777-4788) and incubated with streptomycin / spectinomycin selection. To transform tobacco plants (Nicotiana tabacum cv. Samsun NN with clone nt002035041r), an overnight culture of a positively transformed agrobacterial colony diluted in YEB medium to OD600 = 0.8-1.6 was used. Leaf disks of sterile plants (each about 1 cm2) were placed in a Petri dish incubated with the agrobacterial dilution for 5-10 minutes, followed by a 2-day incubation in the dark at 25 ° C. on Murashige-Skoog medium (Physiol. Plant. 15 (1962), 473) with 2% sucrose (2MS medium) 0.8% Bacto agar The cultivation was continued after 2 days with 16 hours of light / 8 hours of darkness and in a weekly rhythm on MS medium with 500 mg / l claforan (cefotaxime sodium), 50 mg / l kanamycin, 1 mg / l Benzylaminopurin (BAP), 0.2mg / l naphthylacetic acid and 1.6g / l glucose continued Sprouts were transferred to MS medium with 2% sucrose, 250 mg / 1 Claforan and 0.8% Bacto agar. Regenerated sprouts were obtained on 2MS medium with kanamycin and claforan. In this way, transgenic plants of the line E_0000005869 were generated.

The integration of the cDNA of the clones into the genome of the transgenic lines was verified via PCR with the oligonucleotides G1 and G2 (see Table 2) and genomic DNA which was prepared from the corresponding transgenic lines. For this purpose, TAKARA Taq-DNA polymerase was preferably used according to the manufacturer's instructions (MoBi-Tee, Göttingen). The cDNA clone of the binary cDNA bank used for the transformation served as a positive control as a template for a PCR reaction. PCR products of identical size or possibly the same cleavage pattern, which were obtained after cleavage with different restriction enzymes, served as evidence of the integration of the corresponding cDNA. The insert of the clone nt002035041 r was detected in this way in the transgenic plants with the above-mentioned phenotypes.

After transfer of the shoots to soil, the plants were observed for 2-20 weeks in the greenhouse for the expression of phenotypes. It turned out that transgenic plants of the line E_0000005869, in which the insert of nt002035041 r was detected, had similar phenotypes. After 2 weeks, these plants showed strong growth retardations, as well as chlorotic leaves and occasional necrosis.

Example 3 Sequence analysis of the clone

To isolate a full length cDNA sequence of nt002035041r (SEQ ID NO: 2) the Smart-RACE kit (Clontech) according to the manufacturer's instructions was used. SEQ ID NO: 2 of 1513 bp in length codes on an open reading name (nt 148-1221) of 960 nt in length for a protein of 320 amino acids.

It was shown for the first time and surprisingly that the natural expression of glyoxysomal MDH-coding sequences is essential for plants and that a reduced expression leads to damage in accordance with the phenotypes listed in Example 2. It follows that malate dehydrogenase, preferably MDH, is suitable as a target for herbicides.

The greatest homology is found with glyoxysomal MDHs from watermelon (P19446, Swissprot) with 77.5% identity at the nucleotide level (90.7% similarity or 85.4% identity at the protein level), cucumber, Arabidopsis, soya and rice. The N-termi- 38 amino acids probably represent a glyoxysomal transit sequence.

Example 4: Expression in E. coli

To generate active Nt-MDH protein with plant-based MDH activity, the coding region of Nt-MDH and MDH from Arabidopsis (Genbank: At2g22780) was overexpressed in E. coli bacteria. For this purpose, Nt-MDH fragments were amplified by means of special oligonucleotides and Pfu-Ultra Polymerase (Stratagene) by PCR and ligated into the expression vector pCR T7 / CT TOPO (Invitrogen) (constructs Nt1 and At1, Table 7). In this way, encoded fusion proteins with C-terminal hexahistidine tag were generated. During the cloning, an N-terminally shortened version of the MDH was generated by using the specified oligonucleotides in order to exclude the glyoxysomal transit sequence which could hinder functional expression. The transit sequence of the glyoxysomal MDH from watermelon was assumed (Gietl. Et al. 1996, BBA 1274, 48-58). The PCR was carried out according to standard conditions (eg according to Sambrook, J. et al. (1989) "Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press) in 36 cycles, the annealing temperatures being between 44 and 55 ° C and for the polymerization time was 60sec per 1000bp. The templates and the primers used for the respective templates as well as annealing temperatures are listed in Table 7.

Table 7

(1) Template: Nicotiana tabacuum cDNA-Bank

(2) Template: Arabidopsis thaliana cDNA library

The products obtained in the PCR were ligated into the vector pCR T7 / CT TOP and transformed into E. coli. Expression was carried out in E. coli BL21 (DE3) strains such as BL21 (DE3) pLysS or BL21 (DE3) pLysE (Invitrogen), BL21-CodonPlus (DE3) or BL21-CodonPlus (DE3) RIL (Stratagene) after induction with IPTG , To was followed according to standard protocols (Invitrogen).

The expression products Nt-gMDH-E1 and At-gMDH-E1 were purified by affinity chromatography using Ni agarose. This was done according to the manufacturer's instructions (Qiaagen).

Example 5: in vitro test systems

The enzyme activity of the gMDH can be determined according to Huang et al. (1974, Plant Physiol 54, 364-368) can be measured from preparations of plant microbodies. However, it is advisable to use the proteins expressed and purified in E. coli as described above in order to minimize background activities of cytosolic and mitochondrial MDHs.

The measurement of the enzymatic activity of the gMDH from example 3 was carried out photometrically at 340 nm by the conversion of NAD + to NADH according to Gietl et al. (1996, BBA 1274, 48-58). This assay can be carried out on a microtiter plate scale.

Claims

claims

1. Use of malate dehydrogenase in a method of identifying herbicides.

2. Use according to claim 1, characterized in that the malate dehydrogenase is encoded by a nucleic acid sequence which:

c) a 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: 3 which has an identity with SEQ ID NO: 3 of at least 63%;

includes.

3. Use according to claim 1 or 2, characterized in that the malate dehydrogenase is a glyoxysomal malate dehydrogenase which is encoded by a nucleic acid sequence comprising:

a) a nucleic acid sequence with that shown in SEQ ID NO: 1

nucleic acid sequence; or

d) a nucleic acid sequence which can be determined on the basis of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent of SEQ ID NO: 3, which has an identity with the SEQ ID NO: 3 of at least 66%, can be derived.

4. Plant nucleic acid sequences encoding a glyoxysomal malate dehydrogenase comprising:

c) functional equivalents of the nucleic acid sequence SEQ ID NO: 2 with an identity of at least 79% to SEQ ID NO: 2; or

d) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 87%.

5. polypeptide with the biological activity of a glyoxysomal malate dehydrogenase as a target for herbicides encoded by a nucleic acid molecule according to claim 4.

6. Method for the detection of functional analogues of SEQ ID NO: 2

a) by preparation of a probe followed by subsequent searching of a genomic or cDNA library of the corresponding species; or

b) a computer search for analog sequences in electronic databases.

7. Includes expression cassette

a) genetic control sequences in functional linkage with a nucleic acid sequence according to claim 4; or

b) additional functional elements; or c) a combination of a) and b).

8. Vector comprising an expression cassette according to claim 7.

9. Transgenic organism comprising at least one nucleic acid sequence coding for a polypeptide with the biological activity of a glyoxysomal malate dehydrogenase according to claim 4, an expression cassette according to claim 7 or a vector according to claim 8 selected from bacteria, yeast, fungi, animal or plant cells.

10. A method for identifying substances with herbicidal activity comprising the following steps:

i. Contacting malate dehydrogenase with one or more test compounds under conditions which allow the test compound (s) to bind to the nucleic acid molecule or to the glyoxysomal malate dehydrogenase; and

ii. Evidence of whether the test compound binds to the malate dehydrogenase from i); or

iii. Evidence of whether the test compound reduces or blocks the enzymatic or biological activity of the malate dehydrogenase from i); or

iv. Evidence of whether the test compound has transcription. Translation or expression of the malate dehydrogenase from i) reduced or blocked.

11. The method according to claim 10, characterized in that the malate dehydrogenase is encoded by a nucleic acid sequence which

a) a nucleic acid sequence according to claim 4; or

b) a nucleic acid sequence which, owing to the degenerate genetic code, is converted back by translating the amino acid sequence into a functional equivalent of SEQ ID NO: 3, which has an identity with SEQ ID

NO: 3 of at least 63%, can be derived;

includes.

12. The method according to claim 10, characterized in that the malate dehydrogenase is a glyoxysomal malate dehydrogenase and by a nucleic acid which sequence is encoded

a) a nucleic acid sequence according to claim 4; or

b) a 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: 3, which has an identity with SEQ ID NO: 3 of at least 66%;

includes.

13. The method according to claim 10, 11 or 12, characterized in that a test compound is selected which reduces or blocks the enzymatic or biological activity of the glyoxysomal malate dehydrogenase.

14. The method according to claim 10, 11, 12 or 13, characterized in that

i. Malate dehydrogenase is either expressed in a transgenic organism or an organism which naturally contains malate dehydrogenase is cultivated;

ii. the malate dehydrogenase 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; and

iii. a test compound is selected which reduces or blocks the enzymatic activity of the malate dehydrogenase from step a).

15. The method according to claim 10, 11, 12, 13 or 14, characterized in that it comprises the following steps:

i. Production of a transgenic organism according to claim 7 or a transgenic organism containing a nucleic acid sequence coding for a malate dehydrogenase comprising a nucleic acid sequence which, owing to the degenerate genetic code, is converted back by the amino acid sequence of a functional equivalent of SEQ ID NO: 3, which has an identity with SEQ ID NO: 3 derives from at least 63%;

wherein malate dehydrogenase is overexpressed in the transgenic organism; ii. Applying a test substance to the transgenic organism according to i) and to a non-transgenic organism of the same genotype; and

iii. Determining the growth or viability of the transgenic and non-transgenic organisms after application of the test substance; and

iv. Selection of test substances that cause a reduced growth or a limited survivability of the non-transgenic organism compared to the growth of the transgenic organism.

16. The method according to claim 15, characterized in that it is carried out in a plant organism, a cyanobacterium or a proteobacterium.

17. A method for identifying substances with a growth regulatory effect, characterized in that it comprises the following steps:

i. Production of a transgenic plant comprising a nucleic acid sequence coding for a polypeptide with the biological activity of a glyoxysomal malate dehydrogenase comprising

a) a nucleic acid sequence according to claim 4; or

b) a 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: 3 which has an identity with SEQ ID NO: 3 of at least 63%;

wherein malate dehydrogenase is overexpressed in the transgenic organism; ii. Applying a test substance to the transgenic plant according to i) and to a non-transgenic plant of the same variety;

iv. Selection of test substances that cause an altered growth of the non-transgenic plant compared to the growth of the transgenic a plant.

18. The method according to any one of claims 10 to 17, characterized in that the identification of the substances is carried out in a high-throughput screening.

19. Carrier containing one or more of the nucleic acid molecules according to claim 4, or one or more expression cassettes according to claim 7, one or more vectors according to claim 8, one or more organisms according to claim 9 or one or more (poly) peptides according to claim 5 has.

20. The method according to any one of claims 10 to 18, characterized in that the identification of the substances is carried out in a high-throughput screening using a carrier according to claim 19.

21. Compounds with herbicidal activity identified via one of the methods according to one of claims 10 to 1 _. 6, 18 and 20.

22. Compounds with growth regulatory activity identified by the method according to claim 17, 18 or 20.

23. A method for producing an agrochemical composition, characterized in that

a) a compound with herbicidal activity via one of the processes according to

Claims 10 to 16, 18 and 20 or a compound with growth regulatory activity according to claim 17, 18 or 20 identified; and

b) this compound is formulated together with suitable auxiliaries to give crop protection agents having a herbicidal or growth-regulating action.

24. A method for controlling undesirable plant growth and / or for regulating the growth of plants, characterized in that at least one malate dehydrogenase inhibitor according to claim 21 or

22 or an agrochemical composition containing an inhibitor of malate dehydrogenase according to claim 21 or 22 on plants, their habitat and / or on seeds.

25. Use of an inhibitor of malate dehydrogenase according to claim 21 or 22 or an agrochemical composition containing an inhibitor of Malate dehydrogenase according to claim 21 or 22 in a method according to claim 24.

26. A method for generating nucleic acid sequences which code for malate de-> hydrogenase, which is not inhibited by substances according to claim 21, wherein the nucleic acid sequence comprises a nucleic acid sequence which, based on the degenerate genetic code, is converted back by the amino acid sequence of a functional equivalent of the SEQ ID NO: 3, which has an identity with SEQ ID NO: 3 of at least 63%, can be derived;

characterized in that it comprises the following process steps:

a) expression of the protein encoded by the nucleic acid sequence according to i) in a heterologous system or in a cell-free system;

c) measuring the interaction of the modified gene product with the herbicide;

d) identification of derivatives of the protein which have less interaction;

e) testing the biological activity of the protein after application of the herbicide; and

27. The method according to claim 26, characterized in that the sequences selected according to claim 26 f) are introduced into an organism.

28. A method for producing transgenic plants which are resistant to substances according to claim 21, characterized in that in these plants a nucleic acid sequence coding for a malate dehydrogenase, which

a) a nucleic acid sequence according to claim 4; or

b) a functional equivalent of the nucleic acid sequence of SEQ ID NO: 3, which has an identity with SEQ ID NO: 3 of at least 63%; or includes, is overexpressed.

29. Transgenic plant produced by a method according to claim 28.