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EP0970231A1 - Expression von herbizid-bindenden polypeptiden in pflanzen zur erzeugung von herbizidtoleranz - Google Patents

Expression von herbizid-bindenden polypeptiden in pflanzen zur erzeugung von herbizidtoleranz

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Publication number
EP0970231A1
EP0970231A1 EP98924077A EP98924077A EP0970231A1 EP 0970231 A1 EP0970231 A1 EP 0970231A1 EP 98924077 A EP98924077 A EP 98924077A EP 98924077 A EP98924077 A EP 98924077A EP 0970231 A1 EP0970231 A1 EP 0970231A1
Authority
EP
European Patent Office
Prior art keywords
herbicide
plant
polypeptide
plants
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98924077A
Other languages
German (de)
English (en)
French (fr)
Inventor
Jens Lerchl
Achim Möller
Ralf-Michael Schmidt
Helmut Schiffer
Udo Rabe
Udo Conrad
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
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Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP0970231A1 publication Critical patent/EP0970231A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a method for producing herbicide-tolerant plants by expressing an exogenous herbicide-binding polypeptide in plants or parts of plants.
  • the invention further relates to the use of the corresponding nucleic acids coding for a polypeptide, an antibody or parts of an antibody with herbicide-binding properties in transgenic plants and the plant itself transformed in this way.
  • Transgenic plants are currently used in various biotechnological areas. Examples are insect-resistant plants (Vaek et al. Plant Cell 5 (1987), 159-169), virus-resistant plants (Powell et al. Science 232 (1986), 738-743) and ozone-resistant plants (Van Camp et al. BioTech 12: 165-168 (1994). Examples of genetically engineered quality increases are: increasing the shelf life of fruit (Oeller et al. Science 254 (1991), 437-439), increasing the starch production in potato tubers (Stark et al.
  • the herbicide tolerance is characterized by a type or level of increased tolerance of the plant or of parts of plants to the applied herbicide. This can be done in different ways.
  • the known methods are the use of a metabolism gene such as, for example, the pat gene in connection with glufosinate resistance (WO 8705629) or a target enzyme which is resistant to the herbicide, such as in the case of enolpyruvylshiki-mat-3-phosphate synthase ( WO 9204449), which is resistant to glyphosate, and the use of a herbicide in cell and tissue culture for the selection of tolerant plant cells and from them sulting resistant plants as described for acetyl CoA carboxylase inhibitors (US 5162602, US 5290696).
  • a metabolism gene such as, for example, the pat gene in connection with glufosinate resistance (WO 8705629) or a target enzyme which is resistant to the herbicide, such as in the case of enolpyruvylshiki
  • Antibodies are proteins that are part of the immune system. Common to all 5 antibodies is their spatial, globular structure, the structure of light and heavy chains as well as their fundamental ability to bind molecules or parts of a molecular structure with high specificity (Alberts et al., In: Molecular Biology of the Cell, 2nd edition 1990 , VCH Verlag, ISBN 3-527-27983-0,
  • hybrid somatic cell lines as a source of antibodies against specific antigens is based on ar-
  • each hybridoma cell is derived from a single B cell as a clone, all of the antibody molecules produced have the same structure, including the antigen binding site. This method has greatly promoted the use of antibodies because now antibodies with a single, known
  • phage display method 40 for producing antibodies in which the immune system and the various immunizations in the animal are bypassed, has existed for some years.
  • the affinity and specificity of the antibody is tailored in vitro (Winter et al., Ann. Rev. I Munol. 12 (1994), 433-455; Hoogenboom TIBTech Vol 15 (1997), 62-70).
  • Gene segments which contain the 45 coding sequence of the variable region of antibodies, ie the antigen binding site are fused with genes for the coat protein of a bacteriophage. Then you infect bacterial with phages containing such fusion genes.
  • the resulting phage particles now have envelopes with the antibody-like fusion protein, with the antibody-binding domain pointing outwards.
  • the phage which contains the desired antibody fragment and specifically binds to a specific antigen can now be isolated from such a phage display library.
  • Each phage isolated in this way produces a monoclonal, antigen-binding polypeptide which corresponds to a monoclonal antibody.
  • the genes for the antigen binding site which are unique for each phage, can be isolated from the phage DNA and used to construct complete antibody genes.
  • This resistance-imparting gene is isolated from such a microorganism, cloned into suitable vectors and then, after successful transformation, expressed in herbicide-sensitive crop plants (WO 96/38567).
  • the object of the present invention was to develop a novel, generally applicable, genetic engineering method for producing herbicide-tolerant transgenic plants.
  • This object was surprisingly achieved by a method of expressing an exogenous polypeptide, antibody or parts of an antibody with herbicide-binding properties in the plants.
  • a first subject of the present invention relates to the production of a herbicide-binding antibody and the cloning of the associated gene or gene fragment.
  • a suitable antibody is first generated that binds the herbicide. This can include by immunizing a vertebrate, usually a mouse, rat, dog, horse, donkey or goat with an antigen.
  • the antigen is a herbicidally active compound which is linked or associated via a functional group to a higher molecular weight carrier such as bovine serum albumin (BSA), chicken egg white (ovalbumin), keyhole limpet hemocyanin (KLH) or other carriers.
  • BSA bovine serum albumin
  • ovalbumin chicken egg white
  • KLH keyhole limpet hemocyanin
  • This approach initially provides a polyclonal serum that contains antibodies with different specificities.
  • the first approach uses the fusion of antibody-producing cells with cancer cells to form a hybridoma cell culture that continuously produces antibodies. By separating the clones it contains, it ultimately leads to a homogeneous cell line that produces a defined monoclonal antibody.
  • the cDNA is the so-called single-chain anti ⁇ body (Single chain antibody - scFv) for the antibody or parts of the antibody from such a monoclonal cell line. Isolated. These cDNA sequences can then be cloned into expression cassettes and used for functional expression in prokaryotic and eukaryotic organisms, including plants.
  • a herbicide-resistant plant can also be produced by cloning the gene of this catalytic antibody and expressing it in a plant.
  • the invention particularly relates to expression cassettes, the coding sequence of which codes for a herbicide-binding polypeptide or its functional equivalent, and the use thereof for the production of a herbicide-tolerant plant.
  • the nucleic acid sequence can e.g. be a DNA or a cDNA sequence.
  • Coding sequences suitable for insertion into an expression cassette according to the invention are, for example, those which contain a DNA sequence from a hybridoma cell which codes for a polypeptide with herbicide-binding properties and which impart resistance to inhibitors of plant enzymes to the host.
  • an expression cassette according to the invention also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell.
  • an expression cassette according to the invention comprises upstream, i.e. a promoter at the 5 'end of the coding sequence and downstream, i.e. at the 3 'end a polyadenylation signal and optionally further regulatory elements which are operatively linked to the intervening coding sequence for the polypeptide with herbicide-binding properties and / or transit peptide.
  • An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can perform its function as intended in the expression of the coding sequence.
  • sequences preferred but not limited to the operative linkage are targeting sequences to ensure subcellular localization in the apoplast, in the plasma membrane, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil - corpuscles or other compartments and translation enhancers such as the 5 'guiding sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987) 8693-8711).
  • any promoter that can control the expression of foreign genes is suitable as promoters of the expression cassette according to the invention.
  • a plant promoter or a promoter derived from a plant virus is preferably used in particular.
  • the CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980) 285-294).
  • This promoter contains different identification sequences for transcriptional effectors, which in their entirety lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989) 2195-2202).
  • the expression cassette according to the invention can also contain a chemically inducible promoter, by means of which the expression of the exogenous polypeptide in the plant can be controlled at a specific point in time.
  • a chemically inducible promoter by means of which the expression of the exogenous polypeptide in the plant can be controlled at a specific point in time.
  • promoters as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter induced by salicylic acid (WO 95/1919443), a promoter induced by benzenesufonamide (EP 388186), a by Scisic acid-inducible (EP335528) or an ethanol- or cyclohexanone-inducible (W09321334) promoter are described in the literature and can be used inter alia be used.
  • promoters are particularly preferred which ensure expression in tissues or parts of plants in which the herbicidal action develops. Promoters which ensure leaf-specific expression should be mentioned in particular.
  • the promoter of the cytosolic FBPase from potatoes or the ST-LSI promoter from potatoes should be mentioned (Stockhaus et al., EMBO J. 8 (1989) 2445-245).
  • the expression cassette according to the invention can therefore contain, for example, a seed-specific promoter (preferably the USP or LEB4 promoter, the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
  • a seed-specific promoter preferably the USP or LEB4 promoter, the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
  • the structure of the cassette is shown in FIG. 1 using the example of a single chain Antibody (scFv gene) is shown schematically as an example.
  • An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable polypeptide DNA and preferably a DNA coding for a chloroplast-specific transit peptide inserted between the promoter and polypeptide DNA and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, EF Fritsch and J.
  • sequences which target in the apoplasts, plastids, the vacuole, in the plasma membrane, the mitochondrium, the endoplasmic reticulum (ER) or due to a lack of corresponding operative sequences remaining in the compartment of formation, the cytosol ensure (Kermode, Crit. Rev. Plant Sei. 15, 4 (1996), 285-423). Localization in the ER and the cell wall has proven particularly beneficial for the amount of protein accumulation in transgenic plants
  • the invention also relates to expression cassettes whose coding sequence codes for a herbicide-binding fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
  • Chloroplast-specific transit peptides are particularly preferred which, after translocation of the herbicide-binding polypeptide into the plant chloroplasts, are enzymatically split off from the herbicide-binding polypeptide part.
  • the transit peptide is particularly preferably derived from plastidic transketolase (TK) or a functional equivalent of this transit peptide (e.g. the transit peptide of the small subunit of the Rubisco or the ferredoxin NADP oxidoreductase).
  • the polypeptide DNA or cDNA required for the production of expression cassettes according to the invention is preferably amplified with the aid of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Methods for DNA amplification by means of PCR are known, for example from Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press (1990).
  • the PCR-generated DNA fragments can expediently be checked by sequence analysis to avoid polymerase errors in constructs to be expressed.
  • the inserted nucleotide sequence coding for a herbicide-binding polypeptide can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components.
  • synthetic nucleotide sequences are generated with codons that are preferred by plants. These codons preferred by plants can be determined from codons with the highest protein frequency, which are described in most interesting plant species can be expressed.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions according to the invention should expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp within the regulatory areas, often less than 60 bp, but at least 5 bp.
  • the promoter according to the invention can be both native or homologous and foreign or heterologous to the host plant.
  • the expression cassette according to the invention contains, in the 5 '-3' transcription direction, the promoter according to the invention, any sequence and a region for the transcriptional termination. Different termination areas are interchangeable.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially contain T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid correspond to pTiACH ⁇ (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents.
  • An expression cassette according to the invention can contain, for example, a constitutive promoter (preferably the CaMV 35 S promoter), the LeB4 signal peptide, the gene to be expressed and the ER retention signal.
  • a constitutive promoter preferably the CaMV 35 S promoter
  • the structure of the cassette is shown schematically in Figure 2 using the example of a single-chain antibody (scFv gene).
  • the amino acid sequence KDEL lysine, aspartic acid, glutamic acid, leucine
  • KDEL lysine, aspartic acid, glutamic acid, leucine
  • the fused expression cassette which codes for a polypeptide with herbicide-binding properties, is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens.
  • Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as e.g. of tobacco plants can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the transformation of plants by agrobacteria is known, among other things, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu,
  • transgenic plants can be regenerated from the transformed cells of the wounded leaves or leaf pieces which contain a gene integrated into the expression cassette according to the invention for the expression of a polypeptide with herbicide-binding properties.
  • an expression cassette according to the invention is inserted as an insert in a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
  • Suitable vectors are inter alia in "Method in Plant Molecular Biology and Biotechnology” (CRC Press), Chap. 6/7, p.71-119 (1993).
  • the expression cassettes according to the invention can be cloned into suitable vectors which enable their multiplication, for example in E. coli.
  • suitable vectors include pBR332, pUC series, M13mp series and pACYC184.
  • Another object of the invention relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or parts of plants.
  • the aim of the use is preferably to impart resistance to inhibitors of plant enzymes.
  • the expression can take place specifically in the leaves, in the seeds or in other parts of the plant.
  • Such transgenic plants, their reproductive material and their plant cells, tissue or parts thereof are a further subject of the present invention.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation. There are the described
  • Plant tissues or plant cells used for transient or stable transformation are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene gun, electroporation,
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res.
  • Agrobacteria transformed with an expression cassette according to the invention can then be used in a known manner to transform plants, in particular crop plants, such as cereals,
  • Functionally equivalent sequences which code for a herbicide-binding polypeptide are, according to the invention, those sequences which, despite a different nucleotide sequence, still have the desired functions. Functional equivalents thus include naturally occurring variants of the sequences described here, as well as artificial nucleotide sequences, for example those obtained by chemical synthesis and adapted to the codon usage of a plant.
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations of an originally isolated sequence encoding the herbicide-binding polypeptide, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
  • the present invention also encompasses those nucleotide sequences which are obtained by modification of this nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also the insertion of further restriction enzyme interfaces.
  • Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
  • artificial DNA sequences are suitable as long as they induce the desired resistance to herbicides, as described above.
  • Such artificial DNA sequences can be determined, for example, by back-translating proteins constructed using molecular modeling, which have herbicide-binding activity, or by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable. The specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.
  • Sequences which code for fusion proteins are to be mentioned as further suitable nucleic acid sequences according to the invention, part of the fusion protein being a non-plant herbicide-binding polypeptide or a functionally equivalent part thereof.
  • the second part of the fusion protein can be, for example, another polypeptide with enzymatic activity or an antigenic polypeptide sequence with the aid of which it is possible to detect scFvs expression (for example mye-tag or his-tag).
  • this is preferably a regulatory protein sequence, such as a signal or transit peptide, which directs the polypeptide with herbicide-binding properties to the desired site of action.
  • the invention also relates to the expression products produced according to the invention and to fusion proteins composed of a transit peptide and a polypeptide with herbicide-binding properties.
  • Resistance or tolerance in the context of the present invention means the artificially acquired resistance to the action of plant enzyme inhibitors. It includes the partial and, in particular, the complete insensitivity to these inhibitors for at least one generation of plants.
  • the primary site of action of herbicides is generally leaf tissue, so that leaf-specific expression of the exogenous herbicide-binding polypeptide can offer adequate protection.
  • the action of a herbicide need not be limited to the leaf tissue, but can also be tissue-specific in all other parts of the plant.
  • constitutive expression of the exogenous herbicide-binding polypeptide is advantageous.
  • inducible expression may also appear desirable.
  • the effectiveness of the transgenically expressed polypeptide with herbicide-binding properties can be determined, for example, in vitro by increasing the number of shoots on herbicide-containing medium by means of graduated concentration series or by means of seed germination tests.
  • a change in the type and level of herbicide tolerance of a test plant can be tested in greenhouse experiments.
  • the invention also relates to transgenic plants transformed with an expression cassette according to the invention, and to transgenic cells, tissues, parts and propagation material of such plants.
  • Transgenic crop plants such as e.g. Cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species.
  • transgenic plants, plant cells, tissues or parts can be treated with an active ingredient which inhibits the plant enzymes, as a result of which those which have not been successfully transformed Plants, cells, tissues or parts of plants die.
  • suitable active ingredients are, in particular, 5- (2-chloro-4- (trifluoromethylphenoxy) -2-nitrobenzoic acid (acifluorfen) and 7-chloro-3-methylquinoline-8-carboxylic acid (Quinmerae), and also metabolites and functional derivatives of these compounds DNA inserted into the expression cassettes according to the invention and coding for a polypeptide with herbicide-binding properties can thus also be used as a selection marker.
  • the present invention offers the advantage that, after induction of a selective resistance of the crop plant to plant enzyme inhibitors, these inhibitors can be used as specific herbicides against non-resistant plants.
  • these inhibitors can be used as specific herbicides against non-resistant plants.
  • the following herbicidal compounds from groups bl - b41 can be mentioned as non-limiting examples of such inhibitors:
  • b2 amides allidochlor (CDAA), benzoylprop-ethyl, bromobutide, chlorothiamide, dimepiperate, dimethenamid, diphenamid, etobenzanid (benzchlomet), flamprop-methyl, fosamin, isoxaben, monalide, naptalame, pronamid (propyzamid), propanil
  • b3 aminophosphoric acids bilanafos, (bialaphos), buminafos, glufosinate-ammonium, glyphosate, sulfosate
  • 2,4-D, 2,4-DB clomeprop, dichlorprop, dichlorprop-P, dichlorprpp-P (2,4-DP-P), fenoprop (2,4,5-TP), fluoroxypyr, MCPA, MCPB, mecoprop, mecoprop-P, napropamide, napropanilide, tri-clopyr
  • b9 Bleacher clomazone (dimethazone), diflufeniean, fluorochloridone, flupoxam, fluridone, pyrazolate, sulcotrione (chloromesulone)
  • BlO carbamates asulam, barban, butylate, carbetamide, chlorobufam, chloropropam, cyeloate, desmedipham, dialallate, EPTC, esproearb, molinate, orbencarb, pebulate, phenisopham, phenmedipham, propam, prosulfocarb, pyributicarb, sulfallate (CD ), terbu-carb, thiobencarb (benthiocarb), tiocarbazil, triallate, vernolate
  • bl2 chloroacetanilides acetochlor, alachlor, butachlor, butenachlor, diethatyl ethyl, dimethachlor, metazachlor, metolachlor, pretilachlor, propachlor, prynachlor, terbuchlor, thenylchlor, xylachlor
  • bl3 cyclohexenones alloxydim, caloxydim, clethodim, cloproxydim, cycloxydim, sethoxydim, tralkoxydim, 2- ⁇ 1- [2- (4-chlorophenoxy) propyloxyi - minolbutyl ⁇ '3-hydroxy-5- (2H-tetrahydrothiopyran-3-yl) - 2-cyclohexen-l-one
  • bl7 Dinitroani1ine benefin, butralin, dinitramine, ethalfluralin, fluchloralin, isopropalin, nitralin, oryzalin, pendimethalin, prodiamine, profluralin, trifluralin
  • bl8 dinitrophenols bro ofenoxim, dinoseb, dinoseb-acetate, dinoterb, DNOC bl9
  • Diphenyl ether acifluorfen-sodium, aclonifen, bifenox, chloronitrofen (CNP), difenoxuron, ethoxyfen, fluorodifen, fluoroglycofen-ethyl, fomesafen, furyloxyfen, lactofen, nitrofen, nitrofluorfen, oxyfluorfen
  • b21 ureas benzthiazuron, buturon, chlorbromuron, chloroxuron, chlorto- luron, cumyluron, dibenzyluron, cycluron, dimefuron, diuron, dymron, ethidimuron, fenuron, fluormeturon, isoproturon, isouron, karbutilate, linuron, methabenzthiazuron, metobenzu- ron, metoxuron, monolinuron , monuron, neburon, siduron, tebutiuron, trimeturon
  • Imidazolinones imazamethapyr, imazapyr, imazaquin, imazethabenz-methyl (imazame), imazethapyr
  • b26 phenols bromoxynil, ioxynil b27 phenoxyphenoxypropionic acid esters: clodinafop, cyhalofop-butyl, diclofop-methyl, fenoxaprop-ethyl, fenoxaprop-p-ethyl, fenthiapropethyl, fluazifop-butyl, fluazifop-p-butyl, haloxyfoxy-ethopethyl-ethoxyethyl -p-methyl, isoxapyrifop, propaquizafop, quizalofop-ethyl, quizalofop-p-ethyl, quizalofop-tefuryl
  • b29 phenylpropionic acids chlorophenprop-methyl b30
  • Protoporphyrinogen IX oxidase inhibitors benzofenap, cinidon-ethyl, flumiclorac-pentyl, flumioxazin, flumipropyn, flupropacil, fluthiacet-ethyl, pyrazoxyfen, sulfentrazone, thidiazimin
  • b32 pyridazines chloridazon, maleic hydrazide, norflurazon, pyridate
  • pyridinecarboxylic acids clopyralid, dithiopyr, picloram, thiazopyr
  • b3 pyrimidyl ethers pyrithiobac acid, pyrithiobac sodium, KIH-2023, KIH-6127
  • Sulfonylureas amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorosulfuron, cinosulfuron, cyclosulfamuron, ethamet- sulfuron methyl, ethoxysulfuron, flazasulfuron, halosulfuron-methyl, imazosulfuron, pyrosulfuron, metsulfuron, metsulfuron, metsulfuron ethyl, rimsulfuron, sulfometuron-methyl, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, triflusulfuron-methyl
  • b37 triazines ametryn, atrazin, aziprotryn, cyanazine, cyprazine, desme- tryn, dimethamethryn, dipropetryn, eglinazin-ethyl, hexazinone, procyazine, prometon, prometryn, propazin, secbumeton, simazin, simetryn, terbumeton, terbutryn, terbutryn, terbutryn, terbutryn, terbutryn - magazine
  • b40 üracile bromacil, lenacil, terbacil b41
  • Functionally equivalent derivatives of plant enzyme inhibitors have a comparable spectrum of activity to the specifically named substances, with lower, equal or higher inhibitory activity (e.g. expressed in g inhibitor per hectare of cultivated area, required to completely suppress the growth of non-resistant plants).
  • cloning steps carried out in the context of the present invention such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were carried out as with Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).
  • the bacterial strains used below (E. coli, XL-I Blue) were obtained from Stratagene.
  • the Agrobacterium strain used for plant transformation (Agrobacterium tumefaeiens, C58C1 with the plasmid pGV2260 or pGV3850kan) was developed by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777).
  • the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used.
  • the vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBlues- script SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl Acids Res. 12 (1984) 8711-8720) and pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).
  • nucleotides 11749-11939 was used as PvuII-
  • the HindIII fragment was isolated and cloned after addition of Sphl linkers to the PvuII site between the SpHI-HindII site of the vector, resulting in the plasmid pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).
  • herbicides are not immunogenic, they have to be attached to a carrier material such as e.g. KLH can be coupled. If there is a reactive group in the molecule, this coupling can take place directly, otherwise a functional group is introduced during the synthesis of the herbicide or a reactive precursor is selected during the synthesis in order to couple these molecules to the carrier molecule in a simple reaction step. Examples of couplings are described by Miroslavic Ferencik in "Handbook of Immunochemistry", 1993, Chapman & Hall, in the chapter Antigens, pages 20-49.
  • this modified carrier molecule e.g. Balb / c mice immunized.
  • the spleen cells of these animals are removed and fused with myeloma cells in order to cultivate hybrids.
  • "herbicide-modified BSA” is also used as an antigen in order to distinguish the immune response against the hapten from the KLH response.
  • the starting point for the investigation was a monoclonal antibody which specifically recognizes the herbicide Quinmerae and which also has a high binding affinity.
  • the selected hybridoma cell line is characterized in that the secreted monoclonal antibodies directed against the herbicide antigen Quinmerae have a high affinity and the specific sequences of the immunoglobulins are available (Berek, C. et al., Nature 10 316, 412-418 (1985)).
  • This monoclonal antibody against Quinmerae was the starting point for the construction of the single-chain antibody fragment (scFv-antiQuinmerac).
  • mRNA was isolated from the hybridoma cells and in cDNA
  • variable immunoglobulin genes VH and VK served as a template for the amplification of the variable immunoglobulin genes VH and VK with the specific primers VH1 BACK and VH FOR-2 for the heavy chain and VK2 BACK and MJK5 FON X for the light chain (Clackson et al., Nature 352, 624 -628 (1991)).
  • the functional characterization (antigen binding activity) of the constructed scFv-antiQuinmerac gene was carried out after expression in a bacterial system.
  • the scFv-antiQuinmerac was developed using the method of Hoogenboom, H.R. et al., Nucleic Acids
  • the scFv-antiQuinmerac gene was cloned downstream of the LeB4 promoter.
  • the LeB4 promoter isolated from Vicia faba shows a strictly seed-specific expression of various foreign genes in tobacco (Bäumlein, H. et al., Mol. Gen.
  • the constructed expression cassette was cloned into the binary vector pGSGLUC 1 (Saito et al., 1990) and transferred into the Agrobacterium strain EHA 101 by electroporation. Recombinant agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum. 70-140 tobacco plants were regenerated per construct. After self-fertilization, seeds from various developmental stages were harvested from the regenerated transgenic tobacco plants. The soluble proteins were obtained from these seeds after extraction in an aqueous buffer system.
  • the constructed scFv-antiQuinmerac gene had a size of approx. 735 bp.
  • the variable domains were fused together in the order VH-L-VL.
  • the starting point for the investigations was a single-chain antibody fragment against the herbicide Quinmerae (scFv-anti Quinmerae).
  • the functional characterization (antigen binding activity) of this constructed scFv-anti- Quinmerae gene was carried out after expression in a bacterial system and after expression in tobacco leaves. The activity and the specificity of the antibody fragment constructed was checked in ELISA tests.
  • the scFv-antiQuinmerac gene was cloned downstream of the USP promoter.
  • the USP promoter isolated from Vicia faba shows a strictly seed-specific expression of various foreign genes in tobacco (Fiedler, U. et al., Plant Mol. Biol. 22, 669-679 (1993)).
  • scFv-anti-Quinmerac polypeptide By transporting the scFv-anti-Quinmerac polypeptide into the endoplasmic reticulum, a stable accumulation of large amounts of antibody fragments was achieved.
  • the scFv-antiQuinmerac gene was identified with a signal peptide sequence that prevents entry into the endoplasmic see the reticulum and the ER retention signal SEKDEL, which ensures that it remains in the ER (Wandelt et al., 1992), merges (Fig. 1).
  • the constructed expression cassette was cloned into the binary vector pGSGLUCl (Saito et al., 1990) and transferred into the Agrobacterium strain EHA 101 by electroporation. Recombinant agrobacteria clones were used for the subsequent transformation of Nicotiana tabacum. After self-fertilization, seeds of various developmental stages were harvested from the regenerated transgenic tobacco plants. The soluble proteins were obtained from these seeds after extraction in an aqueous buffer system.
  • the scFv-anti- Quinmerac gene was cloned downstream of the CaMV 35 S promoter. This strong constitutive promoter mediates expression of foreign genes in almost all plant tissues (Benfey and Chua, Science 250 (1990), 956-966). By transporting the scFv-anti-Quinmerac protein into the endoplasmic reticulum, a stable accumulation of large amounts of antibody fragments in the leaf material was achieved.
  • the scFv-antiQuinmerac gene was first fused with a signal peptide sequence which ensures entry into the endoplasmic reticulum and the ER retention signal KDEL, which ensures that it remains in the ER (Wandelt et al., Plant J. 2 (1992), 181-192) .
  • the constructed expression cassette was cloned into the binary vector pGSGLUC 1 (Saito et al., Plant Cell Rep. 8 (1990), 718-721) and transferred into the Agrobacterium strain EHA 101 by electroporation. Recombinant agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum. About 100 tobacco plants were regenerated.
  • Leaf material from various stages of development was removed from the regenerated transgenic tobacco plants.
  • the soluble proteins were obtained from this leaf material after extraction in an aqueous buffer system. Subsequent analyzes (Western blot analyzes and ELISA tests) showed that a maximum accumulation of greater than 2% of biologically active, antigen-binding scFv-antiQuinmerac polypeptide could be achieved in the leaves.
  • the high expression values were The antibody fragment was found in green leaves, but also in senescent leaf material.
  • the PCR amplification of the one-chain antibody cDNA was carried out in a DNA thermal cycler from Perkin Elmer.
  • the reaction mixtures contained 8 ng / ⁇ l single-stranded template cDNA, 0.5 ⁇ M of the corresponding oligonucleotides, 200 ⁇ M nucleotides (Pharmacia), 50 mM KC1, 10 mM Tris-HCl (pH 8.3
  • the plasmid pGSGLUC 1 was transformed into Agrobacterium tumefaeiens C58Cl: pGV2260.
  • tobacco plants Nicotiana fcaJbacum cv. Samsun NN
  • the starting point of the investigations was a single-chain antibody fragment expressed in tobacco plants against the herbicide Quinmerae (scFv-anti Quinmerae).
  • the amount and activity of the synthesized scFv-antiQuinmerac polypeptide were determined in Western blot analyzes and ELISA tests.
  • the foreign gene was controlled under the control of the CaMV 53S promoter as a translation fusion with the LeB4 signal peptide (N-terminal) and the ER retention signal KDEL (C- terminal).
  • the scFv-antiQuinmerac polypeptide was transported into the endoplasmic reticulum, a stable accumulation of high amounts of active antibody fragment was achieved. After harvesting the leaf material, pieces were frozen at -20 ° C (1), lyophilized (2) or dried at room temperature (3).
  • soluble proteins were obtained from the respective leaf material by extraction in an aqueous buffer and the scFv-antiQuinmerac polpypeptide was purified by affinity chromatography. Equal amounts of purified scFv-anti-Quinmerac polypeptide (frozen, lyophilized and dried) were used to determine the activity of the antibody fragment (Fig. 6).
  • Fig. 6 A shows the antigen binding activity of the scFv-antiQuinmerac polypeptide purified from fresh (1), lyophilized (2) and dried leaves (3).
  • Fig. 6 B the respective amounts of scFv-anti-Quinmerac protein (about 100 ng) that were used for the ELISA analyzes are determined by means of Western blot analyzes. The sizes of the protein molecular weight standards are shown on the left. The same antigen binding activities were found.

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