CA2264677A1 - Adenylosuccinate synthetase - Google Patents

Abstract

Expression cartridges are disclosed which confer to plants, plant cells, tissues or parts a resistance against inhibitors of the vegetable adenylosuccinate synthetases. Also disclosed is the use of the expression cartridges in appropriate vectors to transform plants, plant cells, tissues or parts.

Description

10152025303540450050/47289(2)Adenylosuccinate synthetaseThe present invention relates to expression cassettes coding fornon—plant adenylosuccinate synthetases (ADSS) which conferresistance to inhibitors of plant ADSS on the plants; vectors andmicroorganisms comprising such expression cassettes; transgenicplants transformed therewith; the corresponding expressionproducts and nucleic acid sequences and an expression kit to beused for transforming a plant host.Plants, as photoautotrophic organisms, are able to synthesizetheir cellular components from carbon dioxide, water andinorganic salts. This process is possible only by usingbiochemical reactions for synthesizing organic substances. Inparticular, it is necessary for plants to synthesize de novo alsothe nucleotides as constituents of nucleic acids.It is to be assumed that efficient production, use anddistribution of the nucleotides greatly influence cell divisionand growth of a plant. Since plants depend on a functioning denovo nucleotide biosynthesis, this biosynthetic pathway appearsan appropriate target for the use of herbicides. The complexreactions which ensure nucleotide biosynthesis are divided intothe biosynthesis of purines and of pyrimidines.The enzyme reactions in purine biosynthesis starting fromphosphoribosyl pyrophosphate (PRPP) can be divided into the'following steps:a) synthesis of the pyrimidine ringb) synthesis of the purine ringc) branching from IMP to AMP or GMPThe reaction sequence of step c) is depicted diagrammatically inappended Figure 1.Some of the enzymes involved in purine biosynthesis representpotential points of attack for herbicidal agents. ADSS occupies aspecial position. The enzyme catalyzes the following reaction:-‘——IMP + L—aspartate + GTP _+adenylosuccinate + GDP + PiThe ADSS from E. coli has been purified to homogeneity (Bass etal., 1987, Arch. Biochem. Biophys., 256, 335-342), the crystalstructure has been determined (Poland et al., 1993, J. Biol.Chem., 268, 25334-25342) and kinetic properties of the enzymeCA 02264677 1999-02-2610152025303540450050/472892have been characterized (Kang and Fromm, 1995, J. Biol. Chem.,270, 15539-15544). The enzyme has also been purified fromDictyostelium discoideum (Jahngen and Rossomando, 1984, Arch.Biochem. Biophys., 229, 145-154) and rabbit muscle (J. Biol.Chem., 1974, 249(2), 459-464). Plant adenylosuccinate synthetasehas been isolated from wheat seedlings (Hatch, 1967,Phytochemistry, 6, 115-119) and from corn (siehl et al.,Plant Physiol., 110, 753-758) for determining the enzymeactivity.1996,The adenylosuccinate synthetase in E. coli is present as a dimerof two 48 kD polypeptides. No data have yet been published onplant ADSS.Genes which code for ADSS have to date been isolated fromEscherichia coli (EMBL Accession Number JO4199), Bacillussubtilis (J02732), Haemophilus influenzae (L46263), Saccharomycespombe (L22185), Schizosaccharomyces pombe (M98805), Dictyosteliumdiscoideum (M58471), Homo sapiens (X65503) and Mus musculus(M74495). It is possible by comparisons of data to find expressedsequence tags, called test sequences, from Arabidopsis thaliana(T42641) and Oryza sativa (D15352) with distinct similarity tosaid ADSSs.Complete cDNA sequences of plant adenylosuccinate synthetasesfrom Arabidopsis thaliana and corn (US—A-5519125), and from wheat(W096/19576), have also been described.No cofactors are necessary for ADSS activity measurement.However, agents which inhibit the enzyme activity of plant ADSShave been described in the form of hadacidin and alanosine(Stayton et al., 1983, Curr. Top. Cell. Regul., 22, 103-141) andhydantocidin and its metabolite 5'-phosphohydantocidin (Siehl etal., 1996, Plant Physiol., 110, 753-758).The phytotoxic agent hydantocidin was isolated from Streptomyceshygroscopicus (strain SANK 63584) (Nakajima et al., 1991, J.Antibiot., 44, 293-300). It was shown that5’-phosphohydantocidin, a metabolite which is produced in theplant, is the actual inhibitor of ADSS (Siehl et al., 1996, PlantPhysiol., 110, 753-758). Hydantocidin displays its toxic effectonly on plants, has low mammalian toxicity and has no effect onvarious microorganisms investigated (Nakajima et al., 1991, J.Antibiot., 44, 293-300). It would be desirable to find ways, ifpossible, to alter the response of plants to ADSS inhibitors in atargeted manner.CA 02264677‘1§99-02-26CA10152025303540450050/472893It is accordingly an object of the present invention to providemeans with whose aid the response of plants to ADSS inhibitorscan be modified in a targeted manner. The particular intentionwas that this modification be possible by genetic manipulation.We have found that this object is achieved by providing anexpression cassette comprising, under the genetic control ofregulatory nucleic acid sequences, the coding nucleic acidsequence for a protein which confers resistance to inhibitors ofplant adenylosuccinate synthetase on a plant host.The invention is now explained in detail with reference to thefollowing figures, which showFigure 1 de novo purine biosynthesis in plants;Figure 2 the nucleic acid sequence of the adenylosuccinatesynthetase from Escherichia coli, including the 5’- and3'—terminal BamHI cleavage sites;Figure 3 the amino acid sequence of the adenylosuccinatesynthetase from Escherichia coli;Figure 4 oligonucleotide sequences for isolating the nucleicacid sequence of adenylosuccinate synthetase fromEscherichia coli;Figure 5 the nucleic acid sequence of the transit peptide of theplastid transketolase in three reading frames;Figure 6 the construction of the expression cassettes andtransformation vectors pTPO9, pTP1O and pTP11;Figure 7 the construction of the expression cassettes and thetransformation vector pTP09-ASS.The present invention firstly relates to expression cassetteswhich are suitable for transforming a plant host and comprise acoding sequence which confers resistance to inhibitors of plantADSS on the host.For the purpose of the present invention, resistance means theartificially acquired ability to withstand the effect of plantADSS inhibitors. It comprises partial and, in particular,complete insensitivity to these inhibitors for the duration of atleast one plant generation.In a preferred embodiment of the invention, expression cassettesare provided for a plant host selected from whole plants, plantcells, plant tissues or parts of plants such as leaves, roots,fruit. The plant host is selected in particular from crop plants,and cells, tissues or parts derived therefrom. Nonlimitingexamples of suitable crop plants which may be mentioned are:02264677 1999-02-26101520V25303540450050/472894cereals, corn, soybean, rice, cotton, sugar beet, canola,sunflower, flax, potato, tobacco, tomato, oilseed rape, alfalfa,lettuce and the various tree, nut and vine species.The present invention has the advantage especially for cropplants that, after induction of selective resistance of the cropplant to plant ADSS inhibitors, these inhibitors can be employedas specific herbicides for nonresistant plants. Nonlimitingexamples of such inhibitors which may be mentioned are alanosine,hadacidin, hydantocidin and metabolites and functionallyequivalent derivatives thereof. Functionally equivalentderivatives of plant ADSS inhibitors have a spectrum of actionwhich is comparable to that of the specifically mentionedsubstances, while the inhibitory activity may be lower, the sameor higher (eg. expressed in g of inhibitor per hectare ofcultivated area necessary for complete suppression of growth ofnonresistant plants).The invention particularly relates to expression cassettes whosecoding sequence comprises a non-plant ADSS nucleic acid sequencewhich is tolerant of plant ADSS inhibitors, or a functionalequivalent thereof. The ADSS nucleic acid sequence may be, forexample, a DNA or a CDNA sequence.Examples of coding sequences suitable for insertion into anexpression cassette according to the invention are those whichessentially comprise a microbial DNA sequence which codes for anadenylosuccinate synthetase from microorganisms of the generaEscherichia, Bacillus, Haemophilus, Dictyostelium, Saccharomycesor Schizosaccharomyces and, in particular, from Escherichia coli,Bacillus subtilis, Haemophilus influenzae, Dictyosteliumdiscoideum, Saccharomyces pombe or Schizosaccharomyces pombe. Aparticularly suitable ADSS sequence essentially corresponds tothe DNA sequence from E. coli depicted in Figure 2 (SEQ ID NO:1).Artificial DNA sequences are also suitable as long as they inducethe required resistance as described above. Such artificial DNAsequences can be found, for example, by translating back fromproteins constructed by molecular modeling and havingadenylosuccinate synthetase activity or by in vitro selection.DNA sequences which code for ADSS and have been obtained bytranslation back from an ADSS polypeptide sequence in accordancewith the codon usage specific for the host plant are particularlysuitable. The specific codon usage can easily be found by askilled worker familiar with methods of plant genetics byCA 02264677 1999-02-2610150050/472895computer analyses of other genes known for the plant to betransformed.The invention also relates to sequences which are functionallyequivalent to the above nucleic acid sequences and to the aminoacid sequences derived therefrom. According to the invention,functional equivalents are those sequences which essentiallycorrespond to one another. This means, in particular, sequencevariants resulting from natural or artificial mutations of anADSS sequence as long as the required ADSS activity necessary formaintaining plant metabolism is retained during this. Theexogenous ADSS activity transformed into the plant host can thusbe higher than, comparable to or somewhat less than that of theendogenous plant ADSS. Mutations comprise substitutions,deletions, transpositions or insertions of one or more nucleotideor amino acid residues.Substitutions mean exchanges of nucleotides or amino acids. What. are called silent or conservative exchanges are preferred. A'202530354045silent nucleotide exchange makes no alteration in the amino acidsequence. A conservative exchange brings about a substitution ofan amino acid by an amino acid residue with comparableproperties, such as size, charge, polarity, solubility. Examplesof pairs of amino acids with similar properties are Glu and Asp,Val and Ile, Ser and Thr.According to the invention, a deletion means the removal ofleast one nucleotide or at least one amino acid residue.Preferred positions for deletions are the DNA regions whichfor the termini of the polypeptide and the linkages betweenindividual protein domains. DNA frequencies which areparticularly preferred according to the invention are thosecoding for ADSS proteins produced by N—terminal truncations of upto about 100, such as 20 to 100, amino acids from the sequencedepicted in Figure 3 (SEQ ID NO:2).atcodetheAccording to the invention, insertions comprise introductions ofat least one nucleotide or amino acid residue into one of thesequences according to the invention.Further functionally equivalent nucleic acid sequences accordingto the invention which should be mentioned are sequences whichcode for fusion proteins, where a non-plant ADSS polypeptide or afunctionally equivalent part thereof forms part of the fusionprotein. The second part of the fusion protein can be, forexample, another peptide with the same or different enzymeactivity. However, it is preferably a regulatory protein sequenceCA 022646777 V1999-02-2610152025303540450050/472896such as a signal or transit peptide which leads ADSS to therequired site of action.The invention thus also relates to expression cassettes whosecoding sequence codes for an ADSS fusion protein, where part ofthe fusion protein is a transit peptide which controls thetranslocation of the ADSS sequence. Particularly preferredtransit peptides are those which are specific for chloroplastsand which, after translocation of the ADSS sequence into theplant chloroplasts (main site of purine biosynthesis in plants),are cleaved off enzymatically from the ADSS part. The transitpeptide is particularly preferably derived from plastidtransketolase (TK) or a functional equivalent of this transitpeptide. An expression cassette which comprises a transit peptideDNA sequence depicted in Figure 5 (SEQ ID NOs:3,4,5) isparticularly preferred.The expression cassettes according to the invention additionallycomprise regulatory nucleic acid sequences which control theexpression of the coding sequence in the host cell. In apreferred embodiment, an expression cassette according to theinvention comprises a promoter upstream, ie. at the 5’ end, ofthe coding sequence, and a polyadenylation signal downstream, ie.at the 3' end, with or without other regulatory elements whichare operatively linked to the coding sequence lying between themfor ADSS and/or transit peptide. Operative linkage means thesequential arrangement of said regulatory elements in such a waythat each of the regulatory elements is able to execute itsfunction in the expression of the coding sequence.A suitable promoter for the expression cassette according to theinvention is in principle every promoter able to control theexpression of foreign genes in plants. A plant promoter isparticularly preferably used. The 358 CaMV promoter fromcauliflower mosaic virus (Franck et al. (1980) Cell 21, 285-294)is particularly preferred. This promoter contains variousrecognition sequences for transcriptional effectors which, intheir totality, result in constitutive expression of the insertedgene (Benfey et al. (1989) EMBO J. 8, 2195-2202).Preferred polyadenylation signals are plant polyadenylationsignals, preferably those which essentially correspond to T—DNApolyadenylation signals from Agrobacterium tumefaciens,especially to the polyadenylation signal of gene 3 of the T—DNAof the Ti plasmid pTiACHS.CA 0226hB77“1999-02-26io152025303540450050/472897An expression cassette according to the invention is prepared byfusing a suitable promoter to a suitable ADSS DNA and preferablyto a DNA which is inserted between the promoter and ADSS DNA andcodes for a chloroplast—specific transit peptide, and to apolyadenylation signal, by conventional recombination and cloningtechniques 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) andin T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY (1984) and in Ausubel, F.M. et al., Current Protocols inMolecular Biology, Greene Publishing Assoc. andWiley-Interscience (1987).The ADSS DNA or CDNA required to prepare expression cassettesaccording to the invention is preferably amplified by thepolymerase chain reaction (PCR). Processes for DNA amplificationusing the PCR are known, for example from Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press(1990). It is possible and expedient for the PCR—generated DNAfragments to be checked by sequence analysis to avoid polymeraseerrors in constructs to be expressed.The invention also relates to an expression kit which comprisesat least three expression cassettes, where at least two comprisea variable region or frame shift sequence, which differ from oneanother by insertion or deletion, preferably insertion, of anumber of nucleotides which is not divisible by three, and thusbring about a shift of the reading frame for a sequence inserteddownstream by one and two nucleotides respectively. The 5’ end ofthe frame shift sequence is linked to the transit peptidesequence. The frame shift sequence comprises a restrictioncleavage site at the 3’ end, into which, for example, the ADSSDNA sequence is inserted. The provision of at least three vectorswhich each comprise an expression cassette of the kit withdifferent reading frames for the inserted gene ensures that thefusion constructs from any DNA sequence and from a DNA sequencecoding for a transit peptide can be expressed in three differentreading frames, and at least one of the constructs results inexpression of a functional gene product (such as ADSS). Theconstruction diagrams for an expression kit according to theinvention which comprises three expression cassettes and isparticularly suitable for transforming plants are shown inappended Figure 6.CA 0226£éff‘f§99-02-260050/472898The invention also relates to the use of such kits fortransforming a plant host.It is possible by use of the recombination and cloning techniques5 cited above to clone the expression cassettes according to theinvention into suitable vectors which make their replicationpossible, for example in E. coli. Suitable cloning vectors are,inter alia, pBR332, pUC series, M13mp series and pACYC184. Binaryvectors able to replicate both in E. coli and in agrobacteria,10 such as pBinl9 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711)are particularly suitable.The invention therefore also relates to recombinant vectors, suchas plasmids or viruses, comprising at least one expression15 cassette according to the invention. A particularly preferredrecombinant plasmid, called pTPO9—ASS, comprises the geneconstruct depicted in Figure 7 and confers resistance to plantADSS inhibitors, such as hydantocidin on the plant hosttransformed therewith.20For transfection of a host plant with an ADSS DNA, an expressioncassette according to the invention is incorporated as insertinto a recombinant vector whose vector DNA comprises additionalfunctional regulatory signals, for example sequences for25 replication or integration. Suitable vectors are described, interalia, in "Methods in Plant Molecular Biology and Biotechnology"(CRC Press), Chapters 6/7, pages 71-119.The vectors according to the invention can be used for the30 transformation of plants and cells, tissues or parts of plants.The DNA constructs fused according to the invention can also betransferred into plant genomes by various other known processes.Examples of suitable processes are protoplast transformation by35 polyethylene glycol—induced DNA uptake, electroporation,sonication or microinjection and the transformation of intactcells or tissue by micro- or macroinjection into tissue orembryos, tissue electroporation, incubation of dry embryos inDNA-containing solution, biolistic gene transfer and,40 particularly preferably, agrobacterium transformation. Saidprocesses are described, for example in B. Jenes et al.,Techniques for Gene Transfer; in Transgenic Plants, Vol. 1,Engineering and Utilization, edited by S.D. Kung and R. Wu,Academic Press, 1993, pages 128-143 and in Potrykus (1991) Annu.45 Rev. Plant Physiol. Plant Molec. Biol. 42, 205-225).CA 02264b77’I§99-02-261015202530354045CA 0226460050/472899The fused construct is preferably cloned into a vector, forexample pBin19, which is suitable for transforming Agrobacteriumtumefaciens. Agrobacteria transformed with such a vector can thenbe used in a known manner for transforming plants, especiallycrop plants, such as tobacco plants, by, for example, injuredleaves or pieces of leaf being bathed in an agrobacteriumsolution and subsequently cultivated in suitable media.Transformation of plants by agrobacteria is disclosed inter aliain F.F. White, Vectors for Gene Transfer in Higher Plants; inTransgenic Plants, Vol. 1, Engineering and Utilization, edited bys.D. Kung and R. Wu, Academic Press, 1993, pages 15-38 and inS.B. Gelvin, Molecular Genetics of T—DNA Transfer fromAgrobacterium to Plants, likewise in Transgenic Plants, pages49-78. Transgenic plants which express the ADSS DNA integrated inthe expression cassette according to the invention can beregenerated in a known manner from the transformed cells of theinjured leaves or pieces of leaf.The activity of the transgenically expressed ADSS can be assayedfor example in vitro by an enzyme assay as described in Baugheret al., Biochem. Res. Commun., 94 (l980):123—l29; Stayton et al.,Curr. Top. Cell. Regul., 22 (1983):l03—l41; und Bass et al.,Arch. Biochem. Biophys., 256 (1987):335—342.The invention additionally relates to microorganisms, such asbacteria or fungi, which comprise a recombinant vector accordingto the invention.The invention additionally relates to transgenic plantstransformed with a vector or microorganism according to theinvention, and to transgenic cells, tissue, parts and propagationmaterial of such plants. Particularly preferred in thisconnection are transgenic crop plants such as cereals, corn,soybean, rice, cotton, sugar beet, canola, sunflower, flax,potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and thevarious tree, nut and vine species.The transgenic plants and cells, tissue or parts of plants can betreated with an agent which inhibits the plant ADSS, resulting indeath of unsuccessfully transformed plants or cells, tissue orparts of plants. Examples of suitable agents are alanosine,hadacidin and, in particular, hydantocidin, and metabolites andfunctional derivatives of these compounds. The ADSS DNA insertedinto the expression cassettes according to the invention can thusbe used as selection marker.'lA’I’1"I'!'I"I7 1 99-02-261015202530354045CA 02260050/4728910The invention thus further relates to the use of vectors ormicroorganisms transformed therewith for the transformation ofplants or cells, tissues or parts of plants, in particular forthe expression of exogenous proteins, glycoproteins or fusionproteins. The aim of the use is preferably to confer resistanceto inhibitors of plant ADSS.However, the invention also relates to the expression productsproduced according to the invention, especially the fusionproteins composed of transit peptide and protein part withnon-plant ADSS activity.The invention is explained by the examples which now follow butis not restricted to their use:Example 1: PCR amplification of the Escherichia coliadenylosuccinate synthetase using syntheticoligonucleotidesThe PCR amplification of the Escherichia coli ADSS was carriedout in a Perkin Elmer DNA thermal cycler. The oligonucleotidesused (SEQ ID NO:6,7) are depicted in Figure 4 and have been takenfrom the published sequence. The reaction mixtures contained8 ng/ul genomic DNA from Escherichia coli, 0.5 uM of the‘appropriate oligonucleotides, 200 uM nucleotides (Pharmacia),50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25°C), 1.5 mM MgCl2 and0.02 U/ul taq polymerase (Perkin Elmer). The amplificationconditions were set as follows:Annealing temperature: 45°C, 1 minDenaturing temperature: 92°C, 1 minElongation temperature: 72°C, 1.5 minNumber of cycles: 40A fragment of 1300 base pairs resulted and was ligated into thevector pGEM—T (Promega). The ligation mixture was used totransform E. coli XL—I Blue to result in the plasmid pGEM-ASS.Example 2: Generation of plant expression cassettesA 35S CaMV promoter was inserted as EcoRI—KpnI fragment(corresponding to nucleotides 6909-7437 of cauliflower mosaicvirus (Franck et al. (1980) Cell 21, 285) into the plasmid pBinl9(Bevan et al. (1980) Nucl. Acids Res. 12, 8711) (commerciallyobtainable from Clontech, Palo Alto, CA, USA). Thepolyadenylation signal of gene 3 of the T—DNA of the Ti plasmidpTiACH5 (Gielen et al., (1984) EMBO J. 3, 835), nucleotides4é77“399-02-2610152025303540450050/472891111749-11939, derived from the octopine synthase (OCS) gene, wasisolated as PvuII—HindIII fragment and, after addition of Sphllinkers to the PvuII cleavage site, cloned between the SphI andHindIII cleavage sites of the vector to result in the plasmidpBinAR (Hofgen und Willmitzer (1990) Plant Science 66, 221-230).The CDNA sequence of the transit peptide (TP) of thetransketolase (TK) was removed from the plasmid pBluescript TK—26(DE-A—1950l906) and inserted with the aid of syntheticoligonucleotides as KpnI-BamHI fragment by a polymerase chainreaction into the plasmid pBinAR. Three vectors were obtained asplant expression cassettes (pTP09, pTP10, pTPl1, see Figures 5and 6) by varying the 3'—specific oligonucleotide used, and thesepermit chimeric gene constructs to be produced with the CDNAtransit sequence of the plastid transketolase in three differentreading frames.Example 3: Production of a plant expression cassette foradenylosuccinate synthetaseThe DNA fragment coding for adenylosuccinate synthetase wascloned as BamHI fragment into the vector pTP09 to result in theplasmid pTP09—ASS (see Figure 7). Fusion of the transit peptide(TP/TK) to the adenylosuccinate synthetase ensured import of theprotein into the chloroplasts.Example 4: Sequence analysis of recombinant DNARecombinant DNA molecules were sequenced using a Pharmacia laserfluorescence DNA sequencer by the method of Sanger (Sanger et al.(1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467). Fragmentsresulting from a polymerase chain reaction were sequenced andchecked to avoid polymerase errors in constructs to be expressed.Example 5: Production of transgenic tobacco plants comprising amicrobial adenylosuccinate synthetase in thechloroplastThe plasmid pTP09~ASS was transformed into Agrobacteriumtumefaciens C58Cl:pGV2260. The transformation of Agrobacteriumtumefaciens was carried out by the method of Hofgen andWillmitzer (Nucl. Acid Res. (1988) 16, 9877). The agrobacteriawere cultured in YEB medium (Vervliet et al., J. Gen. Virol.(1975) 26, 33). Tobacco plants (Nicotiana tabacum cv. Samsun NN)were transformed using a 1:50 dilution of an overnight culture ofa positively transformed agrobacterium colony in Murashige—SkoogMedium ((1962) Physiol. Plant. 15, 473) with 2% sucrose (2MSCA 02264677’7fli§99-02-261015202530354045CA0050/4728912medium). Leaf disks from sterile plants (each about 1 cm?) wereincubated with a 1:50 agrobacteria dilution in a Petri dish for5 to 10 minutes. This was followed by incubation on 2MS mediumwith 0.8% Bacto agar at 25°C in the dark for 2 days. Thecultivation was continued after 2 days with 16 hours oflight/8 hours of dark and continued in a weekly rhythm on MSmedium with 500 mg/l Claforan (cefotaxime sodium), 50 mg/lkanamycin, 1 mg/l benzylaminopurine (BAP), 0.2 mg/l naphthyl-acetic acid and 1.6 g/l glucose. Growing shoots were transferredonto MS medium with 2% sucrose, 250 mg/1 Claforan and 0.8% Bactoagar.The procedure for a second transformation corresponded but220 mg/1 hydantocidin was added as antibiotic.Example 6: Analysis of complete RNA from plant tissuesFor detailed investigation of the expression, the complete RNAwas isolated from tobacco plants as described by Logemann et al.(Anal. Biochem. (1987) 163,21). For the analysis, in each case20 ug of RNA were fractionated in a formaldehyde—containing 1.5%agarose gel. Fractionation of the RNA molecules byelectrophoresis was followed by capillary transfer of the RNA toa nylon membrane. Specific transcripts were detected as describedby Amasino (Anal. Biochem. (1986) 152, 304). The CDNA fragmentsemployed as probe were radiolabeled with a random primer DNAlabeling kit (Boehringer, Mannheim).Example 7:Enzyme assay for adenylosuccinate synthetase isolatedfrom transgenic tobacco plantsThe assay mixture contained 14 mM Tris—HCl pH 8.3; 6 mM MgCl2;0.4 mM IMP; 0.1 mM GTP; 0.5 mM phosphoenolpyruvate; 0.1 mM ATP;2 U/ml pyruvate kinase; 3 mM aspartate and from 10 to 100 pl ofenzyme preparation in a 1 ml assay mixture. Incubation took placeat 25°C in the presence or absence of the inhibitor hydantocidinfor 5 to 20 min, followed by measurement in a dual beam photometerat 280 nm (absorption of adenylosuccinate) with a mixture withoutaspartate as reference. The recombinant ADSS showed no inhibitionby hydantocidin.Example 8: Testing of hydantocidin resistant tobacco plantsTobacco plants transformed with the plasmid pTP09-ASS were grownin tissue culture on 2MS medium with 0.8% Bacto agar, 250 mg/lClaforan, 50 mg/1 kanamycin and axial shoots were transferred tocorresponding medium with 220 mg/1 hydantocidin. Untransformed02264677 1999-02-260050/4728913plants died within a few weeks, while resistant plants continuedto grow.1015202530354045CA 02264677 1999-02-262264677.seqSEQUENCE LISTING(1) GENERAL INFORMATION:(i) APPLICANT:(A) NAME: BASF Aktiengesellschaft(B) STREET: --(C) CITY: Ludwigshafen(E) COUNTRY: Germany(F) POSTAL CODE (ZIP): D-67056(ii) TITLE OF INVENTION: ADENYLOSUCCINATE SYNTHETASE(iii) NUMBER OF SEQUENCES: 7(iv) CORRESPONDENCE ADDRESS:(A) ADDRESSEE: Robic(B) STREET: 55 St—Jacques(C) CITY: Montréal(D) STATE: QC(E) COUNTRY: Canada(F) ZIP: H2Y 3x2(G) TELEPHONE: 514-987-6242(H) TELEFAX: 514-845-7874(v) COMPUTER READABLE FORM:(A) MEDIUM TYPE: Disk 3.5" / 1.44 MB(B) COMPUTER: IBM PC compatible(C) OPERATING SYSTEM: PC-DOS/MS—DOS(D) SOFTWARE: TXT ASCII(Vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER: 2,264,677(B) FILING DATE: O4—SEPT-1997(Vii) PRIOR APPLICATION DATA:(A) APPLICATION NUMBER: PCT/EP97/04812(B) FILING DATE: O4-SEPT—1997(2) INFORMATION FOR SEQ ID NO: 1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1311 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)Page 1CA 02264677 1999-05-112264677.seq(iii) HYPOTHETICAL: NO(iv) ANTI—SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Escherichia coli(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:1..6(D) OTHER INFORMATION:/function: "BamHI Schnittstelle"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION:7..l302(D) OTHER INFORMATION:/product: "AdenylosuccinateSynthetase"(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:1303..1305(D) OTHER INFORMATION:/function: "Stop codon"(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:l306..l3l1(D) OTHER INFORMATION:/function: "BamHI Schnittstelle"(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:GGATCC ATG GGT AAC AAC GTC GTC GTA CTG GGC ACC CAA TGG GGT GAC 48Met Gly Asn Asn Val Val Val Leu Gly Thr Gln Trp Gly Asp1 5 10GAA GGT AAA GGT AAG ATC GTC GAT CTT CTG ACT GAA CGG GCT AAA TAT 96Glu Gly Lys Gly Lys Ile Val Asp Leu Leu Thr Glu Arg Ala Lys Tyr15 20 25 30GTT GTA CGC TAC CAG GGC GGT CAC AAC GCA GGC CAT ACT CTC GTA ATC 144Val Val Arg Tyr Gln Gly Gly His Asn Ala Gly His Thr Leu Val Ile35 40 45AAC GGT GAA AAA ACC GTT CTC CAT CTT ATT CCA TCA GGT ATT CTC CGC 192Asn Gly Glu Lys Thr Val Leu His Leu Ile Pro Ser Gly Ile Leu Arg50 55 60GAG AAT GTA ACC AGC ATC ATC GGT AAC GGT GTT GTG CTG TCT CCG GCC 240Glu Asn Val Thr Ser Ile Ile Gly Asn Gly Val Val Leu Ser Pro Ala65 70 75Page 2CA 02264677 1999-05-112264677.seqGCG CTG ATG AAA GAG ATG AAA GAA CTG GAA GAC CGT GGC ATC CCC GTT 288Ala Leu Met Lys Glu Met Lys Glu Leu Glu Asp Arg Gly Ile Pro Val80 85 90CGT GAG CGT CTG CTG CTG TCT GAA GCA TGT CCG CTG ATC CTT GAT TAT 336Arg Glu Arg Leu Leu Leu Ser Glu Ala Cys Pro Leu Ile Leu Asp Tyr95 100 105 110CAC GTT GCG CTG GAT AAC GCG CGT GAG AAA GCG CGT GGC GCG AAA GCG 384His Val Ala Leu Asp Asn Ala Arg Glu Lys Ala Arg Gly Ala Lys Ala115 120 125ATC GGC ACC ACC GGT CGT GGT ATC GGG CCT GCT TAT GAA GAT AAA GTA 432Ile Gly Thr Thr Gly Arg Gly Ile Gly Pro Ala Tyr Glu Asp Lys Val130 135 140GCA CGT CGC GGT CTG CGT GTT GGC GAC CTT TTC GAC AAA GAA ACC TTC 480Ala Arg Arg Gly Leu Arg Val Gly Asp Leu Phe Asp Lys Glu Thr Phe145 150 155GCT GAA AAA CTG AAA GAA GTG ATG GAA TAT CAC AAC TTC CAG TTG GTT 528Ala Glu Lys Leu Lys Glu Val Met Glu Tyr His Asn Phe Gln Leu Val160 165 170AAC TAC TAC AAA GCT GAA GCG GTT GAT TAC CAG AAA GTT CTG GAT GAT 576Asn Tyr Tyr Lys Ala Glu Ala Val Asp Tyr Gln Lys Val Leu Asp Asp175 180 185 190ACG ATG GCT GTT GCC GAC ATC CTG ACT TCT ATG GTG GTT GAC GTT TCT 624Thr Met Ala Val Ala Asp Ile Leu Thr Ser Met Val Val Asp Val Ser195 200 205GAC CTG CTC GAC CAG GCG CGT CAG CGT GGC GAT TTC GTC ATG TTT GAA 672Asp Leu Leu Asp Gln Ala Arg Gln Arg Gly Asp Phe Val Met Phe Glu210 215 220GGT GCG CAG GGT ACG CTG CTG GAT ATC GAC CAC GGT ACT TAT CCG TAC 720Gly Ala Gln Gly Thr Leu Leu Asp Ile Asp His Gly Thr Tyr Pro Tyr225 230 235GTA ACT TCT TCC AAC ACC ACT GCT GGT GGC GTG GCG ACC GGT TCC GGC 768Val Thr Ser Ser Asn Thr Thr Ala Gly Gly Val Ala Thr Gly Ser Gly240 245 250CTG GGC CCG CGT TAT GTT GAT TAC GTT CTG GGT ATC CTC AAA GCT TAC 816Leu Gly Pro Arg Tyr Val Asp Tyr Val Leu Gly Ile Leu Lys Ala Tyr255 260 265 270TCC ACT CGT GTA GGT GCA GGT CCG TTC CCG ACC GAA CTG TTT GAT GAA 864Ser Thr Arg Val Gly Ala Gly Pro Phe Pro Thr Glu Leu Phe Asp GluPage 3CA 02264677 l999-05- ll2264677.seq275 280 285ACT GGC GAG TTC CTC TGC AAG CAG GGT AAC GAA TTC GGC GCA ACT ACG 912Thr Gly Glu Phe Leu Cys Lys Gln Gly Asn Glu Phe Gly Ala Thr Thr290 295 300GGG CGT CGT CGT CGT ACC GGC TGG CTG GAC ACC GTT GCC GTT CGT CGT 960Gly Arg Arg Arg Arg Thr Gly Trp Leu Asp Thr Val Ala Val Arg Arg305 310 315GCG GTA CAG CTG AAC TCC CTG TCT GGC TTC TGC CTG ACT AAA CTG GAC 1008Ala Val Gln Leu Asn Ser Leu Ser Gly Phe Cys Leu Thr Lys Leu Asp320 325 330GTT CTG GAT GGC CTG AAA GAG GTT AAA CTC TGC GTG GCT TAC CGT ATG 1056Val Leu Asp Gly Leu Lys Glu Val Lys Leu Cys Val Ala Tyr Arg Met335 340 345 350CCG GAT GGT CGC GAA GTG ACT ACC ACT CCG CTG GCA GCT GAC GAC TGG ‘ 1104Pro Asp Gly Arg Glu Val Thr Thr Thr Pro Leu Ala Ala Asp Asp Trp355 360 365AAA GGT GTA GAG CCG ATT TAC GAA ACC ATG CCG GGC TGG TCT GAA TCC 1152Lys Gly Val Glu Pro Ile Tyr Glu Thr Met Pro Gly Trp Ser Glu Ser370 375 380ACC TTC GGC GTG AAA GAT CGT AGC GGC CTG CCG CAG GCG GCG CTG AAC 1200Thr Phe Gly Val Lys Asp Arg Ser Gly Leu Pro Gln Ala Ala Leu Asn385 390 395TAT ATC AAG CGT ATT GAA GAG CTG ACT GGT GTG CCG ATC GAT ATC ATC 1248Tyr Ile Lys Arg Ile Glu Glu Leu Thr Gly Val Pro Ile Asp Ile Ile400 405 410TCT ACC GAT CCG GAT CGT ACT GAA ACC ATG ATT CTG CGC GAC CCG TTC 1296Ser Thr Asp Pro Asp Arg Thr Glu Thr Met Ile Leu Arg Asp Pro Phe415 420 425 430GAC GCG TAAGGATCC 1311Asp Ala(2) INFORMATION FOR SEQ ID NO: 2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 432 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linearPage 4CA 02264677 1999-05-11MetLysArgGluVal65MetArgAlaThrArg145LysTyrAlaLeuGln225Ser(ii) MOLECULE TYPE: protein(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2:2264677.seqGly Asn Asn Val Val Val Leu Gly Thr GlnGlyTyrLys50ThrLysLeuLeuThr130GlyLeuLysValAsp210GlySerCALysGln35ThrSerGluLeuAsp115GlyLeuLysAlaAla195GlnThrAsn02264677 1999-05-llIle20GlyValIleMetLeu100AsnArgArgGluGlu180AspAlaLeuThr5ValGlyLeuIleLys85SerAlaGlyValVal165AlaIleArgLeuThr245AspHisHisGly70GluGluArgIleGly150MetValLeuGlnAsp230AlaLeuAsnLeu55AsnLeuAlaGluGly135AspGluAspThrArg215IleGlyLeuAla40IleGlyGluCysLys120ProLeuTyrTyrSer200GlyAspGlyThr25GlyProValAspPIO105AlaAlaPheHisGln185MetAspHisValPage 510GluHisSerValArg90LeuArgTyrAspAsn170LysValPheGlyAla250ArgThrGlyLeu75GlyIleGlyGluLys155PheValValValThr235ThrTrpAlaLeuIle60SerIleLeuAlaAsp140GluGlnLeuAspMet220TyrGlyGlyLysVal45LeuProProAspLys125LysThrLeuAspVal205PhePIOSerAspTyr30IleArgAlaValTyr110AlaValPheValAsp190SerGluTyrGlyGlu15ValAsnGluAlaArg95HisIleAlaAlaAsn175ThrAspValLeu255GlyValGlyAsnLeu80GluValGlyArgGlu160TyrMetLeuAlaThr240GlyPICArgGluArg305GlnAspGlyValGly385LysAsp(2)ArgValPhe290ArgLeuGlyArgGlu370ValArgProVal260TyrGly Ala275Leu CysArg ThrAsn SerLeu Lys340Glu Val355Pro IleLys AspIle GluAsp Arg420AspGlyLysGlyLeu325GluThrTyrArgGlu405ThrTyrProGlnTrp310SerValThrGluSer390LenGluValPheGly295LeuGlyLysThrThr375GlyThrThrLeuPro280AsnAspPheLeuPro360MetLeuMetINFORMATION FOR SEQ ID NO:(i)(ii)(iii)(iv)(Vi)CA2264677.Gly265ThrGluThrCysCys345LeuProPIOValIle4253:SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)nucleic acidMOLECULE TYPE:CDNAHYPOTHETICAL: NOANTI-SENSE: NOORIGINAL SOURCE:02264677 1999-05-11Page 6IleGluPheValLeu330ValAlaGlyGlnPro410LeuLENGTH: 258 base pairsTYPE:STRANDEDNESS: singleTOPOLOGY: linearseqLeuLeuGlyAla315ThrAlaAlaTrpAla395IleArgLysPheAla300ValLysTyrAspSer380AlaAspAspAlaAsp285ThrArgLeuArgAsp365GluLeuIlePIOTyr Ser Thr270GluThrArgAspMet350TrpSerAsnIlePhe430ThrGlyAlaVal335PIOLysThrTyrSer415AspArgVal320LeuAspGlyPheIle400ThrAla2264677.seq(I) ORGANELLE: Chloroplast(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:1..6(D) OTHER INFORMATION:/function= "KnpI Schnittstelle"(ix) FEATURE:(A) NAME/KEY: transit_peptide(B) LOCATION:7..252(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:253..258(D) OTHER INFORMATION:/function= "BamHI Schnittstelle"(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:GGTACCATGG CGTCTTCTTC TTCTCTCACT CTCTCTCAAG CTATCCTCTC TCGTTCTGTCCCTCGCCATG GCTCTGCCTC TTCTTCTCAA CTTTCCCCTT CTTCTCTCAC TTTTTCCGGCCTTAAATCCA ATCCCAATAT CACCACCTCC CGCCGCCGTA CTCCTTCCTC CGCCGCCGCCGCCGCCGTCG TAAGGTCACC GGCGATTCGT GCCTCAGCTG CAACCGAAAC CATAGAGAAAACTGAGACTG CGGGATCC(2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 260 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(I) ORGANELLE: Chloroplast(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:1..6(D) OTHER INFORMATION:/function= "Kpnl Schnittstelle"Page 7CA 02264677 1999-05-11601201802402582264677.seq(ix) FEATURE:(A) NAME/KEY: transit_peptide(B) LOCATION:7..252(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:253..254(D) OTHER INFORMATION:/function: "frame shift insert"(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:255..260(D) OTHER INFORMATION:/function: "BamHI Schnittstelle"(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:GGTACCATGG CGTCTTCTTC TTCTCTCACT CTCTCTCAAG CTATCCTCTC TCGTTCTGTCCCTCGCCATG GCTCTGCCTC TTCTTCTCAA CTTTCCCCTT CTTCTCTCAC TTTTTCCGGCCTTAAATCCA ATCCCAATAT CACCACCTCC CGCCGCCGTA CTCCTTCCTC CGCCGCCGCCGCCGCCGTCG TAAGGTCACC GGCGATTCGT GCCTCAGCTG CAACCGAAAC CATAGAGAAAACTGAGACTG CGCTGGATCC(2) INFORMATION FOR SEQ ID NO: 5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 259 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(Vi) ORIGINAL SOURCE:(I) ORGANELLE: Chloroplast(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:l..6(D) OTHER INFORMATION:/function: "KpnI Schnittstelle"Page 8CA 02264677 1999-05-11601201802402602264677.seq(ix) FEATURE:(A) NAME/KEY: transit_peptide(B) LOCATION:7..252(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:253(D) OTHER INFORMATION:/product: "Frame shift insert"(ix) FEATURE:(A) NAME/KEY: misc_feature(B) LOCATION:254..259(D) OTHER INFORMATION:/function: "BamHI Schnittstelle"(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:GGTACCATGG CGTCTTCTTC TTCTCTCACT CTCTCTCAAG CTATCCTCTC TCGTTCTGTC 60CCTCGCCATG GCTCTGCCTC TTCTTCTCAA CTTTCCCCTT CTTCTCTCAC TTTTTCCGGC 120CTTAAATCCA ATCCCAATAT CACCACCTCC CGCCGCCGTA CTCCTTCCTC CGCCGCCGCC 180GCCGCCGTCG TAAGGTCACC GGCGATTCGT GCCTCAGCTG CAACCGAAAC CATAGAGAAA 240ACTGAGACTG CGGGGATCC 259(2) INFORMATION FOR SEQ ID NO: 6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = "synthetic"(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:AAGGATCCAT GGGTAACAAC GTCGTCGTAC TGG 33(2) INFORMATION FOR SEQ ID NO: 7:Page 9CA 02264677 1999-05-11(iii)(iv)(xi)2264677.seqSEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linearMOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = "synthetic"HYPOTHETICAL: NOANTI-SENSE: NOSEQUENCE DESCRIPTION: SEQ ID NO: 7:AAGGATCCCG TACCAGAATT ACGCAPage 1002264677 1999-05-1123

Claims (22)

We claim:

1. An expression cassette for transformation of a plant host comprising, under the genetic control of regulatory nucleic acid sequences, the coding nucleic acid sequence for a non-plant adenylosuccinate synthetase (ADSS) or a functional equivalent thereof as well as for a regulatory protein sequence which leads ADSS to the required site of action.

2. An expression cassette as claimed in claim 1, wherein the non-plant adenylosuccinate synthetase is derived from a microbial adenylosuccinate synthetase.

3. An expression cassette as claimed in claim 2, wherein the microbial adenylosuccinate synthetase is derived from Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Dictyostelium discoideum, Saccharomyces pombe or Schizosaccharomyces pombe.

4. An expression cassette as claimed in any of the preceding claims, wherein the coding nucleic acid sequence codes for a protein comprising an amino acid sequence as specified in SEQ
ID NO:2 or a functional equivalent thereof, or comprises a nucleic acid sequence from residue + 7 to + 1305 as specified in SEQ ID NO:1 or a functional equivalent thereof.

5. An expression cassette as claimed in any of the preceding claims, wherein the regulatory protein sequence is a chloroplast-specific transit peptide.

6. An expression cassette as claimed in claim 5, wherein the transit peptide is plastid transketolase or a functional equivalent thereof.

7. A recombinant vector, comprising an expression cassette as claimed in any of claims 1 to 6.

8. A vector as claimed in claim 7, which essentially corresponds to the vector pTP09-ASS.

9. A microorganism comprising a recombinant vector as claimed in claim 7 or 8.

10. A microorganism as claimed in claim 9 from the genus Agrobacterium and, in particular, the species Agrobacterium tumefaciens.

11. The use of a vector as claimed in either of claims 7 and 8 or of a microorganism as claimed in either of claims 9 and 10 for the transformation of plants and cells, tissues or parts of plants.

12. The use as claimed in claim 11, wherein resistance to inhibitors of plant adenylosuccinate synthetase is conferred on the plants or cells, tissues or parts of plants.

13. The use of a coding nucleic acid sequence as defined in any of claims l to 4 as selection or marker gene in plants and cells, tissues or parts of plants.

14. A transgenic plant transformed with a vector as claimed in either of claims 7 and 8 or with a microorganism as claimed in either of claims 9 and 10, or transgenic cells, tissues, parts or transgenic propagation material thereof.

15. A transgenic plant as claimed in claim 14, selected from crop plants such as cereals, corn, soybean, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species.

16. A process for the production of transgenic plants as claimed in either of claims 14 and 15, which comprises transforming cells, tissue or parts of plants or protoplasts with a vector as claimed in either of claims 7 and 8 or with a microorganism as claimed in either of claims 9 and 10, cultivating the transformed cells, tissues or parts of plants or protoplasts in a growth medium and, where appropriate, regenerating plants from the culture.

17. An expression product of an expression cassette defined in any of claims 1 to 6.

18. An expression product as claimed in claim 17, which essentially comprises an amino acid sequence from amino acid + 1 to + 432 as specified in SEQ ID NO:2 or a functional equivalent thereof.

19. An expression kit for expressing a foreign gene in a plant host, which comprises at least three expression cassettes, where at least two of these expression cassettes under the genetic control of regulatory nucleic acid sequences each comprise a frame shift sequence, which differ from one another by insertion or deletion of a number of nucleotides which is not divisible by 3, and shift the reading frame for a coding nucleic acid sequence, which is located downstream, by one and two nucleotides respectively.

20. An expression kit as claimed in claim 19, wherein each expression cassette comprises a coding sequence for a chloroplast-specific transit peptide, and the frame shift sequence which is present where appropriate follows the 3' end thereof.

21. An expression kit as claimed in claim 20, comprising expression cassettes which in each case comprise one of the following nucleotide sequences:
SEQ ID NO:3 from nucleic acid residue +7 to +252 SEQ ID NO:4 from nucleic acid residue +7 to +254 SEQ ID NO:5 from nucleic acid residue +7 to +253 or a functional equivalent thereof.

22. The use of an expression kit as claimed in any of claims 19 to 21 for transforming a plant host.