IL85143A - Resistance gene against phosphinothricin - Google Patents

Abstract

A resistance gene coding for the protein of amino acid sequence l (annex), characterized in that ATG is used as start codon and TGA is used as stop codon, and the GC content of the gene is adapted to that in plants.

Description

85143/5 RESISTANCE GENE AGAINST PHOSPHINOTHRICIN Eitan, Pearl, Latzer & Cohen-Zedek P-55049-IL HOECHST AKTIENGESELLSCHAFT HOE 87/F 333 J Dr. KL/mu Spec if ication where Phosph inothricin-res istance agile active plants, and. ts i¾se underlined .1.88 Genes from Streptomycetes have a very high proportion of G + C, the adenine (A) + thymine (T) : guanine (G) + cytosine (C) ratio being about 30 : 70. The proportion of GC in plant genes is far lower, being about 50%. For this reason, in a further development of the inventive idea, the ONA sequence of the resistance gene has been optimized, by de novo synthesis, to a codon usage favorable for plant RNA polymerase II.

The invention relates to a modification of the resistance gene which is proposed in German Patent Application P 36 28 747.4 and the additional application P 36 42 829.9, namely an adaptation to the codon usage in plants. The corresponding amino acid sequence is depicted in the annex. Further embodiments of the invention are defined in the patent claims or are explained hereinafter.

As is known, the genetics code is degenerate, i.e. only 2 amino acids are coded for by a single triplet, whereas the remaining 18 genetically codable amino acids are assigned to 2 to 6 triplets. Thus, theoretically, a wide variety of codon combinations can be chosen for the synthesis of the gene. Since the said relative proportion of the individual nucleotides in the total DNA sequence exerts an influence, it was used as one of the criteria on which the sequence optimization was based.

The following modifications were made to the sequenced gene : 1. The St rep tomycetes gene start codon GTG (position 258-260 in the sequence in the additional applica- tion) was replaced by the start codon ATG which is used by plant RNA polymerase II. 2. Within the gene, the St reptomycetes gene codons were changed in such a way that they resulted in codons suitable in plant genes (G/C ratio). 3. The TGA stop codon was placed at the end of the sequence to terminate the translation process. 4. The beginning and end of the gene sequence were provided with protruding ends of restriction sites in order to be able to amplify the gene and ligate it between plant regulation sequences.

. Palindromic sequences were reduced to a minimum.

The ONA sequence I according to the invention (with the corresponding amino acid sequence) is depicted in the annex .

Three internal unique cleavage sites for the restriction enzymes Xbal (position 152), BamHI (312) and Xmal (436) make possible the subcloning of part-sequences which can be incorporated in we 11 - i nves t i ga ted cloning vectors such as, for example, pUC18 or pUd9. In addition, a number of other unique recognition sequences for restriction enzymes were incorporated within the gene, and these, on the one hand, provide access to part-sequences of acetyl-transferase and, on the other hand, allow modifications to be made: Restriction enzyme Cut after nucleotide No. (codings strand) BspMII 11 SacII 64 EcoRV 74 Hpal 80 Aatll 99 BstXI 139 Apa I 232 Seal 272 Avrll 308 Af III 336 Stul 385 BssHII 449 Fokl 487 Bgll 536 Bglll 550 The construction of part-sequences by chemical synthesis and enzymatic ligation reactions is carried out in a manner known per se (EP-A 0,133,282, 0,136,472, 0,155,590, 0,161 ,504, 0,163,249, 0,171,024, 0,173,149 or 0,177,827). Details, such as restriction analyses, ligation of DNA fragments and transformation of plasmids in E . col i , are described at length in the textbook of Maniatis (Mole-cular Cloning, Maniatis et al., Cold Spring Harbor, 1982) .

The gene sequence which has been cloned in this way is then introduced into plants, under the control of plant regulation signals, and its expression is induced.

EP-A 0,122,791 reviews known methods. In this way are obtained PTC-res istant plant cells (i.e. a selection feature for transformed cells is available), plants or parts of plants and seeds.

Some embodiments of the invention are explained in detail in the examples which follow. Unless otherwise indicated, percentage data therein relate to weight.

Examples The following media were used: a) for bacteria: YT medium: 0.5% yeast extract, 0.8% Bacto tryp- tone, 0.5% NaCl LB medium: 0.5% yeast extract, 1% Bacto tryptone, 1% NaCl solid medium: addition of 1.5% agar to each b) for plants: M+S medium: see Murashige and Skoog, Physiologica Plantarum 15 (1962) 473 2MS medium: M+S medium containing 2% sucrose MSC10 medium: M+S medium containing 2% sucrose, 500 mg/l cefotaxime, 0.1 mg/l naphthy I acet c acid (NAA), 1 mg/l benzylaminopur ine (BAP), 100 mg/l kanamycin MSC15 medium: M+S medium containing 2% sucrose, 500 mg/L cefotaxime, 100 mg/L kanamycin.

Chemical synthesis of a single-stranded oligonucleotide The synthesis of fragment II, one of the four part-fragments I - IV, started from the terminal oligonucleotide lie (nucleotide No. 219 to 312 in the coding strand of DNA sequence I). For the solid-phase synthesis, the nucleoside at the 3' end, that is to say guanosine (nucleotide No. 312) in the present case, is covalently bonded via the 3'-hydroxyl group to a support. The support material is CPG (controlled pore glass) functional ized with long-chain amino-alkyl radicals. Otherwise, the synthesis follows the known (from the said EP-As) methods.

The plan of synthesis is indicated in DNA sequence II (annex ), -.wh ich- otherwise corresponds to DNA sequence I.

Enzymatic linkage of the single-stranded oligonucleotides to give gene fragment II For the phosphorylation of the oligonucleotides at the 5' end, 1 nmol of each of oligonucleotides lib and lie was treated with 5 nmol of adenosine triphosphate and 4 units of T4 polynucleotide kinase in μΐ of 50 mM tris-HCl buffer (pH 7.6), 10 mM magnesium chloride and 10 mM dithiothreitol (DTT) at 37°C for 30 minutes. The enzyme is inactivated by heating at 95°C for 5 minutes. Oligonucleotides Ha and lid, which form the "protruding" sequence in DNA fragment II, are not phosphorylated. This prevents the formation, during the subsequent ligation, of larger subfragments than correspond to DNA fragment II. 01 igonuc Leot ides II (a-d) are Ligated to give sub-fragment II as follows: 1 nmol of each of oligonucleotides Ila and lid and the 5 · -phosphates of lib and lie are together dissolved in 45 μΐ of buffer containing 50 mM tris-HCl (pH 7.6), 20 mM magnesium chloride, 25 mM potassium chloride and 10 mM DTT.

For the annealing of the ol gonucleotides corresponding to DN A fragment II, the solution of the oligonucleotides is heated at 95°C for 2 minutes and then slowly cooled (2-3 hours) to 20°C. Then, for the enzymatic linkage, 2 μΐ of 0.1 M DTT, 8 μΐ of 2.5 mM adenosine triphosphate (pH 7) and 5 μΐ of T4 DNA ligase (2000 units) are added, and the mixture is incubated at 22°C for 16 hours.

The gene fragment II is purified by gel electrophoresis on a 10% polyacrylamide gel (without addition of urea, 20 x 40 cm, 1 mm thick), the marker substance used being jffX 174 ONA (from BRL) cut with Hinf I, or pBR322 cut wi th Haelll . - ■ Gene fragments I, III and IV are prepared analogously, although the "protruding" sequences are, before the annealing, converted into the 5 · -phosphates because no ligation step is necessary.

Preparation of hybrid plasmids containing gene fragments I, II, III and IV. a) Incorporation of gene fragment I in pUC18 The commercially available plasmid pUCl8 is opened in a known manner using the restriction endo nuclea ses Sail and Xbal in accordance with the manufacturers' instructions. The digestion mixture is frac tionated by electrophores s in a known manner on a 1% agarose gel, and the fragments are visualized by staining with ethidium bromide. The plasmid band (about 2.6 kb) is then cut out of the agarose get and removed from the agarose by electro- elut ion. 1 ug of plasmid, opened with Xbal and Sail, is then Ligated with 10 ng of DNA fragment I at 16°C overnight. b) Incorporation of gene fragment II in pUCl8.

In analogy to a), pUCl8 is cut open with Xbal and BamHI and Ligated with gene fragment II which has previously been phosphor I ated at the protruding ends as described in Example 2. c) Incorporation of gene fragment III in pUCl8 In analogy to a), pUCl8 is cut open with BamHI and Xmalll and ligated with gene fragment III. d) Incorporat ion of gene -fragment IV in pUC 18 In analogy to a), pUC18 is cut with Xmalll and Sail and ligated with gene fragment IV.

Construction of the complete gene and cloning in a pUC plasmid a) Trans ormation and amplif cation of gene fragments I - IV The hybrid plasmids obtained in this way are transformed into E . col i . For this purpose, the strain E . col i K 12 is made competent by treatment with a 70 mM calcium chloride solution, and the suspension of the hybrid plasmid in 10 mM tris-HCl buffer (pH 7.5), which is 70 mM in calcium chloride, is added. The transformed strains are selected as is customary, utilizing the antibiotic resistances or sensiti ities conferred by the plasmid, and the hybrid vectors are amplified. After the cells have been killed, the hybrid plas-mids are isolated and cut open with the restriction enzymes originally used, and gene fragments I, II, III and IV are isolated by gel electrophoresis.

Linkage of gene fragments I, II, III and IV to give the total gene Sub ragmen ts I and II obtained by amplification are linked as follows. 100 ng of each of the isolated fragments I and II are dissolved together in 10 μΐ of buffer containing 50 mM tris-HCL (pH 7.6), 20 mM magnesium chloride and 10 mM DTT, and this solution is heated at 57°C for 5 minutes. After the solution has cooled to room temperature, 1 μΐ of 10 mM adenosine triphosphate (pH 7) and 1 μΐ of T4 ligase (400 units) are added, and the mixture is incubated at room temperature for 16 hours. After subsequent cutting with the restriction enzymes Sail and BamHI, the desired 312 bp fragment (nucleotides 1-312, Sall-BamHI) is purified by gel electrophoresis on an 8% polyacryl-amide gel, the marker substance used being JGTX 174 RF DNA (from BRL) cut with the restriction enzyme Haelll.

Gene fragments III and IV are linked together in the same way, there being obtained after purification a 246 bp fragment (nucleotides 313-558, BamHI-Sal I ) . The marker used for the gel electrophoresis is pBR322 cut with the restriction enzyme Mspl.

To contruct the total gene (DNA sequence I), 15 ng of the 312 bp fragment and 12 ng of the 246 bp fragment are ligated, as described above, with 1 g of the commercially available plasmid pUCl8 which has previously been cut open with the restriction enzyme Sail and enzymat ical ly dephosphory- lated at the ends. After transformation and amp i fication (as described in Example 4a), the correct clone having the 558 bp fragment corresponding to DNA sequence I is identified by Sail digestion.

Transformation of the hybrid plasmids Competent E . col i cells are transformed with 0.1 to 1 ig of the hybrid plasmid containing DNA sequence I, and are plated out on ampl i c i 11 i n-con t a i n i ng agar plates. It is then possible to identify clones which contain the correctly integrated sequences in the plasmid by rapid DNA analysis (Maniatis loc. c i t . ) .

Fusion of the synthetic gene to regulation signals which are recognized in plants.

The optimized resistance gene which had been provided at the ends with Sail cleavage sites was ligated in the Sail cleavage site of the polylinker sequence of the plasmid pDH51 (Pietrzak et al., Nucleic Acids Res. 14 (1986) 5857). The promoter and terminator of the 35S transcript from cauliflower mosaic virus, which are recognized by the plant transcription apparatus, are located on this plasmid. The ligation of the resistance gene resulted in it being inserted downstream of the promoter and upstream of the termin ator of the 35S transcript. The correct orientation of the gene was confirmed by restriction analyses.

The promoter of the ST-LS1 gene from Solanum tuberosum (Eckes et al., Mol. Gen. Genet. 205 (1986) 14) was likewise used for the expression of the optimized acetyltransferase gene in plants.

Insertion of the resistance gene having the regulation sequences into Ag robac te r i um tumefaciens a) Cointegrate method The entire transcription unit comprising promoter, optimized resistance gene and terminator (Example 6) was cut out with the restriction enzyme Eco I and ligated in the EcoRI cleavage site of the intermediary E . col i vector pMPK110 (Peter Eckes, Thesis, Univ. Cologne, 1985, pages 91 et seq.). This intermediary vector was necessary for the transfer of the resistance gene with its regulation sequences into the Ti plasmid of Agrobac te r i um tumefaciens. Th s so-called conjugation was carried out by the method described by Van Haute et al. (EMBO J. 2 (1983) 411). This entailed the gene with its regulation signals being integrated in the Ti plasmid by homologous .. recombinat on via the - sequences of the standard vector pBR322 which are present in the pMPKHO vector and in the Ti plasmid pGV3850kanR (Jones et al., EMBO J. 4 (1985) 2411).

For this purpose, 50 μΐ of fresh liquid cultures of each of the E . col i strains DH1 (host strain of the pMP 110 derivative) and GJ23 (Van Haute et al., Nucleic Acids Res. 14 (1986) 5857) were mixed on a dry YT-agar plate and incubated at 37°C for one hour. The bacteria were resuspended in 3 ml of 10 mM MgSO^ and plated out on antibiotic- agar plates (spectinomyc in 50 ug/ml: selection for pMPK 110; tetracycline 10 ug/ml: selection for R64drd11; kanamycin 50 ug/ml: selection for pGJ28). The bacteria growing on the selective agar plates contained the three plasmids and were grown for the conjugation with Agrobac te r i um tume- f ac iens in YT liquid medium at 37°C. The Agro- bacteria were cultivated in LB medium at 28°C. 50 ul of each bacterium suspension were mixed on a dry YT-agar plate and incubated at 28°C for 12 to 16 hours. The bacteria were resuspended in 3 ml of 10 mM MgS0 and plated out on antibiotic plates (erythromycin 0.05 g/l, chloramphenicol 0.025 g/l: selection for the Agrobacter ium strain; streptomycin 0.03 g/l and spect inomyc in 0.1 g/l: selection for integration of pMPK110 in the Ti plasmid). Only Agrobacteria in which the pMPK110 derivative has been integrated into the bacterial Ti plasmid by homologous recombination are able to grow on these selected plates.

Besides the gene for resistance to the antibiotic kanamycin, which is active in plants and was already present from the outset, the PTC-res istance gene was now also located on the Ti plasmid pGV3850kan . Before these Agrobacter um clones were used for transformation, a Southern blot experiment was carried out to "check whether the desired integration had taken place.

Binary vector method The binary vector system described by Koncz et al. (MoL. Gen. Genet. 204 (1986) 383) was used. The vector pPCV701 described by Koncz et al.

(PNAS 84 (1987) 131) was modified in the following way: the restriction enzymes BamHI and Hindlll were used to delete from the vector a fragment on which are located, inter alia, the TR1 and TR2 promoters. The resulting plasmid was recir-cularized. Into the EcoRI cleavage site present on this vector was inserted a fragment from the vector pOH51 which is about 800 base-pairs in length and on which were located the promoter and terminator of the 35S transcript from cauliflower mosaic virus (Pietrzak et al., Nucleic Acids Res. 14 (1986) 5858). The resulting plasmid pPCV801 had a unique Sail cleavage site between the 35S promoter and terminator. The optimized PTC- resistance gene was inserted into this cleavage site. Its expression was now under the control of the 35S transcript regulation sequences.

This plasmid (pPCV801Ac) was transformed into the E. co i strain SM10 (Simon et al., Bio/Technology 1 (1983) 784). For the transfer of the plasmid pPCV801Ac into Agrobac ter i urn tumefaciens, 50 μΐ of both the SM10 culture and a C58 Agrobac ter ium culture (GV3101, Van Larebeke et al., Nature 252 (197A) 169) were mixed with the Ti plasmid pMP90RK (Koncz et al., Ι*>1· Gen. Genet. 0^ ( 198b) 383 as helper plasmid on agar plate, and the mixture was incubated at 28°C for about 16 hours. The bacteria were then resus- pended in 3 ml of 1 mfl MgSO^ and plated out on antibiotic plates (rifampicin 0.1 g/l: selection for GV3101, kanamycin 0.025 g/l: selection for PKP90R , carbenicill n 0.1 g/l: selection for pPCV801Ac). Only Agrobacteria which contained both plasmids (pMP90RK and pPCV80 Ac) are able to grow on these plates. Before these Agrobacteria were used for the plant trans ormation, Southern blotting was carried out to check that the plasmid pPCV801Ac is present in its correct form in the Agrobacteria.

Transformation of Nlcotlna tabacum by Agrobacterium tumefaciens The optimized resistance gene was transferred into tobacco plants using the so-called leaf disk trans formation method.

The Agrobacteria were cultured in 30 ml of LB medium containing the appropriate antibiotics at 28°C, shaking continuously (about 5 days). The bacteria were then sedimented by centr i f ugat ion at 7000 rpm in a Christ centrifuge for 10 minutes, and were washed once with 20 mL of 10 mM MgS0 . After a further centrifugation, the bacteria were suspended in 20 mL of 10 mM MgS0 and transferred into a Petri dish. Leaves of Wisconsin 38 tobacco pLants growing on 2MS medium in steriLe cuLture were used for the Leaf disk infection. ALL the steriLe cultures were maintained at 25 to 27°C in a 16 hours light/8 hours dark rhythm under white light.

Tobacco leaves were cut off, and the leaf surfaces were Lacerated with sandpaper. After the laceration, the Leaves were cut into smaLLer pieces and dipped in the bacterium culture. The leaf pieces were then transferred to M+S medium and maintained under normal cuLture conditions for two days. After the 2-day infection with the bacteria, the leaf pieces were washed in liquid M+S medium and transferred to MSC10-agar plates. Transformed shoots were selected on the bas s of the resistance to t he ant b i ot i c kanamycin which had also been transferred. The first shoots became visible 3 to 6 weeks Later. Individual shoots were further cultivated on MSC15 medium in glass jars. In the weeks which followed, some of the shoots which had been cut off developed roots at the site of the cut.

It was also possible to select transformed plants directly on PTC-containing plant media. The presence and the expression of the PTC-res istance gene was demonstrated by DNA analysis (Southern blotting) and RNA analysis (Northern blotting) of the transformed pL ants .

Demonstration of the PTC-res i s tance of the transformed plants To check the functioning of the resistance gene in transformed plants, leaf fragments from transformed and non-trans ormed plants were transferred to M+S nutrient media containing 1 x 10 -4 M L-PTC. The fragments from non-transformed plants died, while the fragments from transformed plants were able to regenerate new shoots. Transformed shoots took root and grew without difficulty on M+S nutrient media containing 1 x 10"^ M L-PTC. Transformed plants were, from sterile conditions, potted in soil and sprayed with 2 kg/ha and 5 kg/ha PTC. Whereas non-transformed plants did not survive this herbicide treatment, transformed plants showed no damage brought about by the herbicide. The appearance and growth behavior of the sprayed transformed plants was at least as good as that of unsprayed control plants.

AcetyLtransferase assay to demonstrate acetylation of PTC in transgenic PTC-res i s tant plants About 100 mg of leaf tissue from transgenic PTC-res istant tobacco plants or from non-transformed tobacco plants were homogenized in a buffer composed of: 50 mM tris-HCl, pH 7.5; 2 mM E0TA; 0.1 mg/ml leupeptin; 0.3 mg/ml bovine serum albumin; 0.3 mg/ml DTT; 0.15 mg/ml phenylmethylsuLfonyl fluoride (PMSF).

After subsequent cent r i fugat i on, 20 μΐ of the clear supernatant were incubated with 1 μΐ of 10 mM radio-labelled D, L-PTC and 1 yl of 100 mM acetyl-CoA at 37°C for 20 minutes. 25 yl of 12% trichloroacetic acid were then added to the reaction mixture, followed by centrifugat ion. 7 μΐ of the supernatant were transferred to a thin-layer chromatography plate and subjected to ascending development twice in a mixture of pyridine : n-butanol : acetic acid : water (50 : 75 : 15 : 60 parts by volume). PTC and acetyl-PTC were separated from one another in this way, and could be detected by autoradiography. Non-transformed plants exhibited no conversion of PTC into acetyl-PTC, whereas transgenic resistant plants were capable of this.

MET SER PRO GL G TC GAC ATG TCT CCG GAG AGG AGA CCA GTT GAG ATT AGG CCA GCT ACA GCA GCT GAT ATG GCC GCG GTT G TAC AGA GGC CTC TCC TCT GGT CAA CTC TAA TCC GGT CGA TGT CGT CGA CTA TAC CGG CGC CAA ILE GLU THR SER THR VAL ASN PHE ARG THR GLU PRO GLN THR PRO GLN GLU TRP ILE ASP ATT GAG ACG TCT ACA GTG AAC TTT AGG ACA GAG CCA CAA ACA CCA CAA GAG TGG ATT GAT TAA CTC TGC AGA TGT CAC TTG AAA TCC TGT CTC GGT GTT TGT GGT GTT CTC ACC TAA CTA 1 100 TRP LEU VAL ALA GLU VAL GLU GLY VAL VAL ALA GLY ILE ALA TYR ALA GLY PRO TRP LYS ALA ARG ASN TGG TTG GTT GCT GAG GTT GAG GGT GTT GTG GCT GGT ATT GCT TAC GCT GGG CCC TGG AAG GCT AGG AAC ACC AAC CAA CGA CTC CAA CTC CCA CAA CAC CGA CCA TAA CGA ATG CGA CCC GGG ACC TTC CGA TCC TTG ZOO SER THR VAL TYR VAL SER HIS ARO HIS GLN ARG LEU GLY LEU GLY SER THR LEU TYR THR HIS LEU LEU AGT ACT GTT TAC GTG TCA CAT AGG CAT CAA AGG TTG GGC CTA GGA TCC ACA TTG TAC ACA CAT TTG CTT TCA TGA CAA ATG CAC AGT GTA TCC GTA GTT TCC AAC CCG GAT CCT AGG TGT AAC ATG TGT GTA AAC GAA 300 PHE LYS SER VAL VAL ALA VAL ILE GLY LEU PRO ASN ASP PRO SER VAL ARG LEU HIS GLU ALA LEU GLY TTT AAG TCT GTG GTT GCT GTT ATA GGC CTT CCA AAC GAT CCA TCT GTT AGG TTG CAT GAG GCT TTG GGA AAA TTC AGA CAC CAA CGA CAA TAT CCG GAA GGT TTG CTA GGT AGA CAA TCC AAC GTA CTC CGA AAC CCT liO ARG ALA ALA GLY TYR LYS HIS GLY GLY TRP HIS ASP VAL GLY PHE TRP GLN ARG ASP PHE GLU LEU PRO CGC GCA GCT GGA TAC AAG CAT GGT GGA TGG CAT GAT GTT GGT TTT TGG CAA AGG GAT TTT GAG TTG CCA GCG CGT CGA CCT ATG TTC GTA CCA CCT ACC GTA CTA CAA CCA AAA ACC GTT TCC CTA AAA CTC AAC GGT 1 • sew PRO VAL THR GLN ILE ---CCA GTT ACC CAG ATC TCA G GGT CAA TGG GTC TAG ACT CAG CT Amino acid and DNA sequence I MET SER PRO OLD A G TC OAC ATO TCT CCG GAG AGO AGA CCA GTT GAQ ATT AGO CCA GCT ACA GCA GCT GAT ATG GCC GCO GTT Q TAC AGA GGC CTC TCC TCT GGT CAA CTC TAA TCC GGT CGA TGTfCGT COA CTA TAC COG CGC CAA 4 PHE LYS SER VAL VAL ALA VAL ILE QLY LEU PRO ASN ASP PRO SER VAL ARQ LEU HIS GLU ALA LEU GLY TTT AAQ TCT GTO GTT GCT GTT ATA GGC CTT CCA AAC OAT CCA TCT OTT AOO TTO CAT GAG GCT TTG GOA AAA TTC AGA CAC CAA CGA CAA TAT CCO GAA GGT TTG CTA OCT AGA CAA TCC AAC GTA CTC CGA AAC CCT ARO ALA ALA OLY TYR LYS HIS OLY OLY TRP HIS ASP VAL QLY PHE TRP GLN ARG ASP PHE GLU LEU PRO CGC GCA CCT GGA TAC AAG CAT GGT GGA TOO CAT GAT GTT GGT TTT TGG CAA AGG GAT TTT GAQ TTG CCA GCG CGT CGA CCT ATO TTC GTA CCA CCT ACC OTA CTA CAA CCA AAA ACC GTT TCC CTA AAA CTC AAC GGT PRO VAL THR GLN ILE CCA GTT ACC CAO ATC TOA 0 GGT CAA TGG GTC TAG ACT CAG CT . . .. . „„. TT Amino acid and DNA sequence II

Claims (10)

85143/4 -17- CLAIMS:

1. A resistance gene coding for the protein of amino acid sequence I (annex), characterized in that ATG is used as start codon and TGA is used as stop codon, and the GC content of the gene is adapted to that in plants.

2. The resistance gene as claimed in claim 1 , having DNA sequenve I (annex, nucleotide positions 9-554).

3. A gene structure according to claim 1 having DNA sequence I (annex) coupled to regulation and expression signals active in plants.

4. A vector containing the resistance gene as claimed in claim 1 or 2.

5. A vector containing a gene structure as claimed in claim 3.

6. Vectors containing a resistance gene according to claim 1 , wherein said resistance gene comprises one or more of DNA sequences (annex) selected from fragment I (nucleotide No. 1 to 152), fragment II (nucleotide No. 153 to 312), fragment 111 (nucleotide No. 313 to 436) or fragment IV (nucleotide No 437 to 558).

7. A host cell containing a vector as claimed in claim 4, 5 or 6.

8. A plant cell containing a gene as claimed in claim 1 , 2 or 3.

9. Plants, their parts and seeds, containing a gene as claimed in claim 1 , 2 or 3.

10. The use of the gene as claimed in claim 1 or 2 or of the gene structure as claimed in claim 3 for generating phosphinotricin-resistant plant cells, parts of plants, plants and seeds, substantially as described in the specification.