AU609082B2 - Phosphinothricin-resistance gene active in plants, and its use - Google Patents

Description

HOECHST AKTIENGESELLSCHAFT HOE 87/F 333 J Dr. KL/mu Specification Phosphinothricin-resistance gene active in plants, pr y its use Non prior-published German Patent Application P 36 28 747.4 ("main application") proposes a phosphinothricin (PTC)-resistance gene which can be obtained from the total DNA of Strertomyces viridochromogenes DSM 40736 (general collection) or DSM 4112 (deposition under the Budapest Treaty), which has been selected for phosphinothricyl-alanyl-alanine (PTT)-resistance, by cutting with BamHI, cloning of a fragment 4.0 kb in size, and selection for PTT-resistance, as well as the use of this S o gene for the production of PTC-resistant plants, as PTTt 15 resistance marker in bacteria and PTC-resistance marker in plant cells. The BamHI fragment which is 4 kb in size and on which the resistance gene is located is defined in detail by a restriction map (Figure 1).

k t 20 The position of the coding region has been located more accurately by cloning part-regions of this 4 kb fragment.

a.It emerged from this that the resistance gene is located on the 1.6 kb SstII-SstI fragment (positions 0.55 to 2.15 in Fig. 1 of the main application). Digestion with BglII 25 results in a fragment 0.8 kb in size which confers PTTooo resistance after incorporation in a plasmid and transformation of S. Lividans. This resistance depends on the N-acetylation of PTC. Hence the resistance gene codes for an acetyltransferase.

The DNA sequence of the abovementioned 0.8 kb fragment is reproduced in the German application for a patent of addition P 36 42 829.9.

It is possible to determine from the sequence the start codon and the open reading fraie of the gene sequence.

The last nucleotide is part of the stop codon TGA.

Genes from Streptomycetes have a very high proportion of G C, the adenine thymine guanine 2 cytosine 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 irea, the DNA 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 it gene: 1. The Streptomycetes gene start codon GTG (position 258-260 in the sequence in the additional application) was replaced by the start codon ATG which is used by plant RNA polymerase II.

2. Within the gene, the Streptomycetes 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 lr II -I 3 it between plant regulation sequences.

Palindromic sequences were reduced to a minimum.

The DNA 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 XmaI (436) make possible the subcloning of part-sequences which can be incorporated in well-investigated cloning vectors such as, for example, pUC18 or pUC19. In addition, a number of other unique recognition sequences for restriction enzymes were incorporated within the gene, and these, on 15 the one hand, provide access to part-sequences of acetyltransferase and, on the other hand, allow modifications to be made: a 44 4 a a0 .444r ''44 a a 4 44 Restriction enzyme BspMII SacII EcoR V HpaI AatlI BstXI Apal Scal AvrIl AftII StuI BssHII FokI BgllI BglII Cut after nucleotide No.

(codings strand) 11 64 74 99 139 232 272 308 336 385 449 487 536 550 The construction of part-sequences by chemical synthesis and enzymatic ligation reactionis i carried out in a manner known per se (EP-A 0,133,282, 0,136,472, 0,155,590, 4 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. coli, are described at Length in the textbook of Maniatis (Molecular 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-resistant plant cells a selection feature for transformed cells is available), plants or parts of plants and seeds.

44000 o 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: 0u5% yeast extract, 0.8% Bacto tryptone, 0.5% NaCI LB medium: 0.5% yeast extract, 1% Bacto tryptone, 1% NaCl as solid medium: addition of 1.5% agar to each b) for plantss M+S mediu: see Murashige and Skoog, Physiologita Plantarum 15 (1962) 473 2M3 medium: M+S medium containing 2% sucrose MSC10 medium: M+S medium containing 2% sucrose, 500 mg/L cefotaxime, 0.1 mg/L naphthylacetic acid (NAA), 1 mg/l benzylaminopurine (BAP), 100 mg/L kanamycin M3C15 medium: M+S medium containing 2% sucrnse, o~ -rr- 5 500 mg/L cefotaxime, 100 mg/l kanamycin.

1. Chemical synthesis of a single-stranded oligonucLeotide Th 3 synthesis of fragment II, one of the four partfragments I IV, started from the terminal oligonucleotide IlIc (nucLeotide No. 219 to 312 in the coding strand of DNA sequence 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) functionalized with long-chain amino- 15 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), which otherwise corresponds to DNA sequence I.

2. Enzymatic linkage of the single-stranded oLigonucleotides to give gene fragment II For the phosphorylatioh of the oligonucleotides at the 5' end, 1 nmol of each of oligonucleotides lib and IIc was treated with 5 nmoL of adenosine t-iphosphate and 4 units of T4 polynucleotide kinase in 20 pl of 50 mM tris-HCl buffer (pH 10 mM magnesium chloride and 10 mM dithiothreitol (DTT) at 37 0 C for 30 minutes. The enzyme is inactivated by heating at 95 0 C for 5 minutes. OLigonucLeotides IIa and IId, which form the "protruding" sequence in DNA fragment II, are not phosahorylated. This prevents the formation, during the subsequent Ligation, of larger subfragments than correspond to DNA fragment II.

I

1 ~ra~ l 6- Oligonucleotides II are Ligated to give subfragment II as follows: 1 1nrol of each of oligonucleotides IIa and lid and the 5'-phosphates of IIb and llc are together dissolved in 45 Ui of buffer containing 50 mM tris-HCL (pH 20 mM magnesium chloride, 25 mM potassium chloride and 10 mM DTT.

For the annealing of the oligonuct! ctides corresponding to DNA fragment II the solution of the oligonucleotides is heated at 95 0 C for 2 minutes and then slowly cooled (2-3 hours) to 20°C. Then, for the enzymatic linkage, 2 pl of 0.1 M DTT, 8 pl of mM adenosine triphosphate (pH 7) and 5 p1 of T4 DNA ligase (2000 units) are added, and the mixture is incubated at 22 0 C for 16 hours.

The gene fragment II is purified by gel electropho- ~resis on a 10% polyacrylamide gel (without addition of urea, 20 x 40 cm, 1 mm thick), the marker substance used being ZX 174 DNA (from BRL) cut with Hinfl, or pBR322 cut with HaeIII.

o 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.

3, Preparation of hybrid plasmids containing gene frag- Sments I, II, III and IV.

a) Incorporation of gene fragment I in pUC18 The commercially available plasmid pUC18 is opened in a known manner using the restriction endo nucleases SalJ and Xbal in accordance with the manufacturers' instructions. The digestion mixture i; fractionated by electrophoresis 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 7 gel and removed from the agarose by electroelution.

1 pg of plasmid, opened with XbaI and Sail, is then Ligated with 10 ng of DNA fragment I at 160C overnight.

b) Incorporation of gene fragment II in pUC18.

In analogy to pUC18 is cut open with Xbal and BamHI and Ligated with gene fragment II which has previously been phosphorylated at the protruding ends as described in Example 2.

c) Incorporation of gene fragment III in pUC18 In analogy to pUC18 is cut open with BamHI and XmaIII and ligated with gene fragment III.

d) Incorporation of gene fragment IV in pUC18 In analogy to pUC18 is cut with XmaIIl and SalI and ligated with gene fragment IV.

4. Construction of the complete gene and cloning in a pUC pLasmid a) Transformation and amplification of gene fragments I IV The hybrid plasmids obtained in this way are transformed into E. coli. For this purpose, the strain E. coli 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 which is 70 mO in calcium chloride, is added. The transformed strains are selected as is customary, utilizing the antibiotic resistances or sensitivities conferred by 1 -8the plasmid, and the hybrid vectors are amplified.

After the cells have been killed, the nybrid plasmids are isolated and cut open with the restriction enzymes originally used, and gene fragments I, II, III and IV are isolated by gel electrophoresis.

b) Linkage of gene fragments I, II, III and IV to give the total gene Subfragments 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 10I pl of buffer containing 50 mM tris-HCL (pH 20 mM magnesium chloride and 10 mM DTT, and this solution is heated at 57 0 C for 5 minutes.

After the solution has cooled to room temperature, 1 pl of 10 mM adenosine triphosphate (pH 7) and 1 pl 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, SalI-BamHI) is purified by gel electrophoresis on an 8% polyacrylaide gel, the marker substance used being OX 174 RF DNA (from BRL) cut with the restriction enzyme HaeIII.

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-Sa4L). The marker used for the gel electrophoresis is pBR322 cut with the restriction enzyme Mspl.

To contruct the total gene (DNA sequence 15 ng of the 312 bp fragment and 12 ng of the 246 bp fragment are Ligated, as described above, with 1 Pg of the commercially available plasmid pUC18 9 which has previously been cut open with the restriction enzyme SaLI and enzyiadticall y dephosphory- Lated at the ends. After transformation and amplification (as described in Example 4a), the correct clone having the 558 bp fragment corresponding to DNA sequence I is identified by SalI digestion.

Transformation of the hybrid plasmids Competent E. coli cells are transformed with 0.1 to 1 pg of the hybrid plasmid containing DNA sequence 1, and are plated out on amplicillin-containing agar plates. It is then possible to identify clones which contain the correctly integrated sequences in the plasmid by rapid DNA analysis (Maniatis loc.

cit.).

6. 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 SalI 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 terminator of the 35S transcript. The correct orientation of the gene was confirmed by restriction, analyses.

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

1-- 10 7. Insertion of the resistance gene having the regulation sequences into Agrobacterium tumefaciens a) Cointegrate method The entire transcription unit comprising promoter, optimized resistance gene and terminator (Example 6) was cut out with the restriction enzyme EcoRI and ligated in the EcoRI cleavage site of the intermediary E. coli 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 Agrobaccerium tumefaciens. This so-called conjugation uas 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 recombination via the sequences of the standard vector pBR322 which are present in the pMPK110 vector and in the Ti plasmid p'GV3850kanR (Jones et aL., EMBO J. 4 (1985) 2411).

For this purpose, 50 pl of fresh liquid cultures of each of the E. coli strains DH1 (host strain of the pMPK110 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 0

C

for one hour. The bacteria were resuspended in 3 ml of 10 mM MgSO 4 and plated out on antibioticagar plates (spectinomycin 50 pg/ml: selection for pMPK110; tetracycline 10 g/ml: selection for R64drdll: kanamycin 50 pg/ml: selection for pGJ28). The bacteria growing on the selective agar plates contained the three plasmids and were grown for the conjugation with Agrobacterium tumefaciens in YT Liquid medium at 37°r- The Agrobacteria were cultivated in LB medium at 28 0

C.

11 pl of each bacterium suspension were mixed on a dry YT-agar pLate and incubated at 280C for 12 to 16 hours. The bacteria were resuspended in 3 ml of 10 mM MgSO4 and pLated out on antibiotic plates (erythromycin 0.05 g/L, chLoramphenicol 0.025 g/L: seltution for the Agrobacterium strain; streptomycin 0.03 g/L and spectinomycin 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.

o0 ,o 4 Besides the gene for resistance t a the antibiotic kanamycin, which is active in plants and was al-

L

ready present from the outset, the PTC-resistance o o gene was now also located on the Ti plasmid SpGV3850kanR. Before these Agrobacterium clones were used for transformation, a Southern blot experiment was carried out to check whether the desired integration had taken place.

b) 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 oncz et at.

(PNAS 84 (1987) 131) was modified in the following way: the restriction enzymes BamHI and HindIll were used to delete from the vector a fragment on which are located, inter alia, the TR1 and TR2 promoters. The resulting plasmid was recircularized. Into the EcoRI cleavage site present on this vector was inserted a fragment from the vector pDH51 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 at., Nucleic Acids Res.

14 (1986) 558). The resulting plasmid pPCV801 12 had a unique SalI cleavage site between the promoter and terminator. The optimized PTCresistance gene was inserted into this cleavage site. Its expression vas now under the control of the 35S transcript regulation sequences.

This plasmid (pPCV801Ac) was transformed into the E. coli strain SM10 (Simon et a Bio/Technology 1 (1983) 784). For the transfer of the plasmid pPCV801Ac into Agrobacterium tumefaciens, 50 pl of both the SM10 culture and a C58 Agrobacterium culture (GV3101, Van Larebeke et al., Nature 252 (1974) S169) were mixed with the Ti plasmid pMP90RK (Koncz et.al.Mol.Grn.Genet.204(1986)383 as helper plasmid on a dry YTagar plate, and the mixture was incubated at 280C for about 16 hours. The bacteria were then resuspended in 3 ml of 1 mM MgSO 4 and plated out on antibiot'c plates (rifampicin 0.1 g/l: selection for GV3101, kanamycin 0.025 g/l: selection for o 20 pMP90RK, carbenicillin 0.1 g/l: selection for pPCV801Ac). Only AgrobactPria which contained both plasmids (pMP90RK and pPCV801Ac) are able to grow on these plates. Before these Agrobacteria were used for the plant transformation, Southern blotting was carried out to check that the plasmid o 6 pPCV801Ac is present in its correct form in the Agrobacteria.

8. Transformation of Nicotina tabacum by Agrobacterium tumefaciens The optimized resistance gene was transferred into tobacco plants using the so-called Leaf disk transformation method.

The Agrobacteria were cultured in 30 ml of LB medium containing the appropriate antibiotics at 28 0 C, shaking continuously (about 5 days). The bacteria were then sedimented by centrifuga' ion at 7000 rpm in a 13 Christ centrifuge for 10 minutes, and were washed once with 20 ml of 10 mM MgSO 4 After a further centrifugation, the bacteria were suspended in 20 ml of 10 mM MgS0 4 and transferred into a Petri (:ish.

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 0 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 cf' in the bacterium culture. The leaf pieces were then *noo 15 transferred to M+S medium and maintained under nor- S' mal culture conditions for two days. After the 2day infection with the bacteria, the leaf pieces were washed in liquid M+S medium and transferred to plates. Transformed shoots were selected on the basis of the resistance to the antibiotic kanamycin which had also been transferred. The first shoots became visible 3 to 6 weeks later. Individual shoots were further cultivated on medium in glass jars. tn 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-resistance gene was demonstrated by DNA analysis (Southern blotting) and RNA analysis (Northern blotting) of the transformed plants.

9. Demonstration of the PTC-resistance of the transformed plants To check the functioning of the resistance gene in transformed plants, leaf fragments from transformed

I

14 and non-transformed plants were transferred to M+S nutrient media containing 1 x 10 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 nontransformed 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-resistant plants Abou.t 100 mg of leaf tissue from transgenic PTCresistant tobacco plants or from non-transformed tobacco plants were homogenized in a buffer composed of: 50 mM tris-HCL, pH 7.5; 2 mM EDTA; 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 centrifugation, 20 pl of the clear supernatant were incubated with 1 pl of 10 mM radio- Labelled D,L-PTC and 1 pL of 100 mM acetyl-CoA at 37°C for 20 minutes. 25 pl of 12% trichloroacetic acid were then added to the reaction mixture, followed by centrifugation. 7 pl 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 15 60 parts by volume). PYC 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.

MIET SER PRO GLU ARG ARG PRO VAL GLU ILE AflO PRO ALA T1111 ALA ALA ASP IIET ALA ALA VAL CYS ASP ILE VAL ASH Ills TYR TC GAC ATG TCT CCG GAG AGG AGA CCA GTT GAO ATT AGO CCA GC ACA GCA OCT GAT AIG GCC GCG GII TOT GAT ATC CIT AAC CAT TAC G TAC AGA 600 CTC 7CC 1CT GOT CAA CTC TAA ICC GGT CGA TOT COT CGA CTA TAC CGG CSC CAA ACA CTA TAG CAA TTG GTA ATG -4 ILE GLU AT? GAG TMA CTC 1I111 SER TI;R VAL ASH PilE ARG IIIR GLU PRO CLII THR PRO GUI GLU TAP ILE ASP ACO TCT ACA GTG AAC ITT AGO ACA GAG CCA CAA ACA CCA CAA GAG TGG ATT OAT TGC AGA TOT CAC TIC AMA TCC TGT CTC OUT GTT TOT GOT CIT CTC ACC TAA CIA ASP LEO GLU ARG LEU GUI GAT CIA GAG AGO TTC CAA CIA CAT CIC TCC AAC GTT ASP ARG TYR PRO GAT AGA TAC CCI CIA TCT ATO GGA TAP LEO IGG 116 ACC MAC VAL ALA OLU VAL GLIJ OLT VAL VAL ALA OLI ILE ALA TYR 2tLA GLY PRO TAP LYS ALA ARU 't ALA TYR ASP TAP TIIR VAL GLU OTT GOT GAG OTT GAG G CIT GIG OCT GGT ATT GCC TAC CCI GGG CCC TGG AA GOCT AGr, AC GOT TAO GAT IGG ACA GTl GAG CAA CGA CIC CM. CIC CCA CMA CAC CGA CCA TMA COA ATG, CGA CCC GO ACC TIC CGA TCC TIG COA AIG CIA ACC TGT CAA CTC 1 1 200 VAL TYR VAL SER 111S ARO HIS ULMI ARG LEU OLY LIU GLY SER IIIR LEO Trfl TIJR HIS LEU LEO LYS SER MET GLU ALA GUI GLY OTT TAC GIG ICA CAT ACG CAT CAA AGO ITT GGC CIA GOA TCC ACA TIC TAC ACA CAT ITO CIT MCG TCT ATG GAG GCG CAA GOT CAA ATG CAC AGT CIA TCC GTA CIT TCC MAC CCC OAT CCT AGO TOT AAC ANG TOT CIA MOC GM TIC AGA TAC CIC CCC GTl C' A SEll THR ACT ACT TCA TGA PlE LYS ITT AAG AAA TIC VAL ALA VAL ILE GLY OTT CCI CIT AlA GCC CAA CGA rA TAT CCG ASH ASP PRO SER VAt.

ACGAT CCA ICT CIT IN CIA COT AGA CAA Lio T11R ALA ARC GLY ACA CCC CCC COT TOT CGG GOC COA ARG ALA ALA GLY TYR LYS 11IS OLY GLY TRP 111S ASP VAk, OLY PHlE TRP OLN ARC ASP L'IE ULU LEO PRO ALA PRO PRO ARC PRO VAL ARC CGC GCA OCT OCA TAC AAC CAT OCT OCA TOO CAT GAT OTT GOT TIT TOO CAA AGO OAT TTIA GAO 710 CCA oCr CCd CCA AGO CCA CII AGO 0CC COT CGA CCT A7C TIC OTA CCA COT ACC CIA CIA CAA CCA AAA ACC GTl 7CC CTA AAA CIC AAC 001 CCA GGA GOT ICC OCT CAA TOO I S PRO VAL 71111 GUI ILE-- CCA OTT ACC UAG AIC TGA G OCT CAA TOG CIC TAO ACT CAG CT Amino acid and DNA sequence I WET SER PRO ULUJ TC OAC ATN TCT CCU GAG G TAC AGA GCC CTC ARO ARO AGO AGA 7CC TC? PRO VAL OLU ILK ARO PRO UCA GTT GAG ATT AGO CCA GOT CAA CTC TAA TCC GOT ALA TIM ALA OCT ACA GCA COA TOT COT ALA ASP MElT ALA ALA VAL CYS ASP ILK VAL ASH HIS TYR GCT GAT ATG CCC G TT TOT GAT ATC OTT AAC CAT TAC COA CTA TAC CGG COC CMA ACA CTA TAG CAM TTG GTA ATG .4 ILK OLU TR BER TRR VAL. ASH VHSl AT? GAG ACO TCT ACA 070 AAC TTT TMA CTC TOGC AGA TOT CAC TTO AAA ARO TIIR GLU AGO ACA GAO TCC TGT CTC yb PRO OLM TAR PRO GUI OLU TRP ILE ASP CCA CAA ACA CCA CAA GAO TOO ATT OAT GOT OTT TOT GOT OTT CTC ACC TMA CTA ASP LEOUOLU ABO LEU GUI ASP ARO TYR PRO OAT GSA AGO TTG CAA OAT AGA TAC CCT OT AT C 7CC MAC OTT CTA TCT ATO GGA 1g1b TRF LIII VAL ALA, OLU VAL. ULU OLT VAL. VAL ALA OLT ILE IALA TYR ALA GLY PRO TRP LYS TOO TYG OTT OCT GAO OTT GAGOU 00GTT 070 OCT GOT AT? GC TA GOCT GOO CCC TOO AAO ACC hAG CAA CGA CTC CAA CTC CCA CMA CAC COA CCA TMA CGI OA CCC GOO ACC TTC ALA ARO ASH OCT AGO AAG CGA TCC TTO ALA tR ASP TRP TAR1 VAL OLU OCT TACGOAT TOO ACA OTT GAG COA ATO CTA ACC TOT CAA CTC SER TAn VAL. TR VAL. SIR AG? ACT OTT TACGT07 TCA TCA TOA CAA ATO CAG AGT IJd- F a HIS ARG HIS OLN ARO CAT AGO CAT CMA AGO OTA TCC GTA OTT TCC I- LEOGOLr LIII OLY SER TAR LEOI TR TAR HIS LEII L T GGC CTA W.A TCC ACA T70 TAG ACA CAT TTO C AACCCGGATCCTACPTOT MGC ATO TOT GTA AAC G lp 1..w aL EU LYS SIR MET7 GU ALA GUI OLY TT MOG TCT ATO GAG OCG CMA GOT AA TTG AGA TAG CTC COC GTT CCA 22a o 1111 LYS SIR VAL. VAL ALA TTT MOG TCT 070 OTT OCT MAA TTC AGA CAC CAA COA VAL. ILK OTT ATA CMA TAT OLY LIII PRO GC CT? CCA CCO aAM GO? ASH ASP PRO SEW VAL. ARO LIII HIS ULU ALA LEO OLY TYR THR AAC GAT CCA TCT OTT AGO TTO CAT GAG OCT TTO OOA TAC ACA TTO CTA 00? AGA CMA TCC AAC GTA CTC COA hA CC? ATO TOT ALA ARO OLY TAR LEO GCP COG GOT ACA 770 C~tU UC CCA TOT MAC

TVQ

AR13 ALA ALA OLT TYR LYS.HIS OLT OLY TRP HIS CCC GA OCT GGA TAG AAG CAT GOT OGA TOO CAT OCO CUT COA CCT ATO TTC GTA CCA CC? ACC 0TA ASP YAK. OLY PRE TRP OAT OTT GOT TTT TOO CTA CMA CCA AMAA ACC OLM ARO ASP Pill GU LEO PRO ALA PRO PRO ARO PRO VAK. ARG CAA AGO GA? TTT GAO TTO CCA OCT CCT CCA AGO CCA CTT AGO OTT TCC CTA AAA CTG MAC GOT CGA OGA GOT 7CC OT CMA 7CC PRO VAL. THR CLH ILE CCA 07? ACC CA(U iTC TOA 0 GOT CMA TOO 070 TAO ACT CAG CT Amino acid and DNA sequence II

Claims (7)

1. A resistance gene coding for the protein of amino acid sequence I as herein defined, which gene is adapted to the codon usage in plants.

2. The resistance gene as claimed in claim 1, having DNA sequence I, nucleotide positions 9-554.

3. A gene structure having DNA sequence I as herein defined coupled to regulation and expression signals active in plants. 4, A vector containing the resistance gene as claimed in claim 1 or 2. A vector containing a gene structure as claimed in claim 3.

6. Vectors containing one or more of gene fragments I IV as herein defined.

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. I~ I ;B n~ I i 18 The use of the gene as claimed in claim 1 or 2 or of the gene structure as claimed in claim 3 for generating phosphinothricin-resistant plant cells, parts of plants, plants and seeds. DATED this 24th day of December, 1990 HOECHST AKTIENGESELLSCHAFT WATERMARK Patent Trademark Attorneys "The Atrium" 290 Burwood Road Hawthorn Victoria 3122 AUSTRALIA DBM/JMW:EK(14:10) 'S 'T