DK175800B1 - Resistance gene, gene structure containing the resistance gene, vector containing the resistance gene or gene structure, host cell containing the vector, plant cell, plants ............. - Google Patents

Description

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The present invention relates to a phosphinothricin resistance gene, a gene structure containing the resistance gene, a vector containing the resistance gene or gene structure, a host cell containing the vector, a plant cell, plants, parts thereof and seeds containing the resistance gene, and the use of the resistance gene or gene structure to provide phosphinothricin-resistant plant cells, plant parts, plants and seeds.

West German Patent Application No. 3,628,747.4 20 proposes a resistance gene against phosphinothricin (PTC) obtainable from the entire DNA from phosphinothricyl-alanyl-alanine (PTT) resistance selected by Strepcomyces virichochogenogenes DSM 40736 (from the regular collection) or DSM 4112 (deposited under the Budapest Treaty) at 25 sections with BamHI, cloning a 4.0 kb fragment and selection for PTT resistance, and its use! gene for producing PTC-resistant plants, as PTT-1 resistance marker in bacteria and as PTC-resistance marker I in plant cells. The 4 kb BamHI fragment, in which the 20 resistance gene is located, is more precisely defined by a restriction map (Figure 1).

By cloning subregions of this 4 kb fragment, the location of the code area was more closely located.

Hereby, the resistance gene was found to be in the 1.6 25 kb SstII-SstI fragment (positions 0.55 to 2.15 in Figure .... 1 in the main application). By digestion with BglII, a 0.8 kb fragment is recovered which, upon incorporation into a plasmid and transformation of S. lividans, mediates PTT resistance. This resistance is conditioned by N-acetylation of 30 PTC. Thus, the resistance gene encodes an acetyl transferase.

In West German Patent Application No. 3,642,829.9 the DNA sequence of the 0.8 kb fragment mentioned above is reproduced. From the sequence, the start codon and the gene sequence open reading frame can be determined. The last nucleo-35 time is part of the stop codon TGA.

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Streptomyceter genes have a ratio of I

adenine (A) + thymine (T): guanine (G) + cytosine (C) on I

ca. 30%: 70% a very large proportion of G + C. The GC proportion I

of plant genes lies with approx. 50% far lower. Of these

For five reasons, the DNA sequence of the resistance gene is optimized in I

further embodying the idea of the invention in novel synthesis I

of one favorable for the vegetable RNA polymerase II

codon usage. IN

The invention relates to a modification of the resistance I

In 10 9 © n proposed in German patent applications No. H

I P 3.628.747.4 and P 3.642.829.9, namely an adaptation to I

In codon use in plants I

More particularly, the invention relates to the following:

In a resistance gene encoding the protein with amino-I

In the acid sequence I (listed below), starting as I

In codon, ATG is used and as stop codon TGA, and the gene I

In GC proportion is adjusted GC proportion in plants, I

- a gene structure characterized in that the DNA sequence I

In late I are connected in plants active regulations and I

In 20 expression signals, H

In a vector characterized by resistance H

the gene of the invention, H

a vector characterized by a gene structure

In turn according to the invention, I

In 25 vectors containing one or more DNA sequences H

In friends selected from: I

I I (nucleotide # 1-152) I

I II (Nucleotide Nos. 153-312) I

I III (nucleotide nos. 313-436) or I

I IV (Nucleotide Nos. 437-558), I

I - a host cell characterized by a vector H

In accordance with the invention, H

In 35 - a plant cell or plants, parts thereof and H

In seeds characterized by a gene according to H

And the use of the gene or gene structure of the invention to provide phosphino-thricin-resistant plant cells, plant parts, plants and seeds.

As is known, the genetic code is such that a single triplet encodes only 2 amino acids, while each of the remaining 18 amino acids which can be genetically coded for 10 are assigned 2-6 triplets. Therefore, for the synthesis of the gene, there is theoretically a great diversity of codon combinations. Since the said relative proportion of the individual nucleotides in the entire DNA sequence is of influence, this is the basis of one of the criteria in the sequence optimization.

The following changes have been made to the sequenced gene: 1. The streptomycet gene start codon GTG (position 258-260 in the sequence of the West German patent application) is replaced by the mec * vegetable RNA polymerase II starting codon ATG.

2. Within the gene, the Streptomycet gene codons change so that it results in plant genes suitable codons (G / C ratio).

2 £ 3. To end the translation process, put the TGA stop codon at the end of the sequence.

4. The beginning and end of the gene sequence are provided with imminent termination of restriction sites to amplify the gene and to align the gene between vegetable regulatory sequences.

5. Palindromic sequences are reduced to a minimum.

The DNA sequence I of the invention (with the corresponding amino acid sequence) is set out below.

Three internal singular intersection sites for the restriction enzymes XbaI (position 152), BamHI (312), and Xmal (436) allow subcloning of partial sequences that may be embedded in well-studied cloning vectors such as pUC18 or PUC19. Furthermore, there is within the gene Η

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incorporated a number of additional singular recognition sites

for restriction enzymes which, on the one hand, provide ad- I

to the partial sequences of acetyltransferase and on the other I

H side allows implementation of variations: I

I 5 i

In Section after nucleotide no. IN

Restriction enzyme_ (coding string) _ I

I BspMII 11 I

In SacII 64 I

I 10 EcoRV 74 I

I Hpal 80 I

Aatll 99 I

BstXI 139 I

I I Apal 232 I

15 Seal 272 I

I Avrll 308 I

AfIII 336 I

Chair 385 I

I BssHII 449 I

I 20 Fokl 487 I

I Bgll 536 I

Bglll 550 I

Structure of the partial sequences using chemical I

In 25 synthesis and enzymatic ligation reaction, i and i are performed

H known per se (European Patent Application No. I

0.133.282, 0.136.472, 0.155.590, 0.161.504, 0.163.249, I

I 0.171.024, 0.173.149 or 0.177.827). Details such as I

In restriction assays, ligation of DNA fragments and trans I

The formation of plasmids in E. coli is described in detail

in Maniatis textbook (Molecular Cloning, Maniatis et al., i

Cold Spring Karbor, 1982). IN

The gene clone thus cloned is then inserted. IN

I under the control of vegetable regulation signals in plan I

For 35 hours, it is expressed. The European pa- I

In application No. 0.122.791 gives an overview of known I

In methods. Thus, PTC-resistant plant cells (i.e., I) are obtained

You have a selection characteristic of transformed cells), plants or parts of plants and seeds.

In the following examples, some embodiments of the invention are illustrated in detail. Percentages are 5 based on weight unless otherwise stated.

examples

The following media are used: a) for bacteria: • YT medium: 0.5% yeast extract, 0.8%; bacto-tryptone, 0.5%

NaCl, LB medium: 0.5% yeast extract, 1% bacto-tryptone, 1% NaCl as solid medium: each time addition of 1.5% agar, b) for plants: 15 M + S medium: see Murashige and Skoog, Physiologica

Plantarum 15 (1962) 473, 2MS medium: M + S medium with 2% sucrose, MSC10 medium: M + S medium with 2% sucrose, 500 mg / 1 cefotaxime, 0.1 mg / 1 naphthylacetic acid 20 ( NAA), 1 mg / 1 benzylaminopurine (BAP), 100 mg / 1 kanamycin, MSCl5 medium: M + S medium with 2% sucrose, 500 mg / 1 cefotaxime, 100 mg / 1 kanamycin.

1. Chemical synthesis of a single-stranded oligonucleotide.

As starting material for the synthesis of fragment II, which is one of the four sub-fragments I - IV, the terminated oligonucleotide Ile (nucleotides Nos. 219 to 312 in the coding strand of DNA sequence I) serves. For the solid phase synthesis, the nucleoside at the 3 'end, in the present case, ie guanosine (nucleotide # 312), is used covalently linked to a carrier via the 3' hydroxy function. The carrier material is functionalized CPG ("Controlled Pore Glass") with long chain amino groups. Moreover, the synthesis follows the known methods (from the European patent applications mentioned on page 3, lines 27-28).

The synthesis scheme is plotted in the DNA sequence II which

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otherwise similar to the DNA sequence I. I

2. Enzymatic binding of the single-stranded oligonucleotides

In times to the gene fragment II. IN

For phosphorylation of the oligonucleotides in 5'-I

At the terminal, each 1 nmol of oligonucleotides I is processed

I IIb and Ile with 5 nmol adenosine triphosphate and 4 units I

In T4 polynucleotide kinase in 20 µl of 50 mM Tris-HCl buffer I

(pH 7.6), 10 mM magnesium chloride and 10 mM dithio-I

In threitol (DTT) for 30 minutes at 37 ° C. The enzyme inactive- I

For two minutes, heat at 95 ° C for five minutes. Oligonucleo- I

times Ila and fire forming the "imminent" sequence I

H of the DNA fragment II is not phosphorylated. This will prevent you

I! by the subsequent ligation, formation of larger sub-frag- I

In gents other than those corresponding to the DNA fragment II. IN

The oligonucleotides II (a-d) are ligated as follows to I

In the sub-fragment II: every 1 nmol of the oligonucleotides IIa

I and Ild as well as the 5 'phosphates of Ilb and Ile are dissolved together I

I in 45 µl buffer containing 50 mM tris-HCl {pH I

I 7.6), 20 mM magnesium chloride, 25 mM potassium chloride and 10

20 mM DTT · For annealing the oligonucleotides of DNA-I

In fragment II, the solution of the oligonucleotides is heated

For 2 minutes to 95 ° C, then slowly (2-3 hours) H

cool to 20 ° C. For enzymatic binding, I is then added

In 2 µl 0.1 M DTT, 8 µl 2.5 mM adenosine triphosphate (pH I

7) and 5 µl of T4 DNA ligase (2000 Units) to the mixture, I

Incubate for 16 hours at 22 ° C. IN

I The purification of the gene fragment II occurs by gel leakage

In trophoresis on a 10% polyacrylamide gel (without addition I

of urea, 20 * 40 cm, 1 mm thick), thereby labeling I

In substance 30, ØX 174 DNA (Fa. BRL) cut H is used

In with HinfI, or pBR322, which is incised with Haelll. IN

In the preparation of the gene fragments I, III and IV I

occurs analogously thereto, however, the "imminent" sequences I

You are converted to the 5 'phosphates prior to annealing, since it I

In 35 no ligation step is necessary. IN

7 GB 175800 B1 3. Preparation of hybrid plasmids containing the gene fragments 1, II, III and IV.

a) Incorporation of gene fragment I into pUC18.

The commercially available plasmid pUC18 is opened in known manner 5 with the restriction endonucleases Sall and Xbal as described by the manufacturer. The digestion mixture is separated in a known manner on a 1% agarose gel by electrophoresis and the fragments are made visible by staining with ethidium bromide.

The plasmid bands (about 2.6 kb) are then excised from the agarose gel and separated from the agarose by electroelution.

1 µg of plasmid opened with XbaI and SalI is then ligated with 10 ng of the DNA fragment I overnight at 16 ° C.

b) Incorporation of the gene fragment II into pUC18.

Analogously to a), pUC18 is excised with XbaI and BamHI, and it is ligated with the gene fragment II, previously phosphorylated at the imminent end, as described in Example 2.

c) Incorporation of the gene fragment III into pOC18. ! Analogously to a), pOC18 is cut with BamHI and Xmalll, and then ligated with the gene fragment III.

d) Incorporation of gene fragment IV into pUC18.

Analogously to a), pUC18 is cut with. Xmalll. and Sall, and the gene fragment IV is ligated.

4. Construction of the complete gene and cloning into a pUC plasmid.

(a) Transformation and amplification of the gene fragments I - IV.

The hybrid plasmids thus obtained are transformed into E. coli. To this, the strain E. coli K 12 is made competent by treatment with a 70 mM calcium chloride solution, and to this is added the suspension of the hybrid plasmid in 10 mM tris - HCl buffer (pH 7.5), which is 70 mM with respect to calcium chloride . The transformed strains are commonly selected using the plasmid-transmitted antibiotic resistance or sensitivity, and the hybrid vectors are amplified. After killing the cells, the hybrid plasmids are isolated and cut up with those originally used

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restriction enzymes, after which the gene fragments I, II, III I

I and IV are isolated by gene electrophoresis. IN

I b) Binding of the gene fragments I, II, III and IV to an I

In all gen. IN

I The sub-fragments I and II I obtained by amplification

connected as follows. Every 100 ng of the isolated fragments

Iter I and II are dissolved together in 10 µl buffer containing I

In 50 mM Tris-HCl (pH 7.6), 20 mM magnesium chloride and 10 L

In mM DTT and this solution is heated for 5 minutes to 57 ° C. IN

The solution is then cooled to room temperature, where I

After adding to the mixture, 1 µl of 10 mM adenosine triplet is added

phosphate (pH 7) and 1 μ 1 T4 DNA ligase (400 units), and I

Incubate for 16 hours at room temperature. After you

In the post-cut with the restriction enzymes Sall and BamHI, I is purified

In the desired 312-bp fragment (nucleotides 1-312, SalI-I

I -BamHI) by gel electrophoresis on an 8% polyacrylamide gel, I

In which, as labeling substance, ØX 174 RF DNA I is used

I (Fa. BRL) cut with the restriction enzyme Haelll. IN

In the same way, the gene fragments III and IV I are linked

In 20 with each other, whereupon, after purification, a 246-I

I -bp fragment (nucleotides 313-558, Bam HI-SalI). As in

In marker by gel electrophoresis, pBR322 is used, which is I

In court with the restriction enzyme Mspl. IN

I To construct the entire gene (DNA sequence I), I is ligated

In 25 15 ng of the 312-bp fragment and 12 ng of the 246 bp fragment I

I as described above with 1 µg of the commercially available plasma

In mid pUC18 pre-excised with the restriction enzyme I

In Sal, which is dephosphorylated at the ends. After trans- I

In the formation and amplification (as described in Example I)

In Sala 4a), by SalI digestion, the correct clone I is identified

I with the 558 bp fragment of the DNA sequence I. I

In 5. Transformation of the hybrid plasmids. IN

In Competent E. coli cells, 0.1-1 I are transformed

In µg of the hybrid plasmid containing the DNA sequence I, I

I and the cells are spread on agar plates containing amp. I

In cillin. Next, clones containing the correct I

In integrated sequences in the plasmid, I is identified

9 DK 175800 B1 DNA rapid reprocessing (Maniatis in the aforementioned book).

6. Fusion of the synthesized gene: for regulatory signals recognized in plants.

The optimized resistance gene provided with SalI sites at the ends is ligated to the polylinker sequence.

SalI cross-section of plasmid pDH51 (Pietrzak et al. Nucleic Acids Res. 14 (1986) 5857). On this plasmid is located the initiator and terminator of the 35S transcript from the Cauliflower Mosaic Virus, which is recognizable by the 10 vegetable transcription apparatus.

When ligating the resistance gene, this is inserted behind the initiator and in front of the terminator of the 35S transcript.

The correct orientation of the gene is confirmed by restriction analysis.

The initiator of the ST-LS1 gene from Solanum tuberosum (Eckes et al., Mol. Gen. Gen. 205 (1986) 14) is also used to express the optimized acetyltransferase gene in plants.

7. Insertion of the resistance gene with the regulatory sequences into Agrobacterium tumefaciens.

a) Cointegrate method.

The total transcription unit for the initiator, optimized resistance gene and terminator (Example 6) is excised with the restriction enzyme EcoRI, and ligated into the intermediate E. coli vector pMPK11011 EcoRI cut site (Peter Eckes, Dissertation, Univ. Koln, 1985 , p. 91 f.). This intermediate vector is required to transfer the resistance gene with its regulatory sequences to the Ti plasmid from Agrobacterium tumefaciens. This so-called conjugation 30 is performed according to that of Van Haute et al. (J.0 J. 2 (1983) 411). Hereby, the gene is integrated with its regulation signals by homologous recombination via the sequences 35 of the standard vector pBR322 contained in the Ti plasmid in the pMPKUO vector and in the Ti plasmid pGV3850kanR (Jones et al., EMBO J. 4 (1985) 2411).

To this, every 50 µl of fresh broth cultures of E. coli strains DH1 (pMPKUO derivative

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host strain) and GJ23 (Van Haute et al., Nucleic Acids Res. I

H 14 (1986) 5857) on a dry YT agar plate and incubate I

for 1 hour at 37 ° C. The bacteria are resuspended in 3 ml of 10 L

H mM MgSO 4 and they are spread on antibiotic agar plates I

5 (spectinomycin 50 µg / ml: selection for pMPKUO, tetracycline I

clin 10 pg / ml: selection for R64drdll, kanamycin pg / ml: I

selection for pGJ28). The bacteria that grow on the selec- I

tive agar plates, they contain the three plasmids and they are amplified

are added to the conjugation with Agrobacterium turoefaciens in I

10 YT broth medium at 37 ° C. The agrobacteria are grown in I

H LB medium at 28 ° C. Every 50 µl of bacterial suspension together

mix on a dry YT agar plate and incubate for 12-16 L

hours at 28 ° C. The bacteria are resuspended in 3 ml of 10 mM I

MgSO4 and spread on antibiotic plates (erythromy- I

In 15 cin 0.05 g / l, chloramphenicol 0.025 g / l: selection for I

Agrobacterium strain; streptomycin 0.3 g / l and spectinomycin I

In 0.1 g / l: selection for the integration of pMPKUO into Ti plasm I

bromide). On these selective plates, only I can now grow

agrobacteria, wherein the pMPKUO derivative of a homo- I

20 log recombination is integrated into the bacterial Ti plasmid I

mid. IN

In addition to the pre-existing plants

active resistance gene against the antibiotic kanamy.cin is also .. I

the resistance gene against PTC now located on the Ti plasmid I

25 pGV3850canR. Before using these agrobacterial clones for I

H transformation, it is investigated using a "Southern-I

In the Blot study, check whether the desired integration has occurred

I b) Binary vector method. uL

The binary vector system described in I is used

In 30 by Koncz et al. i.Mol. Gen. Genet. 204 (1986) 383. The one of I

In Koncz et al. (PNAS 84 (1987) 131) described vector pPCV701 I

You change as follows: by means of restriction- I

In the BamHI and HindIII enzymes, a fragment is removed on which I

In the initiations TRI and TR2 i.a. is located, from the vector. IN

The resulting plasmid is cyclized again. In the presence- I

In the EcoRI section of this vector, an approx. 800

base pair long fragment from the vector pDH51, on which I-

11 DK 175800 B1 the putter and terminator of the 35S transcript from the Cauliflower Mosaic Virus is located (Pietrzak et al., Nucleic Acids Res. 14 (1986) 5858). The resulting plasmid pPCV801 has a singular SalI cut site between the 35S initiator and the terminator. In this section, the optimized PTC resistance gene is inserted. Its expression is now under the control of 35S transcript regulation sequences.

This plasmid (pPCV801Ac) is transformed into E. coli strain SM10 (Simon et al., Bio / Technology 1 (1983), 10,784). To transfer the plasmid pPCV801Ac to Agrobacterium tumefaciens, each 50 µl of the SM10 culture and a C58 agrobacterial culture (GV3101, Van Larebeke et al., Nature 252 (1974) 169) are mixed with the Ti plasmid PMP90RK (Koncz et al., Mol. Gen. Genet. 204 (1986) 383) i! 15 as an auxiliary plasmid on a dry YT agar plate and incubated for ca. 16 hours at 28 ° C. The bacteria are then resuspended in 3 ml of 1 mM MgSO 4 and spread on antibiotic plates (rifampicin 0.1 g / 1: selection for GV3101, kanamycin 0.025 g / l, selection for pMP90RK, carbenicillin 20 0.1 g / 1: selection for pPCV80lAc). On these plates, only agrobacteria containing both plasmids (pMP90RK and pPCV801Ac) can grow. Before using these agrobacteria for the plant transformation, it was investigated by Southern Blotting whether the plasmid pPCV801Ac is present in its correct form in the agrobacteria.

8. Transformation of Nicotiana tabacum via Agrobacterium tumefaciens.

The optimized resistance gene is transmitted by the so-called "leaf disc" transformation method to tobacco; 30 plants.

The agrobacteria are cultured in 30 ml of LB medium with the corresponding antibiotics at 28 ° C with still shaking (about 5 days). The bacteria are then sedimented by a 10 minute centrifugation at 7000 rpm in a Christ-35 centrifuge and washed once with 20 ml of 10 mM MgSO4.

After a further centrifugation, the bacteria are suspended in 20 ml of 10 mM MgSO 4 and transferred to a petri dish. To

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leaf disc infection leaves of sterile culture I are used

on 2MS medium growing Wisconsin 38 tobacco plants. All I

In sterile cultures, maintained at 25-27 ° C in a rhythm of 16 hours I

white light / 8 hours of darkness. IN

I 5 Leaves from the tobacco plant are cut off, 09 leaf surface I

wound with sandpaper. After wounding the leaves you cut

In those in smaller pieces, and they are dipped in the bacterial culture. Here

then the leaf pieces are transferred to M + S medium and kept

For 2 days under normal culture conditions. After two. days I

In infection with the bacteria, the leaves are washed in a float

In the M + 'S medium, they are transferred to MSC10 agar plates. Trans- I

In propagated sprouts are selected on the basis of the co-transferred I

In resistance to the antibiotic kanamycin. 3-6 Weeks Later I

You will see the first sprout visible. Some sprouts are re-cultivated

In 15 on MSC15 medium in glass containers. In the following weeks you form

In some of the cut, the root of the cut site sprouts. IN

Transformed plants can also be selected directly I

on PTC-containing plant media. By DNA analysis ("Southern I

Blotting ") and RNA analysis (" Northern Blotting ") of the I

20 transformed plants detect the presence and ex

the depression of the PTC resistance gene. IN

9. Detection of transformed plants PTC resistance. IN

To study the functionality of the resistance gene I

in transformed plants, leaf fragments are transmitted by I

25 transformed and non-transformed plants for M + S- I

-4 I

nutritional media with 1 · 10 M L-PTC. Fragments of non-trans

propagated plants die out, while on fragments of trans- I

propagated plants can regenerate new sprouts. Transform I

nest sprouts form root and grow effortlessly on M + S- I

30 nutrient media with 1 · 10-3 M L-PTC. Transformed plants I

planted under sterile conditions in soil and sprayed

at 2 kg / ha and 5 kg / ha PTC. While non-transformed plan- I

do not survive this herbicide treatment, they show

transformed plants none of the damage caused by the herbicide I

35 effects. The appearance and growth ratio of the sprayed, I

transformed plants are at least as good as they are

unsprayed control plants. H

DK 175800 B1 13 10. Acetyltransferase assay to detect PTC acetylation in transgenic, PTC resistant plants.

Ca. 100 mg of leaf tissue from transgenic, PTC resistant tobacco plants or from non-transformed tobacco plants 5 is homogenized in a buffer consisting of: 50 mM Tris / HCl, pH 7.5; 2 mM EDTA; 0.1 mg / l leupeptin; 0.3 mg / ml oxalic rum albumin; 0.3 mg / ml DTT; 0.15 mg / ml phenylmethylsulfonyl fluoride (PMSF).

After a subsequent centrifugation, 20 10 μΐ of the clear supernatant is incubated with a 1 μΐ 10 mM radiolabeled D, L-PTC and 1 μΐ 100 mM acetyl-CoA for 20 min at 37 ° C. Then, 25 µl of 12% trichloroacetic acid is added to the reaction mixture and frantaxifuged.

From the above liquid, 7 µ 7 is transferred to a thin-layer chromatography plate, and in a mixture of pyridine, n-butanol, acetic acid and water (50: 75: 15: 60 volumes) rise twice. Thus, PTC and acetyl-PTC are separated from each other and can be detected by autoradiography.

Non-transformed plants show no conversion of PTC 20 to acetyl-PTC while transgenic resistant plants are capable of doing so.

25 30 35

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Claims (10)

1. The resistance gene encoding the protein with the amino acid sequence I, using as the start codon ATG and I as the stop codon TGA, and the GC proportion of the gene is matched to the GC proportion of 5 plants. IN 2. A resistance gene according to claim 1, characterized. I B by DNA sequence I (nucleotide position 9-554). IN 3. Gene structure, characterized in that the DNA-I B sequence I is linked to plants active regulation and I10 expression signals. IN A vector characterized by the resistance gene I B according to claim 1 or 2. I The vector, characterized by a gene structure I B according to claim 3. I B 15 Vectors containing one or more DNA sequences IB selected from: IBI (nucleotide # 1-152) I fl I, I (nucleotide # 153-312) IB 'B III (nucleotide # 313-436) or II IV (nucleotide nos. 437-558). I B A host cell, characterized by a vector I B according to claims 4, 5 or 6. I Plant cell, characterized by a gene I B 25 according to claims 1, 2 or 3. I Plants, parts thereof and seeds, characterized in B by a gene according to claims 1, 2 or 3. I Use of the gene of claim 1 or 2 or * I B of the gene structure of claim 3 to provide phos- I B 30 phinothricin-resistant plant cells, plant parts, plants I B and seeds. IN