DE3628747A1 - Phosphinothricin resistance gene and its use - Google Patents

Phosphinothricin resistance gene and its use

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Publication number
DE3628747A1
DE3628747A1 DE19863628747 DE3628747A DE3628747A1 DE 3628747 A1 DE3628747 A1 DE 3628747A1 DE 19863628747 DE19863628747 DE 19863628747 DE 3628747 A DE3628747 A DE 3628747A DE 3628747 A1 DE3628747 A1 DE 3628747A1
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Prior art keywords
ptt
resistance
ptc
gene
fragment
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German (de)
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Eckhard Strauch
Wolfgang Dr Wohlleben
Walter Arnold
Renate Alijah
Alfred Prof Dr Puehler
Gerhard Dr Woehner
Ruediger Dr Marquardt
Susanne Dr Grabley
Dieter Dr Brauer
Klaus Dr Bartsch
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Hoechst AG
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Hoechst AG
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Priority to DE19863628747 priority Critical patent/DE3628747A1/en
Priority to DE19863642829 priority patent/DE3642829A1/en
Priority to DE19873700313 priority patent/DE3700313A1/en
Priority to DE19873701623 priority patent/DE3701623A1/en
Priority to ES198787112023T priority patent/ES2038631T3/en
Priority to AT87112023T priority patent/ATE75776T1/en
Priority to EP87112023A priority patent/EP0257542B1/en
Priority to DE87112023T priority patent/DE3778792D1/en
Priority to FI873610A priority patent/FI100251B/en
Priority to CA000545037A priority patent/CA1337597C/en
Priority to AU77318/87A priority patent/AU604743B2/en
Priority to DK198704378A priority patent/DK175254B1/en
Priority to NZ221526A priority patent/NZ221526A/en
Priority to IL8360487A priority patent/IL83604A/en
Priority to ZA876210A priority patent/ZA876210B/en
Priority to JP62209123A priority patent/JPH0797994B2/en
Priority to CN87105764A priority patent/CN1040772C/en
Priority to HU727/87A priority patent/HU217208B/en
Priority to DE19873737918 priority patent/DE3737918A1/en
Publication of DE3628747A1 publication Critical patent/DE3628747A1/en
Priority to JP2049140A priority patent/JP2749424B2/en
Priority to US07/736,316 priority patent/US5276268A/en
Priority to US07/795,275 priority patent/US5273894A/en
Priority to GR920401371T priority patent/GR3005200T3/el
Priority to JP6249627A priority patent/JPH07147985A/en
Priority to US08/479,717 priority patent/US5879903A/en
Priority to US08/487,695 priority patent/US5637489A/en
Priority to JP8268910A priority patent/JP2815847B2/en
Priority to CN98115017A priority patent/CN1124347C/en
Priority to CL200401481A priority patent/CL2004001481A1/en
Withdrawn legal-status Critical Current

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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8277—Phosphinotricin
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    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines

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Abstract

Selection of Streptomyces viridochromogenes DSM 40736 against phosphinothricyl-alanyl-alanine (PTT) results in PTT-resistant selectants. The DNA fragment which harbours the phosphinothricin (PTC) resistance gene is obtained from the complete DNA of these selectants by cutting with BamHI, cloning of a 4.0 kb fragment and selection for PTT resistance. This gene is suitable for producing PTC-resistant plants as well as resistance markers.

Description

Phosphinothricin (PTC, 2-Amino-4-methylphosphino­ buttersäure) ist ein Glutaminsynthetase-Inhibitor. PTC ist ein "Baustein" des Antibiotikums Phosphinothricyl-alanyl- alanin. Dieses Tripeptid (PTT) ist aktiv gegen Gram-positive und Gram-negative Bakterien und auch gegen den Pilz Botrytis cinerea (Bayer et al., Helv. Chim. Acta 55 (1972) 224). PTT wird von dem Stamm Streptomyces viridochromogenes Tü 494 (DSM 40 736) produziert.Phosphinothricin (PTC, 2-amino-4-methylphosphino butyric acid) is a glutamine synthetase inhibitor. PTC is a "building block" of the antibiotic phosphinothricyl-alanyl alanine. This tripeptide (PTT) is active against Gram-positive and Gram-negative bacteria and also against the fungus Botrytis cinerea (Bayer et al., Helv. Chim. Acta 55 (1972) 224). PTT is from the Streptomyces strain viridochromogenes Tü 494 (DSM 40 736) produced.

Aus der Deutschen Patentschrift 27 17 440 ist es bekannt, daß PTC als Totalherbizid wirkt. In der veröffentlichten PTC-Anmeldung WO 86/02 097 sind Pflanzen beschrieben, deren Resistenz gegen PTC darauf zurückzuführen ist, daß sie Glutaminsynthetase überproduzieren. Solche Überproduktionen, beispielsweise infolge einer Genamplifikation, bergen jedoch die Gefahr der Instabilität in sich. Im Falle einer solchen Instabilität ginge also die Überproduktion an Glutaminsynthetase zurück und die kompetitive Inhibitorwirkung des PTC käme wieder zum Zuge.From German patent specification 27 17 440 it is known that PTC acts as a total herbicide. In the published PTC application WO 86/02 097 describes plants whose Resistance to PTC is due to the fact that it Overproduce glutamine synthetase. Such Overproductions, for example as a result of a Gene amplification, however, carry the risk of instability in itself. In the event of such an instability, that would work Overproduction of glutamine synthetase and the competitive inhibitory effects of the PTC would come into play again.

Die Erfindung, die in den Patentansprüchen definiert ist, bezieht sich demgegenüber auf ein Resistenzgen gegen PTC und seine Verwendung zur Herstellung PTC-resistenter Pflanzen. Darüber hinaus kann dieses Gen auch als Resistenz-Marker Verwendung finden.The invention, which is defined in the claims, in contrast, refers to a resistance gene against PTC and its use in making PTC-resistant Plants. In addition, this gene can also be called Resistance markers are used.

Das erfindungsgemäße Resistenzgen gegen PTC ist erhältlich aus der Gesamt-DNA von auf PTT-Resistenz selektiertem Streptomyces viridochromogenes DSM 40 736 durch Schneiden mit BamHI, Klonieren eines 4,0 kb großen Fragments und Selektion auf PTT-Resistenz. Die Restriktionskarte (Fig. 1) charakterisiert dieses 4,0 kb-Fragment näher.The resistance gene according to the invention against PTC can be obtained from the total DNA of Streptomyces viridochromogenes DSM 40 736 selected for PTT resistance by cutting with BamHI, cloning a 4.0 kb fragment and selection for PTT resistance. The restriction map ( FIG. 1) characterizes this 4.0 kb fragment in more detail.

Das Antibiotikum PTT wird von Bakterien aufgenommen und zu PTC abgebaut. Dieses inhibiert bei Bakterien ebenfalls die Glutaminsynthetase, so daß die Bakterien an Glutaminmangel sterben. PTT-produzierende Bakterien sollten daher einen Mechanismus besitzen, der sie vor der Wirkung des PTT schützt, also entweder die Wiederaufnahme des produzierten PTT verhindert oder eine Modifikation des Abbauproduktes PTC ermöglicht. Überraschenderweise ist der PTT-Produzent S. viridochromogenes DSM 40 736 aber gegen sein eigenes Antibiotikum sensitiv. Unerwarteterweise gelang es aber, durch Selektion auf PTT mit der überraschend hohen Rate von etwa 10-5 Selektanten zu finden, die gegen PTT resistent sind und auch das Untergrundwachstum der benachbarten Kolonien unterdrücken.The antibiotic PTT is absorbed by bacteria and broken down to PTC. This also inhibits glutamine synthetase in bacteria, so that the bacteria die from a lack of glutamine. PTT-producing bacteria should therefore have a mechanism that protects them from the effects of PTT, that is, either prevents the resumption of the PTT produced or enables a modification of the PTC degradation product. Surprisingly, the PTT producer S. viridochromogenes DSM 40 736 is sensitive to its own antibiotic. However, unexpectedly, selection for PTT at the surprisingly high rate of about 10 -5 selectors found that are resistant to PTT and also suppress the background growth of the neighboring colonies.

Aus der DNA dieser Selektanten wurde eine Genbank angelegt, indem die DNA isoliert, mit BamHI gespalten und in einen Streptomycetenvektor ligiert wurde. Das Ligationsgemisch wurde in den handelsüblichen Stamm S. lividans TK 23 transformiert, wobei je 1 µg Ligationsgemisch etwa 5000 bis 10 000 Transformanten mit einem Insert von etwa 1 bis 5 kb erhalten wurden. Unter den Transformanten finden sich PTT-resistente S. lividans-Stämme. Durch Isolierung des Plasmids und Retransformation in S. lividans konnte gezeigt werden, daß die Resistenz plasmidcodiert ist. Das für die Resistenz verantwortliche Gen liegt auf einem 4 kb-BamHI- Fragment (Fig. 1). Der Codierbereich ist wahrscheinlich auf dem 850 bp BglII-Fragment lokalisiert. Das BamHI- Fragment enthält keine Schnittstellen für die Enzyme ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PvuI, PvuII und XhoI.A gene bank was created from the DNA of these selectants by isolating the DNA, cleaving with BamHI and ligating into a streptomycete vector. The ligation mixture was transformed into the commercially available strain S. lividans TK 23, with about 5 to 10 000 transformants with an insert of about 1 to 5 kb being obtained per 1 μg of ligation mixture. PTT-resistant S. lividans strains are among the transformants. Isolation of the plasmid and retransformation in S. lividans showed that the resistance was plasmid-encoded. The gene responsible for the resistance lies on a 4 kb BamHI fragment ( FIG. 1). The coding region is probably located on the 850 bp BglII fragment. The BamHI fragment contains no interfaces for the enzymes ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PvuI, PvuII and XhoI.

Der Vergleich mit der Restriktionskarte eines nicht näher charakterisierten Resistenzgens aus S. hygroscopicus FERM BP-130/ATCC 21 705 (Europäische Patentanmeldung mit der Veröffentlichungsnummer 01 73 327, Fig. 7) zeigt, daß das erfindungsgemäße Resistenzgen von dem bekannten Gen verschieden ist, welches auf der Suche nach den PTT-Biosynthesegenen gefunden wurde. The comparison with the restriction map of an unspecified resistance gene from S. hygroscopicus FERM BP-130 / ATCC 21 705 (European patent application with publication number 01 73 327, Fig. 7) shows that the resistance gene according to the invention is different from the known gene which in search of the PTT biosynthetic genes.

Durch Inkubation von Zellextrakten von S. viridochromogenes DSM 40 736 und S. lividans TK 23 einerseits und der PTT- resistenten S. viridochromogenes-Selektante und einer plasmidtragenden S. lividans-Transformante andererseits mit PTC und Acetyl-Coenzym A konnte gezeigt werden, daß die letztgenannten Zellen eine acetylierende Aktivität zeigen. Chromatographische Befunde zeigen, daß die Acetylierung an der Aminogruppe erfolgt.By incubating cell extracts from S. viridochromogenes DSM 40 736 and S. lividans TK 23 on the one hand and the PTT resistant S. viridochromogenes selectant and one plasmid-bearing S. lividans transformant on the other hand with PTC and acetyl coenzyme A it could be shown that the the latter cells show acetylating activity. Chromatographic findings indicate that acetylation the amino group.

Da auch in E. coli eine PTT-Resistenz festgestellt werden konnte und der Resistenzmechanismus somit auch in Gram- negativen Bakterien funktioniert, kann eine Resistenz auf Grund von Transportphänomenen ausgeschlossen werden. Das erfindungsgemäße Resistenzgen kann somit nach Kopplung an pflanzliche Promotoren mit geeigneten Vektoren in Pflanzen transformiert und es können so PTC-resistente Pflanzen hergestellt werden.Since PTT resistance is also found in E. coli could and the resistance mechanism thus also in gram negative bacteria works, resistance may arise Be excluded due to transport phenomena. The The resistance gene according to the invention can thus be coupled vegetable promoters with suitable vectors in plants transformed and so PTC-resistant plants getting produced.

In den folgenden Beispielen wird die Erfindung näher erläutert. Teile und Prozentangaben beziehen sich auf das Gewicht, sofern keine anderen Angaben gemacht werden.The invention is illustrated in the following examples explained. Parts and percentages refer to the Weight, unless otherwise stated.

Beispiel 1: PTT-resistente SelektantenExample 1: PTT-resistant selectants

Der Stamm S. viridochromogenes DSM 40 736 wurde auf Minmal- Medium (Hopwood et al., Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Norwich, England (1985), S. 233) angezogen und mit PTT in steigenden Konzentrationen versetzt. Bei einer Konzentration von 100 µg/ml wurde pro etwa 105 Kolonien eine resistente Kolonie gefunden.The strain S. viridochromogenes DSM 40 736 was grown on minimal medium (Hopwood et al., Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Norwich, England (1985), p. 233) and increasing with PTT Concentrations added. At a concentration of 100 µg / ml a resistant colony was found for every 10 5 colonies.

Beispiel 2: Herstellung des VektorsExample 2: Preparation of the vector

Das Plasmid pSVH1 (Europäische Patentanmeldung mit der Veröffentlichungsnummer 00 70 522) wird mit BGlII geschnitten, das etwa 7,1 kb große Fragment isoliert und mit dem 1,1 kb BclI-Fragment mit der Thiostrepton-Resistenz (Europäische Patentanmeldung mit der Veröffentlichungsnummer 01 58 201) ligiert. Man erhält das 8,15 kb große Plasmid pEB2 (Fig. 2).The plasmid pSVH1 (European patent application with publication number 00 70 522) is cut with BGlII, the approximately 7.1 kb fragment is isolated and with the 1.1 kb BclI fragment with thiostrepton resistance (European patent application with publication number 01 58 201) ligated. The 8.15 kb plasmid pEB2 is obtained ( FIG. 2).

Beispiel 3: Isolierung des ResistenzgensExample 3: Isolation of the resistance gene

Aus den Selektanten gemäß Beispiel 1 isoliert man die Gesamt-DNA und spaltet sie mit BamHI. Das Plasmid pEB2 wird ebenfalls mit BamHI geöffnet, die beiden Ansätze vereinigt und ligiert. Das Ligationsgemisch wird nach S. lividans TK 23 (erhältlich bei der John Innes Foundation) transformiert, wobei je 1 µg Ligationsgemisch 5000 bis 10 000 Transformanten mit einem Insert von etwa 1-5 kb erhalten werden. Selektion auf PTT ergibt 2 resistente S. lividans-Kolonien. Aus diesen wird das aufgenommene Plasmid isoliert und mit BamHI geschnitten. Man findet ein 4 kb BamHI-Fragment, welches das für die Resistenz verantwortliche Gen trägt. Dieses Plasmid erhielt die Bezeichnung pPR1 (Fig. 3).The total DNA is isolated from the selectants according to Example 1 and cleaved with BamHI. The plasmid pEB2 is also opened with BamHI, the two batches are combined and ligated. The ligation mixture is transformed according to S. lividans TK 23 (available from the John Innes Foundation), whereby 1 μg of ligation mixture gives 5000 to 10,000 transformants with an insert of about 1-5 kb. Selection for PTT results in 2 resistant S. lividans colonies. The plasmid taken up is isolated from these and cut with BamHI. A 4 kb BamHI fragment is found which carries the gene responsible for resistance. This plasmid was named pPR1 ( Fig. 3).

Durch Retransformation in S. lividans TK 23 kann gezeigt werden, daß die PTT-Resistenz plasmidcodiert ist, da die Transformanten auf Minimalmedium wachsen, das 100 µg/ml PTT enthält.By retransformation in S. lividans TK 23 can be shown be that the PTT resistance is plasmid-encoded because the Transformants grow on minimal medium, the 100 µg / ml PTT contains.

Beispiel 4: Nachweis der Inaktivierung von PTC durch N-AcetylierungExample 4: Evidence of inactivation of PTC by N-acetylation

Zum Nachweis der acetylierenden Aktivität des klonierten Fragments wurden folgende Stämme untersucht: S. viridochromogenes DSM 40 736, S. viridochromogenes (PTT- resistente Mutante), S. lividans TK23 und S. lividans TK 23 (pPR1).To demonstrate the acetylating activity of the cloned The following strains were examined: S. viridochromogenes DSM 40 736, S. viridochromogenes (PTT- resistant mutant), S. lividans TK23 and S. lividans TK 23 (pPR1).

Dazu werden die Stämme in Lysemedium A (Europäische Patentanmeldung mit der Veröffentlichungsnummer 01 58 872, S. 6) angeimpft und 2 Tage bei 30°C im Rundschüttler inkubiert. Nach der Ernte wird 1 mg Mycel in einem geeigneten Puffer (z. B. RS-buffer: C. J. Thompson et al., J. Bacetriol. 151 (1982), 678-658) mit Ultraschall aufgeschlossen. Ein typisches Experiment zur Messung des PTC-Abbaus verläuft folgendermaßen:For this purpose, the strains in Lysis medium A (European Patent application with publication number 01 58 872,  P. 6) inoculated and 2 days at 30 ° C in a rotary shaker incubated. After harvesting, 1 mg of mycelium is added in one suitable buffers (e.g. RS buffer: C. J. Thompson et al., J. Bacetriol. 151 (1982), 678-658) with ultrasound open minded. A typical experiment to measure the PTC dismantling proceeds as follows:

250 µl Rohextrakt werden mit 100 µl PTC-Lösung (250 µg/ml) und 50 µl Acetyl-CoA (4 mg/ml) versetzt und 2 Stunden bei 30°C inkubiert. Die dann noch vorhandenen PTC-Mengen wurden durch HPLC gemessen. Dabei ergibt sich folgendes Ergebnis:250 µl crude extract are mixed with 100 µl PTC solution (250 µg / ml) and 50 ul acetyl-CoA (4 mg / ml) and added for 2 hours Incubated at 30 ° C. The remaining PTC quantities were then measured by HPLC. The result is the following:

  • Stamm nicht umgesetztes PTC
    eingesetztes PTC
    S. lividans TK23 100%
    S. viridochromogenes 72%
    (DSM 40 736)
    S. viridochromogenes 7%
    Selektante
    S. lividans TK23 (pPR1) 31%    Strain not converted PTC
    used PTC
    S. lividans TK23 100%
    S. viridochromogenes 72%
    (DSM 40 736)
    S. viridochromogenes 7%
    Selective
    S. lividans TK23 (pPR1) 31%

Daß es sich um eine N-Acetylierung des PTC handelt, kann durch Vergleich mit Referenzsubstanzen in der Dünnschichtchromatographie nachgewiesen werden (keine Anfärbung durch Ninhydrin).That it is an N-acetylation of the PTC can by comparison with reference substances in the Thin layer chromatography can be detected (none Staining with ninhydrin).

Claims (5)

1. Resistenzgen gegen Phosphinothricin (PTC), erhältlich aus der Gesamt-DNA von auf Phosphinothricyl-alanyl-alanin (PTT)-Resistenz selektiertem Streptomyces viridochromogenes DSM 40 736 durch Schneiden mit BamHI, Klonieren eines 4,0 kb großen Fragments und Selektion auf PTT-Resistenz.1. Resistance gene against phosphinothricin (PTC), available from the total DNA from on phosphinothricyl-alanyl-alanine (PTT) resistance of selected Streptomyces viridochromogenic DSM 40 736 by cutting with BamHI, Cloning a 4.0 kb fragment and selection for PTT resistance. 2. Gen nach Anspruch 1, gekennzeichnet durch die Restriktionskarte gemäß Fig. 1.2. Gene according to claim 1, characterized by the restriction map according to FIG. 1. 3. Verwendung des Gens nach Anspruch 1 und 2 zur Herstellung PTC-resistenter Pflanzen.3. Use of the gene according to claim 1 and 2 for Production of PTC-resistant plants. 4. Verwendung des Gens nach Anspruch 1 und 2 als PTT-Resistenz-Marker in Bakterien.4. Use of the gene according to claim 1 and 2 as PTT resistance markers in bacteria. 5. Verwendung des Gens nach Anspruch 1 und 2 als PTC-Resistenzmarker in Pflanzenzellen.5. Use of the gene according to claim 1 and 2 as PTC resistance markers in plant cells.
DE19863628747 1986-08-23 1986-08-23 Phosphinothricin resistance gene and its use Withdrawn DE3628747A1 (en)

Priority Applications (29)

Application Number Priority Date Filing Date Title
DE19863628747 DE3628747A1 (en) 1986-08-23 1986-08-23 Phosphinothricin resistance gene and its use
DE19863642829 DE3642829A1 (en) 1986-08-23 1986-12-16 Phosphinothricin-resistance gene
DE19873700313 DE3700313A1 (en) 1986-08-23 1987-01-08 Use of a phosphinothricin-resistance gene
DE19873701623 DE3701623A1 (en) 1986-08-23 1987-01-21 Phosphinothricin-resistance gene effective in plants and its use
ES198787112023T ES2038631T3 (en) 1986-08-23 1987-08-19 PROCEDURE FOR OBTAINING A RESISTANCE GENE AGAINST PHOSPHINOTRICIN (PTC).
AT87112023T ATE75776T1 (en) 1986-08-23 1987-08-19 RESISTANCE GENE TO PHOSPHINOTHRICIN AND ITS USE.
EP87112023A EP0257542B1 (en) 1986-08-23 1987-08-19 Phosphinotricine resistance gene and its use
DE87112023T DE3778792D1 (en) 1986-08-23 1987-08-19 New gene for resistance to phosphinothricine
FI873610A FI100251B (en) 1986-08-23 1987-08-20 Phosphinothricin resistance gene and its use
CA000545037A CA1337597C (en) 1986-08-23 1987-08-21 Phosphinotricin-resistance gene, and its use
AU77318/87A AU604743B2 (en) 1986-08-23 1987-08-21 Phosphinothricin-resistance gene, and its use
DK198704378A DK175254B1 (en) 1986-08-23 1987-08-21 Phosphinothricin resistance gene sequence and its use
NZ221526A NZ221526A (en) 1986-08-23 1987-08-21 Phosphinothricin resistance gene, cloning and marker
IL8360487A IL83604A (en) 1986-08-23 1987-08-21 Phosphinothricin resistance gene obtainable from streptomyces viridochromogenes and its use
ZA876210A ZA876210B (en) 1986-08-23 1987-08-21 Phosphinothricin-resistance gene,and its use
JP62209123A JPH0797994B2 (en) 1986-08-23 1987-08-22 Phosphinothricin resistance gene and its use
CN87105764A CN1040772C (en) 1986-08-23 1987-08-22 Resistance gene of 2-amino-4-methylphosphine-butyric acid and its application
HU727/87A HU217208B (en) 1986-08-23 1987-08-23 Method for producing phosphinothricin resistence gene
DE19873737918 DE3737918A1 (en) 1986-08-23 1987-11-07 Phosphinothricin resistance gene to which is active in plants, and its use
JP2049140A JP2749424B2 (en) 1986-08-23 1990-02-28 Use of phosphinothricin resistance gene
US07/736,316 US5276268A (en) 1986-08-23 1991-07-29 Phosphinothricin-resistance gene, and its use
US07/795,275 US5273894A (en) 1986-08-23 1991-11-20 Phosphinothricin-resistance gene, and its use
GR920401371T GR3005200T3 (en) 1986-08-23 1992-07-16
JP6249627A JPH07147985A (en) 1986-08-23 1994-10-14 Use of phosphinothricin resistant gene
US08/479,717 US5879903A (en) 1986-08-23 1995-06-07 Phosphinothricin-resistance gene, and its use
US08/487,695 US5637489A (en) 1986-08-23 1995-06-07 Phosphinothricin-resistance gene, and its use
JP8268910A JP2815847B2 (en) 1986-08-23 1996-10-09 Use of phosphinothricin resistance gene
CN98115017A CN1124347C (en) 1986-08-23 1998-06-19 Method for obtg. 2-amino-4-methylphosphine butanoic acid gene
CL200401481A CL2004001481A1 (en) 1986-08-23 2004-06-15 RESISTANCE GENE USED IN PLANTS TO GENERATE PHOSPHINOTRICINE RESISTANCE, GENE STRUCTURE AND VECTOR THAT POSSESSES THE RESISTANCE GENE, VEGETABLE CELLS TRANSFORMED WITH THE RESISTANCE GENE AND PROCEDURES TO GENERATE CELLS AND PLANTS AND S

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