AU616416B2 - A phosphinothricin-resistance gene and the use thereof - Google Patents

A phosphinothricin-resistance gene and the use thereof Download PDF

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AU616416B2
AU616416B2 AU18605/88A AU1860588A AU616416B2 AU 616416 B2 AU616416 B2 AU 616416B2 AU 18605/88 A AU18605/88 A AU 18605/88A AU 1860588 A AU1860588 A AU 1860588A AU 616416 B2 AU616416 B2 AU 616416B2
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gene
resistance
ptc
ptt
plants
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AU18605/88A
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AU1860588A (en
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Alfred Puhler
Eckhard Strauch
Wolfgang Wohlleben
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Hoechst AG
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Hoechst AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Description

I
Form COMMONWEALT'd OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
Class j7 Int. Class Application Number: Lodged: 0 00 00 p 000 0 ao8oComplete Specification Lodged: 04 o )000 AroennteAc o a 0 oon 0 000 a 0 Priorit: 0 0 0009.0 Related Art: Published: 00
II,
I DO: 0 Name of Applicant: o 000 Address of Applicant: Actual Inventor: Address for Service: HOECHST AKTIENGESELLSCHAFT Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republic of Germany ECKHARD STRAUCH, WOLFGANG WOHLLEBEN and ALFRED PUHLER EDWD. WATERS SONS, Go QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the invention entitled: A PHOSPHINOTHRICIN-RESISTANCE GENE AND THE USE THEREOF The following statement is a full description of this invention, including the best method of performing it known to us i, i, ii 1 rspl~ .i;l iLii-.l liil HOECHST AWKTIENGESELLSCHAFT HOE 87/F 290J DrKL/mL A phosphinothricin-resistance gene and the use thereof Phosphinothric in (PTC, 2-amino-4-methyLphoqphinot tric acid) is a glutamine synthetase inhibitor. PTC is a "building block" of the antibiotic phosphinothricyl-aLany- L aLanine. This tripeptide (PTT) is active against Grampositive and Gram-negative bacteria as well as against the fungus Botrytiq cinerea (Bayer et al., Helv. C'im.
Acta 55 (1972) 224). PTT is produced by the strain Strepstomyces viridochromogenes Tu 494. This strain is deposited *jo the Germar microorganism collection, Mascheroder Weg S 10 1 b, D-3300 Bra rachweig, 'n the general collection and 0.0 is generally accessib'e under the number DSM 49736. Under the provisions of the Treaty of Budapest, the same strain o@@o00 was deposited anew on May 13, 1987; it is generally accessible under the number DSM 4112 (and is identified by this K 15 number hereinafter).
German Patent 2,717,440 discloses that PTC acts as a total herbicide. The published PCT application WO 86/02097 describes plants whose resistance to PTC is attributable to the fait that they overproduce glutamine synthetase.
e.r However, overproduction o this nature, for example resulting frorp gene amplification, entails the risk of instability. Thus, if there is such instability the overproduction of glutamine synthetase would decrease, and the competitive inhibitory action of PTC would reappear.
The (not pre-published) European Patent Application with the publication number (EP-A) 0,257,542 has already proposed i PTC-resistance gene which can be obtained from the complete DNA of Streptomyces viridochromogenes DSM 4112 which has been selected for PTT resistance, by cutting with B mHI, cloning of a fragment which is 4.0 kb in size, and selection for PTT resistance.
It has now been found that in the selection for PTT-resistant I .1 110
KC
I115 22 a0 10 C I I C j 20 000 I Y; 2 colonies of S. viridochromogenes DSM 4112, two phenotypes can be distinguished after prolonged incubation 1 week): Type I prevents in every case the background growth of sensitive colonies in its direct environment on PTT-containing medium. The 4.0 kb BamHI fragment proposed in EP-A 0,257,542 can be isolated from the complete DNA of these selectants. In contrast, non-resistant bacteria are able to grow slowly in an area of diameter about 5 mm around type II on selection medium (minimal medium containing PTT). The selectants of type II can be isolated pure by streaking single colonies. These selectants contain tht PTT-resistance ga according to the invention. It emerges from this that, in analogy to EP-A 0,257,542, this gene confers resistance to PTC, so that the term "PTC-resistance" is also used hereinafter. The invention also relates to the use of this gene for the production of PTC-resistant plants and as a marker, specifically as a PTT-resistance marker in bacteria and as a PTC-resistance marker in plant cells.
The invention furthermore relates to plant cells and plants and to the seeds and parts thereof which contain the resistance gene according to the invention.
25 The antibiotic PTT is taken up by bacteria and broken 4 0 down to PTC. The latter likewise inhibits the glutamine synthetase in bacteria, so that the bacteria die of glutamine deficiency. Hence, PTT-producing bacteria should have a mechanism which protects them from the action of PTT, that is to say either prevents the re-uptake of the PTT produced, or makes possible modification of the breakdown product PTC. However, surprisingly, tha PTT-producer S. viridochromogenes DSM 4112 is sensitive to its own antibiotic. It has been possible, however, by selection for PTT-resistance to find from among about 105 seleccants those which are resistant to P'T, inlcluding the selectants of type II according to the invention.
A gene bank was constructed from the DNA of these selectants 3 Sby the DNA being isolated, cleaved with Ncol and ligated into a Streptomycetes vector. The Ligation mixture was transformed into the commercially avaiLable strain S.
Lividans TK 23, resulting in about 5,000 to 10,000 transformants with an insert of about 1 to 5 kb in each 1 ig of Ligation mixture. Among the transformants there are PTT-resistant S. lividans strains. It was possible to show, .1 by isolation of the plasmid and retransformation into S.
Lividans, that the resistance is plasmid-encoded. The gene r' ponsible for the resistance is located on a 2.7 kb Ncol :,-agment (Figure This fragment contains no cleavage sites for the enzymes BamHI, BcLI, ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PstI, SmaI, SphI, SstI and XhoI.
SIt is additionally possible to isolate from the selectants S 15 of type II as proposed in EP-A 0,257,542 the PTC- Sresistance gene defined therein. Conversely, it was not possible under these conditions to isolate the resistance gene according to the invention from the seLectants of i type I.
Comparison with the restriction map of a resistance gene, which has not been characterized in detail, from S. hygroscopicus FERM BP-130/ATCC 21705 (EP-A 0,173,327, Figure 7; of. also the not pre-publishod EP-A 0,242,236 and 0,242,246, and WO 87/05 629, which relate to the same gene) shows that the resistance gene according to the invention is different from the known gene which was found during search for the PTT-biosynthesis genes. The same applies to the resistance gene proposed in EP-A 0,257,542.
The invention is explained in detail, in the Examples which follow. Unless indicated otherwise, parts and percentage data relate to weight.
Example 1: PTT-resistant selectants The strain S. viridochromogenes DSM 4112 was cultured on minimal medium (Hopwood et al., Genetic Manipulation uf Streptomyces, A Laboratory Manual, The John Innes Foundation,
L
,9 ii,
I
~i- 4 Norwich, England (1985), page 233), and increas'ng concentrations of PTT were added. At a concentration of 50 ug/ml two resistant colonies were found in about 105 colonies.
On prolonged incubation (7 days, 30 0 C) two different colony types were evident on the PTT-containing minimal medium plates. Type I intensively inhibits the background growth of sensitive colonies in its environment. The kb BamHI fragment proposed in EP-A 0,257,542 can be isolated from the complete DNA of these mutants.
Besides this, the selection plates also contain a second resistance type (type II). Sensitive colonies are able to grow on PTT-containing medium in the direct environment of these mutants. Resistant mutants can be distinctly 15 and reproducibly differentiated from the wild type which is inhibited even a concentrations of 5 pg/ml.
I I It Example 2: Isolation of the resistance gene 4 t T1.i complete DNA is isolated from the selectants of type II from Example 1, and it is cleaved with NcoI. The plasmid pGM4 (EP-A 0,257,417, Example 1, Figure 4) is likewise opened with NcoI and is treated with alkaline phosphatase. The two mixtures are then combined and Ligated.
o 25 The Ligation mixture is transformed into S. lividans TK 23 0 (obtainable fron the John Innes Foundation), with 5000 to 10,000 transformants with an insert of about 1 5 kb being obtained for each 1 pg of ligation mixture. Selection for PTT-resistance yields S. lividans colonies with the phenotyne II. The plasmid which has been taken up by the latter is isolated and cut with NcoI. A 2.7 kb NcoI fragment which carries the gene responsible for the resistance is found. This plasmid was called p'd81 (Figure 2).
It is 6.9 kb in size, i.e. the 0.55 kb NcoI fragment from pGM4 has been replaced by the 2.7 kb insert with the resistance rene. This insert is incorporated in both orientations and is cctive in each orientation.
It can be shown, by retransformation into S. lividans TK 23 a>: ii _i i ^i i 4 that the PTT-resistance is pLasmid-encoded, because the transformants grow on minimaL medium which contains pig/mL PTT.
0 0 0 aC a 0 a0 0 0 00 0 ii
I

Claims (10)

1. A phosphinothricin (PTC)-resistance gene located on a DNA fragment, obtained from the complete DNA of Streptomyces viridochromogenes DSM 4112 which has been selected for type II resistance, as hereinbefore defined, to phosphinothricy.lalanyl- alanine (PTT), by cutting with Ncol, cloning of a fragment which is 2.7 kb in size, and selection for PTT resistance.
2. A PTC-resistance gene located on a 2.7 kb DNA fragment out of the genome of .Sreptomyces viridochromogenes DSM 4112 of type II phenotype, as hereinbefore defined, which has the restriction map shown in Figure 1.
3. A process for the production of PTC-reslstant plants which comprises incorporating into the genome of the plant a gene as claimed in Claim 1,
4. A process for he production of PTC-resistant plants which comprises incorporating into the genome of the plant a gene as claimed in Claim 2.
5. A PTT-reslstanco marker In bacteria comprising the gone as claimed In Claim 1.
6. A PTT-resistance marker in bacteria comprising the gene as claimed in Claim 2.
7. A PTC-resistance marker In plant cells comprising the gene as claimed in Claim 1.
8. A PTC-resistanco marker In plant cells comprising the gene as claimed in Claim 2.
9. Plant cells, plants, and seeds and parts thereof, which contain a gene as claimed in Claim 1.
10. Plant cells, plants, and seeds and parts thereof, which contain a gene as claimed in Claim 2. DAT.ED this 9th day of August, 1991. HOECHST AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THEATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA DBM:KJS:JL N- i
AU18605/88A 1987-07-02 1988-07-01 A phosphinothricin-resistance gene and the use thereof Ceased AU616416B2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE3721840 1987-07-02
DE3721840 1987-07-02
DE19873732972 DE3732972A1 (en) 1987-07-02 1987-09-30 RESISTANCE GENES TO PHOSPHINOTHRICIN AND ITS USE
DE3732972 1987-09-30

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JP (1) JPS6430589A (en)
AU (1) AU616416B2 (en)
DE (1) DE3732972A1 (en)
DK (1) DK366988A (en)
FI (1) FI883142A (en)
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CN87100603A (en) * 1987-01-21 1988-08-10 昂科公司 Vaccines against melanoma

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU604743B2 (en) * 1986-08-23 1991-01-03 Bayer Cropscience Ag Phosphinothricin-resistance gene, and its use
AU609082B2 (en) * 1987-01-21 1991-04-26 Bayer Cropscience Ag Phosphinothricin-resistance gene active in plants, and its use

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Publication number Priority date Publication date Assignee Title
ES2018274T5 (en) * 1986-03-11 1996-12-16 Plant Genetic Systems Nv VEGETABLE CELLS RESISTANT TO GLUTAMINE SYNTHETASE INHIBITORS, PREPARED BY GENETIC ENGINEERING.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU604743B2 (en) * 1986-08-23 1991-01-03 Bayer Cropscience Ag Phosphinothricin-resistance gene, and its use
AU609082B2 (en) * 1987-01-21 1991-04-26 Bayer Cropscience Ag Phosphinothricin-resistance gene active in plants, and its use

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HUT47320A (en) 1989-02-28
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IL86954A0 (en) 1988-12-30
AU1860588A (en) 1989-01-05
JPS6430589A (en) 1989-02-01
DK366988A (en) 1989-01-03
EP0297618A2 (en) 1989-01-04
EP0297618A3 (en) 1990-02-28

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