EP1070120A1 - Adenylique-desaminase - Google Patents

Adenylique-desaminase

Info

Publication number
EP1070120A1
EP1070120A1 EP99919138A EP99919138A EP1070120A1 EP 1070120 A1 EP1070120 A1 EP 1070120A1 EP 99919138 A EP99919138 A EP 99919138A EP 99919138 A EP99919138 A EP 99919138A EP 1070120 A1 EP1070120 A1 EP 1070120A1
Authority
EP
European Patent Office
Prior art keywords
plant
amp deaminase
expression cassette
sequences
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99919138A
Other languages
German (de)
English (en)
Inventor
Jens Lerchl
Andreas Reindl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1070120A1 publication Critical patent/EP1070120A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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

Definitions

  • a first object of the present invention is a DNA sequence SEQ ID NO: 1 containing the coding region of a plant AMP deaminase from Arabidopsis thaliana (see Figure 2).
  • the invention also relates to functionally equivalent DNA sequences which code for an AMP deaminase gene and which, based on the total length of the gene, have a sequence homology with the DNA sequence SEQ ID NO: 1 of 40 to 100%.
  • the expression can take place specifically in the leaves, in the seeds or in other parts of the plant.
  • Such transgenic plants, their reproductive material as well 10 whose plant cells, tissues or parts are a further subject of the present invention.
  • the expression cassette according to the invention can also be used to transform bacteria, cyanobacteria, yeasts, filamentous fungi and algae with the aim of producing sufficient amounts of the enzyme AMP-deaminase.
  • Another object of the invention is a protein from Arabidopsis thaliana characterized by the amino acid sequence SEQ ID NO: 2 or derivatives or parts of this protein with AMP deaminase activity. Compared to Saccharomyces cerevisiae, the homology at the amino acid level is 43-47% identity (see Figure 4).
  • Vegetable proteins with AMP deaminase activity with an amino acid sequence homology to the Arabidopsis thaliana AMP deaminase of 80-100% identity are particularly preferred.
  • AMP deaminase is a suitable target for herbicides.
  • the complete cDNA sequence of the AMP deaminase from Arabidopis thaliana is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli (see Example 3).
  • the AMP deaminase protein expressed with the aid of the expression cassette according to the invention is particularly suitable for the detection of inhibitors specific for AMP deaminase.
  • test system Using the test system according to the invention, a large number of chemical compounds can be checked quickly and easily for herbicidal properties.
  • the method makes it possible to selectively reproducibly select those with great potency from a large number of substances, in order to subsequently carry out further in-depth tests known to the person skilled in the art.
  • the invention further relates to herbicides which can be identified using the test system described above.
  • the invention also relates to transgenic plants transformed with an expression cassette according to the invention, and to transgenic cells, tissues, parts and propagation material of such plants.
  • Transgenic crop plants such as e.g. Barley, wheat, rye, corn, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species, as well as legumes.
  • increasing the inosine-5 '-phosphate (IMP) content means for at least the artificially acquired ability of an increased IMP biosynthesis by functional overexpression of the AMP deaminase gene in the plant compared to the non-genetically modified plant a generation of plants.
  • the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked in order to avoid polymerase errors in constructs to be expressed.
  • RNA from plant tissues was isolated as in Logemann et al. ((1987) Anal. Biochem. 163, 21). For the analysis, 20 ⁇ g RNA were separated in a 1.5% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond, Amersham). The detection of specific transcripts was carried out as described for Amasino ((1986) Anal. Biochem. 152,
  • the cDNA fragments used as a probe were radioactively labeled with a random primed DNA labeling kit (Boehringer, Mannheim) and hybridized according to standard methods (see Hybond user instructions, Amersham). Hyridization signals were visualized by autoradiography using X-OMAT AR films from Kodak.
  • the agrobacterial strain LBA4404 (Clontech) or other suitable strains can be used.
  • the vectors pUC19 Yanish-Perron, Gene 33 (1985), 103-119) pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711-8720) and pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).
  • the reaction mixtures contained 8 ng / ⁇ l genomic DNA from Escherichia coli, 0.5 ⁇ M of the corresponding oligonucleotides, 200 ⁇ M nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C., 1.5 mM) MgCl 2 ) and 0.02 U / ⁇ l Taq polymerase (Perkin Elmer).
  • the amino acid sequence begins with the third base after the linker sequences (bold ends of the sequence in Figure 2) in the third reading frame and can be translated into an 860 amino acid polypeptide, or from the first methionine start codon into a polypeptide of 839 amino acids (see Figure 3). Alternatively, the methionine in position 46 can be used, so that a polypeptide of 824 amino acids would result.
  • oligonucleotide sequences were derived from the determined sequence and provided with a BamHI restriction site and two overhanging bases.
  • the oligonucleotides are underlined and numbered in Figure 2. Potential methionine start codons are shown in bold.
  • the PCR reaction mixtures contained 8 ng / ⁇ l pBS-AMPl DNA, 0.5 ⁇ M of the corresponding oligonucleotides, 200 ⁇ M nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C., 1, 5 mM MgCl 2 ) and 0.02 U / ⁇ l Taq polymerase (Perkin Elmer).
  • the amplification conditions were set as follows:
  • nucleotides 11749-11939 was isolated as a PvuII-HindiII fragment and after addition of Sphl -Line cloned to the PvuII interface between the SpHI-HindIII interface of the vector.
  • the plasmid pBinAR was produced (Höfgen and Willmitzer (1990) Plant Science 66, 221-230).
  • the PCR fragments I, II and III were cloned into the BamHI site of the vector pBinAR in both orientations and used to transform tobacco plants.
  • Regenerated shoots are obtained on 2MS medium with kanamycin and claforan, transferred to soil after rooting and after cultivation for two weeks in a climatic chamber in a 16 hour light / 8 hour dark rhythm at 60% humidity for foreign gene expression or altered metabolite contents and phenotypic 18th

Landscapes

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

Abstract

L'invention concerne un ADN codant un polypeptide ayant une activité d'adénylique-désaminase (EC 3.5.4.6, adénosine triphosphate aminohydrolase). L'invention concerne en outre l'utilisation de cet acide nucléique pour préparer un système de test.
EP99919138A 1998-04-01 1999-03-25 Adenylique-desaminase Withdrawn EP1070120A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19814512 1998-04-01
DE19814512 1998-04-01
PCT/EP1999/002016 WO1999050400A1 (fr) 1998-04-01 1999-03-25 Adenylique-desaminase

Publications (1)

Publication Number Publication Date
EP1070120A1 true EP1070120A1 (fr) 2001-01-24

Family

ID=7863184

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99919138A Withdrawn EP1070120A1 (fr) 1998-04-01 1999-03-25 Adenylique-desaminase

Country Status (5)

Country Link
EP (1) EP1070120A1 (fr)
JP (1) JP2002527039A (fr)
AU (1) AU3702199A (fr)
CA (1) CA2325054A1 (fr)
WO (1) WO1999050400A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2388851A1 (fr) * 1999-10-25 2001-05-03 Basf Aktiengesellschaft Synthase de formylglycinamidinribotide d'origine vegetale
DE10035084A1 (de) * 2000-07-17 2002-02-07 Aventis Cropscience Gmbh Nucleinsäuremolekül, das für eine pflanzliche AMP-Deaminase codiert
EP1487975A2 (fr) * 2002-03-20 2004-12-22 Basf Aktiengesellschaft Serine hydroxymethyltransferase en tant que cible d'herbicides
CN103805629B (zh) * 2014-01-28 2016-10-05 江南大学 鼠灰链霉菌amp脱氨酶基因的毕赤酵母真核表达方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9950400A1 *

Also Published As

Publication number Publication date
AU3702199A (en) 1999-10-18
CA2325054A1 (fr) 1999-10-07
JP2002527039A (ja) 2002-08-27
WO1999050400A1 (fr) 1999-10-07

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