WO2014183639A1 - Gène de l'oxygénase caceo, et protéine codée et son utilisation - Google Patents

Gène de l'oxygénase caceo, et protéine codée et son utilisation Download PDF

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Publication number
WO2014183639A1
WO2014183639A1 PCT/CN2014/077431 CN2014077431W WO2014183639A1 WO 2014183639 A1 WO2014183639 A1 WO 2014183639A1 CN 2014077431 W CN2014077431 W CN 2014077431W WO 2014183639 A1 WO2014183639 A1 WO 2014183639A1
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WIPO (PCT)
Prior art keywords
caceo
gene
oxygenase
acetochlor
oxygenase gene
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PCT/CN2014/077431
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English (en)
Chinese (zh)
Inventor
何健
陈青
李怡
闫新
蒋建东
洪青
黄星
李顺鹏
朱建春
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南京农业大学
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Publication of WO2014183639A1 publication Critical patent/WO2014183639A1/fr

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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/22—Organic substances containing halogen
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen

Definitions

  • the invention belongs to the field of applied environment microorganisms and agriculture, and relates to an oxygenase gene cace ⁇ and its encoded protein and application. Background technique
  • Chloroacetamide herbicides are a class of highly effective, highly selective contact herbicides that have a very significant killing effect on grass weeds. Chloroamide herbicides are one of the most widely used herbicides in the world. By 2007, the annual production and use area is second only to organophosphorus herbicides, ranking second in the world. Its main representative species is alachlor and B. Oxalamine and butachlor, of which acetochlor is most used. As the agricultural workforce decreases and the way agriculture is cultivated, the demand and use of such herbicides will continue to increase.
  • Chloroacetamide herbicides are chemically stable and have a long residual period in the soil, and such herbicides and their metabolites migrate from the soil into groundwater, causing pollution of domestic water sources. Studies have shown that some varieties of this herbicide are teratogenic and mutagenic. Alachlor, acetochlor and butachlor are classified as B-2 carcinogens by the US Environmental Protection Agency, and metolachlor is classified as C. Carcinogens (USEAP, 1994), therefore, residues of chloroacetamide herbicides in the environment and agricultural products are a serious hazard to human health.
  • Chloroacetamide herbicides are highly toxic to fish and 500-10,000 times more toxic than mammals, so the introduction of such herbicides into water bodies can cause serious damage to fishery resources.
  • Acetochlor and butachlor inhibited the soil microbial quantity and the growth rate of bacteria, actinomycetes and fungi in the soil, and significantly reduced soil microbial diversity.
  • chloroacetamide herbicides such as acetochlor and butachlor have serious phytotoxicity to crops, especially in sand-type soils with low organic matter content, or when the dosage is too large, or after continuous application. In high-humidity weather, it will seriously affect the growth of crops. In recent years, crop phytotoxicity caused by improper use of acetochlor and butachlor in China has occurred frequently, causing serious losses to agriculture.
  • chlorinated acetamide herbicide-degrading strains and degradation genes have the following functions and functions in the research and development of herbicide residues and the elimination of their phytotoxicity technology. (1) Introducing degradation genes into crops through modern biotechnology to construct corresponding degradation Chloroacetamide herbicide-resistant transgenic crops, (ii) herbicide chlorinated B in soil and water Elimination of residual intermediates in amide herbicides, (iii) Biotransformation for useful chemical products and drug synthesis. Therefore, the degradation gene has very important theoretical and practical value in eliminating the herbicide phytotoxicity and biotransformation. Summary of the invention
  • the object of the present invention is to provide a new oxygenase gene cace which degrades various chloroacetamide herbicides such as alachlor, acetochlor and butachlor against the above-mentioned deficiencies of the prior art.
  • Another object of the invention is to provide a protein encoding the gene.
  • a further object of the invention is to provide the use of the gene and its encoded protein.
  • An oxygenase gene caceft which degrades the chloroacetamide herbicide has the nucleotide sequence of SEQ ID NO.
  • the starting strain used in this patent is a bacterial strain capable of degrading chloroacetamide herbicide.
  • Sp. DC-2 (contained in the China-China Type Culture Collection, with the accession number CCTCCNO: M 2012190).
  • a mutant strain DC-2 MUT of this strain was obtained by multiple passages (preservation date 2013. 4.28, accession number: CCTCCNO: M 2013164).
  • the strain DC-2 MUT (CCTCCNO: M 2013164) lost the ability to degrade chloroacetamide herbicides such as alachlor, acetochlor and butachlor.
  • the first step in the degradation of acetochlor by strain DC-2 is the formation of 2-chloro-N-(2-ethyl-6-methylphenyl)acetamide under the catalysis of an oxygenase.
  • the acquisition of the oxygenase gene for the degradation of chloroacetamide herbicides was obtained using modern bioinformatics (technical route is shown in Figure 1). That is, by comparing the genomic information of the wild-type DC-2 and the mutant DC-2 MUT, the oxygenase gene which is present in the wild-type DC-2 genome but is deleted in the mutant DC-2 MUT is sought.
  • the total DNA of the wild-type DC-2 and the mutant DC-2 MUT was first extracted, and the total DNA was subjected to genome sequencing, assembly, and KEGG database alignment. The sequencing results showed that the wild-type DC-2 genome size was 6, 334, 837 bp, 481 scaffold; the mutant DC-2 MUT genome size was 6, 325, 634 bp, 892 scaffold.
  • the 1.2K fragment containing the gene was amplified from the wild strain by PCR and ligated into pBBR1MCS-5 to construct a recombinant vector, which was introduced into £ coli DH5 ⁇ by transformation, and a recombinant strain was obtained: coli DH5 a -CaceO
  • the recombinant vector containing the oxygenase gene was complemented to the mutant DC_2 MUT by triple-parent binding to obtain the recombinant strain DC-2 MUT-Cace0 (see Figure 2 for the technical scheme).
  • the protein Cace encoded by the oxygenase gene cac has an amino acid sequence of SEQ ID NO.
  • the chloroacetamide herbicide described therein is preferably one or more of alachlor, acetochlor or butachlor.
  • the chloroacetamide herbicide described therein is preferably one or more of alachlor, acetochlor or butachlor.
  • the use of the oxygenase gene cace ⁇ in the construction of transgenic crops resistant to chloroacetamide herbicides is preferably one or more of alachlor, acetochlor or butachlor.
  • the use of the oxygenase CaceO in the degradation of chloroacetamide herbicides is preferably one or more of alachlor, acetochlor or butachlor.
  • the chloroacetamide herbicide described therein is preferably one or more of alachlor, acetochlor or butachlor.
  • the starting strain of the present invention is a sphingolipid i Sphingobi DC-2 (CCTCCNO: M 2012190 ), and the strain DC-2 can degrade acetochlor to produce 2-ethyl-6-methylaniline; degrading alachlor and butyl grass The amine produces 2,6-diethylaniline.
  • CTCCNO sphingolipid i Sphingobi DC-2
  • the amine produces 2,6-diethylaniline.
  • the cloning of the oxygenase gene cace from the strain DC-2 to the degradation of alachlor, acetochlor and butachlor in the GenBank showed that the gene was a new gene, from the full length.
  • the start codon to the stop codon is 1047 bp and encodes 348 amino acids.
  • the gene is capable of degrading chloroacetamide herbicides and can be used in the construction of transgenic crops resistant to chloroacetamide herbicides.
  • the encoded protein of the gene can be used in the degradation of chloroacetamide herbicides or in the removal of chloroacetamide herbicide residues in soils and water bodies.
  • DRAWINGS Figure 1 Roadmap for cloning of oxygenated enzymes for degradation of chloroacetamide herbicides.
  • Figure 2 Oxygenase gene force energy verification technology roadmap.
  • Figure 3A is a liquid phase detection spectrum of acetochlor standard
  • B is a liquid phase detection map of the recombinant strain E. coli DH5 a -CaceO degrading acetochlor;
  • Figure C is a mass spectrum of the product in Figure B.
  • E is a liquid phase detection map of the recombinant strain E. coli DH5 a -CaceO degrading alachlor;
  • Figure F is a mass spectrum of the product in Figure E.
  • Figure G is the liquid phase detection spectrum of butachlor standard
  • H is a liquid phase detection map of the recombinant strain E. coli DH5 a -CaceO degrading butachlor;
  • Figure I is a mass spectrum of the product in Figure H. Biomaterial preservation information
  • Chloroacetamide herbicide-degrading bacteria DC-2 classified as Quisquiliarum DC_2, kept at the China Center for Type Culture Collection (CCTCC), Wuhan, China, Wuhan University, with the accession number CCTCC NO: M 2012190, with a deposit date of May 30, 2012.
  • the mutant DC-2 MUT named as ⁇ wV ⁇ sp. DC-2, is stored in the China Center for Type Culture Collection (CCTCC), Wuhan, China, Wuhan University, under the accession number CCTCC NO: M 2013164, date of preservation For April 28, 2013. detailed description
  • the water solubility of acetochlor at room temperature is 225 mg/kg, so the LB plate containing 400 mg/kg acetochlor produces turbidity, and the wild strain DC-2 (CCTCC NO: M 2012190) is streaked on LB plate (containing 400 mg/kg B).
  • LB plate containing 400 mg/kg B.
  • a colony did not produce a transparent circle around it. The colony was selected and cultured in 100 ml of LB liquid medium.
  • Bacterial genome sequencing requires a sample 0D value between 1.8 and 2.0. The higher the concentration, the better, the concentration is not less than 30 ng/ ⁇ , and the fine pattern requires at least 30 ⁇ g of sample. A sufficient amount of prepared DNA samples were sent to Meiji Biotech under dry ice.
  • Sample testing uses: 1. Concentration detection, agarose gel electrophoresis; 2. 0D260: 280 and 0D260/230 detection method: NanoDrop.
  • bioinformatics analysis including genomic sequence assembly, gene composition analysis, gene function annotation, and comparative genomic analysis.
  • the SOAPdenovo assembly software was used to assemble the reads data, and the scaffold sequence was obtained and related basic data statistics were obtained.
  • Gene annotation is based primarily on protein sequence alignments.
  • the sequence of the gene is compared with each database to obtain corresponding functional annotation information. Since each sequence may have many alignment results, in order to ensure its biological significance, we reserve the best results for the comparison as a comment for this gene. All notes are completed using BLAST software in conjunction with the characteristics of each database.
  • the version of BLAST is: blastall 2.2.21, the annotated protein library is: KEGG, GO, etc.
  • the wild-type DC-2 genome size was 6,334,837 bp, 481 scaffold, 388 scaffolds larger than 1000 bp, and 6612 ORFs predicted by ⁇ ? ⁇ ;
  • the mutant DC_2 MUT genome size was 6, 325, 634 bp, 892 scaffold, 704 scaffolds greater than lOOOObp, predicted a total of 6779 0RFs by ⁇ ? ⁇ ⁇ .
  • the 892 scaffold of the strain DC-2 MUT was compared with the wild strain DC_2 genome by 0MIGA3.0, and it was found that 50 fragments of the wild strain DC-2 genome were found in DC-2 but not in T.
  • the 20KB sequence may contain functional fragments that are lost during the passage.
  • the wild type DC-2 colony and the mutant strain DC-2 MUT colony were PCR-amplified according to the primers containing the cace fragment, and the corresponding fragment was amplified only in the wild strain DC-2.
  • the PCR product TA in 1.7 was cloned into pMD-18 T simple vector and sent to Nanjing Jinsui Biotechnology Co., Ltd. for sequencing. The result was 100% homologous to the predicted fragment.
  • the reaction was carried out for 10 hours or more in a 37 ° C water bath.
  • the digested product was subjected to 0.75% agarose gel electrophoresis and recovered.
  • the recombinant pBBR1MCS5_cace0 recombinant plasmid was transformed into E. coli DH5 ⁇ (purchased from TransGen Biotech) to obtain recombinant E. coli DH5 a -CaceO, and positive clones were picked.
  • E. coli DH5 a -CaceO complemented pBBR1MCS5-ca C e0 to the mutant DC-2 MUT (degraded acetochlor trait loss) with the aid of the helper E.
  • coli HB101 (pRK600) (purchased from Novegen) The binder was coated on an LB plate containing 100 mg/kg Str and 50 mg/kg Gm, and the grown single bacteria were verified by the extraction plasmid to obtain a positive clone DC-2 MUT-Cace0.
  • the basic salt medium formula is: 5.
  • the degradation effect was determined by high performance liquid chromatography.
  • the method was as follows: Firstly, an equal volume of dichloromethane was added to the above basic salt medium for extraction, and after shaking vigorously, the layer was allowed to stand, and then 1 ml of the lower layer of dichloromethane was evaporated. After completion, it was dissolved in 1 mL of methanol (chromatographically pure) and filtered through a filter (pore size 0.22 ⁇ m).
  • Liquid Chromatographic Conditions Mobile phase: methanol: water (80: 20, V/V), Zorbax C218 0DS Spherex reversed phase column (5 ⁇ , 4.
  • the ion detection mode is multi-reactive ion detection; the ion polarity is negative ion; the ionization mode is electrospray ionization; the capillary voltage is 4000 volts; the drying gas temperature: 33 CTC; the drying gas flow rate: 10.0 L/min, Atomizing gas pressure: 35 psi, collision voltage: 135 volts; mass scanning range (m/z): 300-500.
  • Secondary ion mass spectrometry conditions Collision voltage: 90 volts; mass scan range (m/z): 30-400.

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Abstract

L'invention porte sur un gène de l'oxygénase caceO et sur une protéine codée et son utilisation, la séquence du gène de l'oxygénase caceO étant représentée par SEQ ID NO:1, et la séquence du produit codé CaceO étant représentée par SEQ ID NO:2. Elle porte aussi sur l'utilisation du gène de l'oxygénase caceO pour la dégradation et la conversion de l'herbicide chloracétamide et la construction de cultures transgéniques, et sur l'utilisation de l'oxygénase CaceO pour la dégradation de l'herbicide chloracétamide et l'élimination de l'herbicide chloracétamide résiduel.
PCT/CN2014/077431 2013-05-16 2014-05-14 Gène de l'oxygénase caceo, et protéine codée et son utilisation WO2014183639A1 (fr)

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CN201310182539.9A CN103255154B (zh) 2013-05-16 2013-05-16 一种加氧酶基因caceO及其编码的蛋白质和应用
CN201310182539.9 2013-05-16

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CN103255154B (zh) * 2013-05-16 2014-07-02 南京农业大学 一种加氧酶基因caceO及其编码的蛋白质和应用
CN110004101A (zh) * 2019-04-15 2019-07-12 南京农业大学 用于为目标蛋白量身构建最优的枯草芽孢杆菌蛋白酶缺失表达宿主的方法
CN111440754B (zh) * 2020-03-19 2022-05-27 南京农业大学 一种利用基因工程改造的甲烷氧化菌消除土壤中有机污染物残留的方法

Citations (2)

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CN101688219A (zh) * 2007-05-09 2010-03-31 美国陶氏益农公司 新除草剂抗性基因
CN103255154A (zh) * 2013-05-16 2013-08-21 南京农业大学 一种加氧酶基因caceO及其编码的蛋白质和应用

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CN101688219A (zh) * 2007-05-09 2010-03-31 美国陶氏益农公司 新除草剂抗性基因
CN103255154A (zh) * 2013-05-16 2013-08-21 南京农业大学 一种加氧酶基因caceO及其编码的蛋白质和应用

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XU JUN ET AL., PROGRESSES ON DEGRADATION OF CHLOROACETANILIDE HERBICIDES, vol. 10, no. 3, 25 June 2004 (2004-06-25), pages 389 - 393 *

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