WO2013013585A1 - 噻吩磺隆水解酶基因tsmE及其应用 - Google Patents

噻吩磺隆水解酶基因tsmE及其应用 Download PDF

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WO2013013585A1
WO2013013585A1 PCT/CN2012/078765 CN2012078765W WO2013013585A1 WO 2013013585 A1 WO2013013585 A1 WO 2013013585A1 CN 2012078765 W CN2012078765 W CN 2012078765W WO 2013013585 A1 WO2013013585 A1 WO 2013013585A1
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thifensulfuron
tsme
gene
hydrolase
methyl
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PCT/CN2012/078765
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French (fr)
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李顺鹏
何健
杭宝建
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南京农业大学
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Priority to US14/234,824 priority Critical patent/US9669247B2/en
Publication of WO2013013585A1 publication Critical patent/WO2013013585A1/zh

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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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/8242—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
    • C12N15/8259—Phytoremediation
    • C—CHEMISTRY; METALLURGY
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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
    • 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
    • C12N15/8278—Sulfonylurea
    • 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)
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • 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
    • A62D2101/26—Organic substances containing nitrogen or phosphorus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/36—Organic compounds containing halogen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/40—Organic compounds containing sulfur

Definitions

  • the invention belongs to the field of applied environment microorganisms and agriculture, and relates to the thifensulfuron hydrolase gene mE and its application.
  • Thiosulfuron is a widely used class of sulfonylurea herbicides.
  • Sulfonylurea herbicides have a long residual period, and accumulation in soil can cause serious phytotoxicity to the crops. It has been found that the residual phytotoxicity of sulfonylurea herbicides can damage rice, soybean, corn and rape. , cotton, beets, flax and sunflowers and other important crops.
  • sulfonylurea herbicide-degrading strains and degradation genes have the following functions and functions in the research and development of herbicide residues and eliminating the phytotoxicity technology. (1) Introducing degradation genes into crops through modern biotechnology to construct corresponding herbicide resistance GM crops, (2) Degradation of sulfonylurea herbicide residues in soil by degrading sulfonylurea herbicide-degrading strains and genes by modern microbial fermentation technology. In addition, sulfonylurea herbicide degradation genes can also be used for biotransformation of useful chemical products and drug synthesis. Therefore, the thifensulfuron-degrading gene has very important theoretical and practical value in eliminating the herbicide phytotoxicity and biotransformation.
  • the sulfonylurea herbicide thifensulfuron has been widely used, but its residual period is longer. Accumulation in the soil will have serious phytotoxic effects on the sorghum crop, and the construction and planting of herbicide-tolerant transgenics will solve the herbicide phytotoxicity. The best way. The current gene that can degrade thifensulfuron has not been reported.
  • the object of the present invention is to provide a thifensulfuron hydrolase gene which can be used for constructing a transgenic crop resistant to thifensulfuron, and can also be used for the herbicide thifensulfuron-methyl and high efficiency in soil and water, aiming at the above-mentioned deficiencies of the prior art.
  • the removal of the grass can be removed and the biotransformation of the drug synthesis.
  • Another object of the invention is to provide the use of this gene.
  • the starting strain used in this patent is a bacterial strain S113 capable of degrading thifensulfuron, which belongs to the class of Methylophilus (Methylophilus sp.), preserved in China General Microorganisms Collection and Management Center, the deposit number is CGMCC 1479, and the deposit date is October 12, 2005. Mass spectrometry analysis showed that the crude enzyme solution of strain S113 could hydrolyze thifensulfuron to sulfinic acid.
  • the strategy used to clone the thienosulfuron hydrolase gene is the shotgun method (see Figure 1).
  • the total DNA of strain S113 was extracted.
  • the total DNA was partially digested and ligated with BflmH/digested plasmid pUC118.
  • the enzyme was ligated into E. coli DH10B competent cells to construct the total DNA library of strain S113, which contained thifensulfuron-degraded.
  • the cloning of the gene can degrade the thifensulfuron in the medium, and the resulting sulfonic acid does not inhibit the growth of Escherichia coli, and the growth of Escherichia coli is inhibited.
  • Clones that do not contain the thifensulfuron-degrading gene are unable to grow due to inhibition by thifensulfuron. This cloning method allows high-throughput screening of the library.
  • the thifensulfuron hydrolase protein TsmE encoded by the thiophenesulfuron hydrolase enzyme gene mE nucleotide sequence has the amino acid sequence of SEQ ID N0.2.
  • the recombinant expression vector preferably inserts the thifensulfuron hydrolase gene tsmE into the NcM of pET-29a(+) and
  • the genetically engineered bacteria preferably have Escherichia coli BL21 (DE3) as a starting strain.
  • thifensulfuron hydrolase gene mE in the construction of a transgenic crop resistant to thifensulfuron.
  • thifensulfuron hydrolase gene mE for the degradation of thifensulfuron and efficient grass cover energy.
  • thifensulfuron hydrolase protein TsmE for degrading thifensulfuron and efficient grass cover energy.
  • thifensulfuron hydrolase protein TsmE for removing herbicide thifensulfuron residues and efficient grass cover energy in soil and water.
  • the thifensulfuron hydrolase gene mE was successfully cloned from strain S113 (CGMCC 1479) by the shotgun method of the present invention.
  • the result of the GenBank alignment indicated that the gene was a novel gene with a full length (from the start codon to the stop codon) of 1194 bp, a G+C content of 51.09%, and a coding sequence of 398 amino acids.
  • the thifensulfuron hydrolase TsmE provided by the present invention can completely degrade 100 mg/L of thifensulfuron in 1 hr and hydrolyze thifensulfuron to the herbicidal product thiophenesulfonic acid (see Figures 4 and 5).
  • mE can be used to construct transgenic crops resistant to thifensulfuron. It can also be used for the removal of herbicide thifensulfuron and high-efficiency herbicide residues in soil and water, and biotransformation of drug synthesis. It has very important theoretical and practical value.
  • Figure 1 Strategy map of the thiophenesulfuron hydrolase gene mE clone.
  • FIG. 2 Schematic diagram of the expression of thifensulfuron hydrolase gene tsmE in BL21 (pET-29a(+) ).
  • Lane 1 is a protein marker and lane 2 is a purified thifensulfuron hydrolase TsmE protein.
  • FIG. 4 Thiosulfuron-methyl and high-efficiency turf hydrolase TsmE degradation of thifensulfuron LC-MS;
  • A liquid chromatogram of thifensulfuron-methyl and high-efficiency turf hydrolase TsmE degradation of thifensulfuron;
  • A First-order mass spectrum of thifensulfuron-methyl and high-efficiency turf hydrolase TsmE degradation.
  • FIG. 7 Thiosulfuron-methyl and high-efficiency turf hydrolase TsmE degrading efficient grass cover.
  • Methylophilus sp. (Methylophilus sp.), kept in China General Microorganisms Collection and Management Center (CGMCC), located at No. 3, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC 1479 The deposit date is October 12, 2005.
  • CGMCC General Microorganisms Collection and Management Center
  • the total DNA after digestion was purified by electrophoresis (TAE buffer), recovered by axygen biosciences (China; 0 collection kit, and the recovered DNA was dissolved in 10 mmol/L of Tris_Cl (pH 8.0), placed in - Deposited at 20 ° C.
  • TAE buffer electrophoresis
  • Tris_Cl pH 8.0
  • E. coli DH10B was purchased from Shanghai Yingjun Biotechnology Co., Ltd. Specific methods for the preparation of highly competent cells are described in F. Osbour, et al., Guide to the Guide to Molecular Biology, P 22-23.
  • the transformant obtained in 1.6 capable of converting thifensulfuron to sulfinic acid is sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing, and the nucleotide sequence of the thifensulfuron hydrolase gene is SEQ ID N0.1, according to The 398 amino acid sequence to which the nucleotide sequence of the thifensulfuron hydrolase gene is derived is SEQ ID N0.2.
  • Example 2 Iridoid expression of thiophenesulfuron hydrolyzed in BL21 (pET-29a(+)) (Fig. 2)
  • Primer star ⁇ $ ( 5 ⁇ 7 ⁇ 1) 0.5 ⁇
  • the reaction was carried out for 3 hours or more in a 37 ° C water bath.
  • the digested product was subjected to 2% agarose gel electrophoresis.
  • the recovered fragment in 2.2 was cleaved with the pET-29a(+) cleaved in 2.3 (Ref. 1.5).
  • the pET-29a(+) recombinant plasmid containing the thienosulfonate hydrolase gene was transformed into the expression host BL21 (DE3) to obtain the recombinant microorganism BL21 (TsmE).
  • BL21 (TsmE) was cultured in LB medium to an OD600nm of 0.6 to IJO.8, and IPTG to a concentration of lmM, and cultured at 30 ° C for 4 hours. Centrifuge 100 ml of the bacterial solution, resuspend the cells in 10 ml (50 mM, pH 7.0) PBS buffer, sonicate (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5 minutes, centrifuge, collect the supernatant, use nickel ions Affinity layer The column was purified by TsmE, and the purified enzyme was subjected to protein electrophoresis, as shown in Fig. 3.
  • Enzyme active reaction system Harvesting phosphate buffer ( ⁇ « ), followed by thiophenesulfuron or subsequent » high-efficiency grass cover, reaction enzyme amount (blocking + purification), 3 ⁇ 4 reaction lift. Each reaction was started with the addition of enzymes. The reaction was terminated with methylene chloride. After separation, the organic phase was dehydrated with anhydrous sodium sulfate. The content of thifensulfuron or high-efficiency grass was determined by reversed-phase HPLC (see » for specific methods).
  • An enzyme activity unit ( ) is defined as: the amount of enzyme required to catalyze the reduction of thifensulfuron or high-efficiency grass at a temperature of « conditions. Degradation tests show that the total degradation can be degraded in the total. Thiosulfuron-methyl or high-efficiency turfgrass, enzymatic tests show that the specific enzyme activity for thifensulfuron-methyl and high-efficiency turfgrass is 3 ⁇ 4 ⁇ 3 ⁇ 43 ⁇ 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
  • the LC-MS chromatogram shows that the product has a retention time of 1.95 min.
  • the first-order mass spectrum shows that it has a m/z of 372.30 molecular anion peak with a m/z of 372.30.
  • the molecular anion of the molecular anion has fragments of m/z 162.10, 188.10, 206.20, which is consistent with sulfonic acid. Therefore, the biochemical reaction of TsmE hydrolyzed thifensulfuron is the conversion of thifensulfuron to sulfinic acid (see Figure 5).
  • tandem mass spectrometry was used to determine the high-efficiency sclerotin degradation of metabolites.
  • the dichloromethane extract of the high-efficiency turfgrass reaction solution in 2ml of 2.6 was dried by nitrogen and dissolved in ⁇ methanol for tandem mass spectrometry.
  • the MS/MS primary mass spectrum (see Figure 6A) has a molecular negative ion peak with m/z of 359.87 and a molecular negative ion with m/z of 359.87.
  • the secondary mass spectrum (see Figure 6B) has a m/z of 287.60. Fragment, which is consistent with the metabolite 2-[4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy]propanoic acid, a highly potent herbicide capable of TsmE hydrolysis.
  • pUC118 ( ⁇ leg HI) was purchased from Bao Bioengineering (Dalian) Co., Ltd.
  • E. coli DH10B was purchased from Shanghai Yingjun Biotechnology Co., Ltd.
  • E. coli high expression vector pET-29a(+) was purchased from Novegen.
  • the expression host Escherichia coli BL21 (DE3) was purchased from Shanghai Yingjun Biotechnology Co., Ltd.

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Abstract

提供了噻吩磺隆水解酶基因tsmE及其应用。噻吩磺隆水解酶基因tsmE，其核苷酸序列为SEQ ID N0:1，全长1194bp，G+C含量为51.09%，其编码全长389个氨基酸的多肽，所述氨基酸序列为SEQ ID NO:2。提供的噻吩磺隆水解酶基因tsmE能在1小时内完全降解100mg/L的噻吩磺隆，并将其水解为无除草活性的产物噻吩磺酸；此外，tsmE还能在1小时内完全降解100mg/L的除草剂高效盖草能。还提供了噻吩磺隆水解酶基因tsmE在构建抗噻吩磺隆的转基因作物中的应用，噻吩磺隆水解酶TsmE在降解噻吩磺隆和高效盖草能中的应用。

Description

说明书
噻吩磺隆水解難因 及其細
技术领域
本发明属于应用环境微生物和农业领域， 涉及噻吩磺隆水解酶基因 mE及其应用。
背景技术
除草剂的使用在减轻农业劳动强度、保证农业正常生产的同时，其残留也带来了严重的作 物药害问题，据统计我国每年农田受除草剂药害面积达到 3000万亩，其中严重药害面积达到 500 万亩， 每年造成几十亿元的损失， 而抗除草剂转基因是解决除草剂药害的最佳途径。磺酰脲类 除草剂在中国使用量大， 研究和应用发展迅速， 已经成为继有机磷、 乙酰胺类除草剂后的第三 大除草剂， 全球年销售额达到 30亿美元以上， 我国磺酰脲类除草剂每年的应用面积已超过 200 万公顷， 并仍呈扩大的趋势， 噻吩磺隆是使用非常广泛的一类磺酰脲类除草剂。磺酰脲类除草 剂残留期较长，在土壤中积累会对下茬作物产生严重的药害作用，现已发现磺酰脲类除草剂的 残留药害可伤及水稻、 大豆、 玉米、 油菜、 棉花、 甜菜、 亚麻和向日葵等多种重要作物。
获得磺酰脲类除草剂降解菌株和降解基因在治理除草剂残留，消除其药害技术研发中具有 以下作用和功能， （一） 通过现代生物技术将降解基因导入作物构建相应的除草剂抗性转基因 作物， （二） 通过现代微生物发酵技术将磺酰脲类除草剂降解菌株和基因制成降解菌剂或酶制 剂将土壤中磺酰脲类除草剂残留降解。此外磺酰脲类除草剂降解基因还可用于有用化工产品及 药物合成的生物转化。因此噻吩磺隆降解基因在消除该类除草剂药害及生物转化领域中具有非 常重要的理论和应用价值。
磺酰脲类除草剂噻吩磺隆应用日益广泛， 但其残留期较长， 在土壤中积累会对下茬作物产 生严重的药害作用， 而构建和种植抗除草剂转基因是解决除草剂药害的最佳途径。 目前能够降 解噻吩磺隆的基因还未见报道。
发明内容
本发明的目的是针对现有技术的上述不足，提供一种噻吩磺隆水解酶基因， 该基因可用于 构建抗噻吩磺隆的转基因作物， 也可用于土壤、水体中除草剂噻吩磺隆和高效盖草能残留的去 除及药物合成的生物转化。
本发明的另一目的是提供该基因的应用。
本发明的目的通过如下技术方案实现：
一种噻吩磺隆水解酶基因 t E, 其核苷酸序列为 SEQ ID N0.1。
本专利所用的出发菌株为一株能够降解噻吩磺隆的细菌菌株 S113 ,在分类上属于嗜甲基菌 (Methylophilus sp. ) ， 保存在中国普通微生物菌种保藏管理中心， 保藏编号为 CGMCC 1479， 保藏日期为 2005年 10月 12日。质谱分析结果表明菌株 S113的粗酶液可以把噻吩磺隆水解为噻磺 酸。
克隆噻吩磺隆水解酶基因采取的策略为鸟枪法 （见图 1 )。 首先提取菌株 S113的总 DNA， 总 DNA采用 部分酶切后和用 BflmH/酶切的质粒 pUC118酶连， 酶连产物转化大肠杆菌 DH10B感受态细胞构建菌株 S113的总 DNA文库， 含有噻吩磺隆降解基因的克隆子能使培养基 中的噻吩磺隆降解， 生成的噻磺酸不抑制大肠杆菌生长， 解除对大肠杆菌的抑制而生长。不含 噻吩磺隆降解基因的克隆子则因受到噻吩磺隆的抑制而不能生长。利用这种克隆方法可以对文 库进行高通量的筛选。
用上面的策略筛选鸟枪法构建的基因文库获得一个能在加入 lOppm噻吩磺隆的基础盐培 养基 (葡萄糖为碳源)上生长的阳性克隆子， 进一步的降解实验表明该阳性克隆子能降解噻吩磺 隆。测序结果表明该阳性克隆子含有 5143个碱基对，其中包含有 18个潜在的 ORF (大于 150bp)， 对这些潜在的 ORF分别进行亚克隆和序列比对分析，最后确定编码噻吩磺隆水解酶的基因的大 小为 1194kb， 命名为 nE。 这是首次克隆到能降解磺酰脲类除草剂的水解酶基因。
所述的噻吩磺隆水解酶酶基因 mE核苷酸序列所编码的噻吩磺隆水解酶蛋白质 TsmE，其 氨基酸序列为： SEQ ID N0.2。
含有所述的噻吩磺隆水解酶基因 t E的重组表达载体。
所述的重组表达载体优选将所述的噻吩磺隆水解酶基因 tsmE插入 pET-29a(+)的 NcM和
H /¾flII位点之间所得。
含有所述的噻吩磺隆水解酶基因 t E的基因工程菌。
所述的基因工程菌优选以大肠杆菌 BL21(DE3) 为出发菌株。
所述噻吩磺隆水解酶基因 mE在构建抗噻吩磺隆的转基因作物中的应用。
所述噻吩磺隆水解酶基因 mE在降解噻吩磺隆和高效盖草能中的应用。
所述噻吩磺隆水解酶蛋白质 TsmE在降解噻吩磺隆和高效盖草能中的应用。
所述噻吩磺隆水解酶蛋白质 TsmE在去除土壤、水体中除草剂噻吩磺隆残留和高效盖草能 中的应用。
本发明的有益效果如下：
1. 本发明用鸟枪法成功的从菌株 S113 ( CGMCC 1479) 中克隆出噻吩磺隆水解酶基因 mE。 在 GenBank比对结果表明该基因为一个新的基因， 全长 （从起始密码子到终止密码子） 为 1194 bp, G+C含量为 51.09%， 编码 398个氨基酸。 2. 本发明提供的噻吩磺隆水解酶 TsmE能在 lhr内完全降解 100 mg/L的噻吩磺隆，并将噻吩 磺隆水解为无除草活性的产物噻吩磺酸（见图 4和图 5)，此外 还能在位 内完全降解 优 的除草剂高效盖草能 （见图 6和图 7)。 mE可用于构建抗噻吩磺隆的转基因作物， 也可 用于土壤、水体中除草剂噻吩磺隆和高效盖草能残留的去除及药物合成的生物转化， 具有非常 重要的理论和应用价值。 附图说明
图 1 噻吩磺隆水解酶基因 mE克隆的策略图。
图 2 噻吩磺隆水解酶基因 tsmE在 BL21 (pET-29a(+) ) 中表达策略图。
图 3 噻吩磺隆水解酶 TsmE蛋白电泳图谱；
其中泳道 1为蛋白质 marker,泳道 2为纯化的噻吩磺隆水解酶 TsmE蛋白。
图 4 噻吩磺隆和高效盖草能水解酶 TsmE降解噻吩磺隆 LC-MS图；
A: 噻吩磺隆和高效盖草能水解酶 TsmE降解噻吩磺隆的液相色谱图；
B : 噻吩磺隆和高效盖草能水解酶 TsmE降解噻吩磺隆的一级质谱图；
C: 噻吩磺隆和高效盖草能水解酶 TsmE降解噻吩磺隆的子离子二级质谱图。
图 5 噻吩磺隆和高效盖草能水解酶 TsmE降解噻吩磺隆的途径。
图 6 噻吩磺隆和高效盖草能水解酶 TsmE降解高效盖草能 MS/MS图。
A: 噻吩磺隆和高效盖草能水解酶 TsmE降解高效盖草能的一级质谱图。
B : 噻吩磺隆和高效盖草能水解酶 TsmE降解高效盖草能的二级质谱图。
图 7 噻吩磺隆和高效盖草能水解酶 TsmE降解高效盖草的途径。 生繊料纖信息
嗜甲基菌 S113 (Methylophilus sp. )，保存在中国普通微生物菌种保藏管理中心（CGMCC) , 地址为北京市朝阳区北辰西路 1号院 3号中国科学院微生物研究所， 保藏编号为 CGMCC 1479, 保藏日期为 2005年 10月 12日。 具体实fc^:
实施例 1. 噻吩磺隆水解 1 ¾因的克隆
1.1细菌基因组总 DNA的提取
S113 ( CGMCC 1479) 大量培养后， 采用 CTAB法提取高纯度、 大片段的 S113的基因组 总 DNA， 溶于 TE缓冲液 （pH8.0)中， 置于 -20°C保藏， 具体方法参考 F_奥斯伯等编的 《精编 分子生物学实验指南》。
1.2总 DNA的酶切 S113 (CGMCC 1479)总 DNA采用 "3AI部分酶切。
1.3 DNA的回收
酶切后的总 DNA通过电泳 （TAE缓冲液）进行纯化， 采用 axygen biosciences(China;0收试剂盒 进行回收， 回收的 DNA溶于 10 mmol/L的 Tris_Cl (pH8.0)中， 置于 -20°C保藏。
1.4麟
建立如下反应体系：
pUC118(£腿 HI) 0.1
总 DNA片段 0.1 μg
lOx连接酶缓冲液 1 μΐ
T4DNA连接酶 0.5 μΐ
加双蒸水至 10 μΐ
16°C温育 12小时。
1.5制备大肠杆菌 DH10B髙效感受态细胞
大肠杆菌 DH10B购自上海英骏生物技术有限公司。高效感受态细胞制备的具体方法参照 F. 奥斯伯等编的 《精编分子生物学实验指南》 P 22-23。
1.6转化
取 10 μ耀每连产物转化 200 μΐ大肠杆菌 DH10B感受态细胞，具体方法参照 F.奥斯伯等编的《精 编分子生物学实验指南》 Ρ 23。 涂布含有 10mg/kg的噻吩磺隆， 100 mg/kg氨苄青霉素的基础 培养基平板， 培养 24 h后挑取生长的菌落， 进一步验证获得一个能将噻吩磺隆转化为噻磺酸的 转化子。 基础盐培养基配方为 5.0 glucose, 1.0 NhkNC , 1.0 Nad, 1.5 ½HP04, 0.5 HHPO^ 0.2 Mg8D4-7H20, pH 7.0。
1.7基因核苷酸序列测定
将 1.6中获得的能将噻吩磺隆转化为噻磺酸的转化子送交上海英骏生物技术有限公司进行 序列测定， 噻吩磺隆水解酶基因的核苷酸序列为 SEQ ID N0.1 , 根据噻吩磺隆水解酶基因核苷 酸序列所推到的 398个氨基酸序列为 SEQ ID N0.2。 实施例 2噻吩磺隆水解難因在 BL21(pET-29a(+))中的髙效表达（图 2)
2.1噻吩磺隆水解 H¾因的 PCR扩增 以正向弓 I物： 5，-TGCAGACATATGGAAACCGATAAAAAAAC-3 ( SEQ ID N0.3 ) 禾口反向 引物： 5，-TGCAGAGAATTCCCTTCCATAAGAGCGCCGAT-3， ( SEQ ID N0.4 )为引物，用 PCR 从 S113 (CGMCC 1479) 基因组 DNA中扩增出噻吩磺隆水解酶基因片段。
扩增体系：
Primer star^$ ( 5ϋ7μ1) 0.5 μΐ
5 PCR Buffer II (Mg2+Plus) 10 μΐ
dNTP Mixture (各 2.5mM) 5 μΐ
模板 DNA 10 ng
正向引物 (20μΜ) Ι μΐ
反向引物 (20μΜ) Ι μΐ
灭菌蒸馏水 至 50 μΐ
PCR扩增程序：
a.95°C 变性 3min;
b.95°C 变性 1.5min， 53°C退火 0.5 min， 72°C延伸 1.5 min， 进行 25个循环；
c.72°C延伸 10 min， 冷却到室温。
2.2 PCR产物用 M和 H«<mi双酶切。
酶切体系：
Nde I 1 μΐ
Hindlll 1 μΐ
DNA <1
灭菌的蒸馏水 加至 20 μΐ
在 37°C水浴中， 反应 3h以上。 酶切产物进行 2%的琼脂糖凝胶电泳切胶回收。
2.3 pET-29a(+)用 和 H«<mi双酶切（参考 2.2)。
2.4转化
2.2中的回收片段和 2.3中酶切好的 pET-29a(+)进行酶连 （参考 1.5 )。 酶连好的含噻吩磺隆水 解酶基因的 pET-29a(+)重组质粒转化到表达宿主菌 BL21(DE3)获得重组微生物 BL21(TsmE)。
2.5 TsmE的表达、 纯化和功能验证
BL21(TsmE)在 LB培养基中培养至 OD600nm为 0.6至 IJO.8之间， 力口 IPTG至浓度 lmM， 30° C 培养 4个小时。 100ml菌液离心， 用 10ml (50 mM, pH 7.0) PBS缓冲液重悬菌体， 超声破碎 (Auto Science, UH-650B ultrasonic processor, 30% intensity) 5分钟， 离心， 收集上清， 用镍离子亲和层 析柱对 TsmE进行了纯化， 纯化后的酶进行蛋白质电泳， 见图 3。
2.6 TsmE活力测定
酶活反应体系： 采料 磷酸缓冲液 （軽 « )， 继纏料 噻吩磺隆或继 » 高效盖草能、 反 应酶量 （攔 »纯化所得） 采 ， ¾ 反应提斗 。 每个反应以加入酶开始计时， 用職斗 二 氯甲烷终止反应， 分层后有机相经无水硫酸钠脱水， 噻吩磺隆或高效盖草能含量用反向 HPLC 测定 （具体方法见 »。 一个酶活力单位 （ ） 定义为： 在 ， 温度«条件下， 每分钟 催化减少^ 噻吩磺隆或高效盖草能所需的酶量。 降解试验表明纯化后的 能在总 内降 解总建瞎 的噻吩磺隆或高效盖草能，酶学试验表明 对噻吩磺隆和高效盖草能的比酶活分 别为 ¾^¾¾斗 。 料
2.7代谢产物的确定
2.7.1 噻吩磺隆降解代谢产物的确定
2.6中的噻吩磺隆的酶反应液过滤， 取 20μί滤液进行 LC-MS, 液相色谱条件： 色谱柱： Agilent Zorbax XDB-C18柱（2.1 χ50ηιηι,3.5μηι)， 流动相： 甲醇： 水 =80: 20， 流速 0.25ml/min; 紫外检测波长 255nm。 一级质谱条件： 离子检测方式为多反应离子检测； 离子极性为负离子； 离子化方式为电喷雾离子化； 毛细管电压为 4000 伏； 干燥气温度： 33CTC ; 干燥气流速： 10.0 L/min, 雾化气压力： 35psi， 碰撞电压： 135 伏； 质量扫描范围 （m/z): 300-500。 二级子离子 质谱条件： 碰撞电压： 90 伏； 质量扫描范围 （m/z): 30-400
LC-MS的液谱图（见图 4A)表明， 其产物的保留时间为 1.95min， 一级质谱图（见图 4B ) 显示其有 m/z为 372.30分子负离子峰， m/z为 372.30的分子负离子其二级质谱图 （见图 4C) 中有 m/z为 162.10， 188.10, 206.20的片段， 这与噻磺酸相符。 因此， TsmE水解噻吩磺隆的 生化反应是就噻吩磺隆转化成噻磺酸 （见图 5)。
2.7.2 高效盖草能降解代谢产物的确定
利用串联质谱测定高效盖草能降解代谢产物， 2ml 2.6中的高效盖草能酶反应液二氯甲烷 提取物经氮气吹干后溶于 ΙΟΟμί甲醇， 进行串联质谱测定， 串联质谱条件： MS/MS (Finnigan TSQ Quantum Ultra AM, Thermal, U.S.A.) ， 采用电喷雾形式离子化， 正负离子同时检测， 质 量扫描范围 ( m/z): 30-1200。
MS/MS的一级质谱图 （见图 6A) 中有 m/z为 359.87的分子负离子峰， m/z为 359.87的 分子负离子其二级质谱 （见图 6B ) 中有 m/z为 287.60的片段， 这与 TsmE水解高效盖草能的 代谢产物 2-[4-(3-氯 -5-三氟甲基 -2-吡啶氧基)苯氧基]丙酸相符。 因此， TsmE水解高效盖草能的 生化反应是就将高效盖草能转化成 2-[4-(3-氯 -5-三氟甲基 -2-吡啶氧基)苯氧基]丙酸 （见图 7)。 以上实施例中使用的微生物来源如
pUC118 (β腿 HI) 购自宝生物工程 （大连） 有限公司， 大肠杆菌 DH10B 购自上海英骏生物技术有限公司， 大肠杆菌高表达载体 pET-29a(+) 购自 Novegen公司，
表达宿主菌大肠杆菌 BL21(DE3) 购自上海英骏生物技术有限公司。

Claims

权利要求书
1. 一种噻吩磺隆水解酶基因 mE， 其核苷酸序列为 SEQ ID N0.1。
2. 权利要求 1所述的噻吩磺隆水解酶酶基因 mE核苷酸序列所编码的噻吩磺隆水解酶蛋 白质 TsmE， 其氨基酸序列为： SEQ ID N0.2。
3. 含有权利要求 1所述的噻吩磺隆水解酶基因 t腿 E的重组表达载体。
4.， 根据权利要求 3所述的重组表达载体， 其特征在于是将权利要求 1所述的噻吩磺隆水 解酶基因 tsmE插入 pET-29a(+)的 M和 ΗίηΛη位点之间所得。
5. 含有权利要求 1所述的噻吩磺隆水解酶基因 tsmE的基因工程菌。
6. 根据权利要求 3所述的重组表达载体， 其特征在于所述的基因工程菌的出发菌株为大 肠杆菌 BL21(DE3)。
7. 权利要求 1所述噻吩磺隆水解酶基因 mE在构建抗噻吩磺隆的转基因作物中的应用。
8. 权利要求 1所述噻吩磺隆水解酶基因 mE在降解噻吩磺隆和高效盖草能中的应用。
9. 权利要求 2所述噻吩磺隆水解酶蛋白质 TsmE在降解噻吩磺隆和高效盖草能中的应用。
10. 权利要求 2所述噻吩磺隆水解酶蛋白质 TsmE在去除土壤、水体中除草剂噻吩磺隆和 高效盖草能残留中的应用。
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