CN108441503B - 麦草畏中间产物3,6-二氯龙胆酸脱氯酶DsmH2及其编码基因的应用 - Google Patents
麦草畏中间产物3,6-二氯龙胆酸脱氯酶DsmH2及其编码基因的应用 Download PDFInfo
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- CN108441503B CN108441503B CN201810242125.3A CN201810242125A CN108441503B CN 108441503 B CN108441503 B CN 108441503B CN 201810242125 A CN201810242125 A CN 201810242125A CN 108441503 B CN108441503 B CN 108441503B
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- Prior art keywords
- dsmh2
- dichloro
- enzyme
- dicamba
- dechlorination
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- 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
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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
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- 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
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- C—CHEMISTRY; METALLURGY
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- C12Y—ENZYMES
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- 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
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- 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
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了麦草畏中间产物3,6‑二氯龙胆酸脱氯酶DsmH2及其编码基因的应用。dsmH2其核苷酸序列为SEQ ID NO.1，全长741bp，编码246个氨基酸，其氨基酸序列为SEQ ID NO.2。DsmH2是首次发现参与麦草畏下游代谢路径的谷胱甘肽硫转移酶类型的还原脱氯酶，能在30min内降解100mg/l的3,6‑二氯龙胆酸。因此，3,6‑二氯龙胆酸脱氯酶基因dsmH2在构建降解麦草畏转基因作物中应用潜能巨大。
Description
技术领域
本发明属于环境微生物和农业领域，涉及参与除草剂麦草畏微生物降解过程中的两个脱氯酶及其编码基因的应用。
背景技术
农药是指应用于防治危害农作物的病虫、杂草等有害生物以及调节植物生长的化学试剂的总称，是保障现代农业生产力的重要手段，合理使用农药可以有效的防治作物病害，提高作物产量。除草剂的使用能有效减轻农业劳动强度，提高作物产量，但是随着除草剂的大量和长期使用，其残留对大气、土壤及水体造成的危害越来越严重，同时对人类健康造成威胁。农药污染修复技术可分为物理降解(热降解、光降解)、化学降解和生物降解。微生物修复技术是一种原位生物修复技术，具有安全温和、效率高、效果好、费用低、无二次污染等多个优点，适合大面积面源污染修复，是土壤有机污染物修复技术的主流和发展方向。而且抗/降解除草剂转基因技术是解决除草剂药害的有效途径，而抗除草剂和降解除草剂的基因一般来自微生物降解基因。
麦草畏(dicamba)(3,6-二氯-2-甲氧基苯甲酸，又名百草敌)是60年代由诺华(现在的先正达)开发的激素类除草剂，对一年生和多年生阔叶杂草有显著防除效果。麦草畏用于苗后喷雾，药剂被被植株吸收、传导，多集中在分生组织及代谢活动旺盛的部位，阻碍植物激素的正常活动，从而使其死亡，能防除200多种杂草，广泛应用于玉米、高粱和小麦等农田杂草防治，目前全球使用量达1.5万吨。麦草畏具有广谱高效、低毒和杂草抗性产生慢等优点，可有效杀死草甘膦抗性杂草，是美国孟山都生物技术公司研发的新一代抗除草剂转基因作物的靶标除草剂。孟山都公司利用麦草畏脱甲基酶基因dmo构建了抗除草剂转基因大豆和棉花，2015年进入商业化阶段，可以预见，随着抗麦草畏转基因作物的商业化推广，麦草畏的使用量会大幅度提高。麦草畏在土壤中稳定，一般能保持40天以上，其在环境中的降解的主力军是微生物，目前已筛选到多种麦草畏的降解菌株，克隆到多个麦草畏降解基因，但是关于麦草畏的微生物降解中间产物3,6-二氯龙胆酸的进一步降解机制，降解基因和酶，目前还没有报道，这严重制约了对麦草畏的环境行为和生态安全方面的研究。因此有必要深入研究麦草畏的微生物代谢的完整降解途径，降解机制，这对研究麦草畏在环境迁移、转化和降解等环境行为及生态安全性具有重要的指导价值。
3,6-二氯龙胆酸(3,6-DCGA)是麦草畏微生物代谢过程中的中间产物，是氯代芳香化合物，脱氯反应一般为脱毒反应，因此3,6-二氯龙胆酸脱氯酶基因和酶的鉴定具有非常重要的应用价值。在治理农药残留中主要具有以下作用，(一)通过现代生物工程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脱氯酶DsmH1和DsmH2制成酶制剂，实现土壤原位修复。综上所述，开展麦草畏降解过程中的降解基因、酶的研究具有非常重要的理论和实际应用价值。
发明内容
本发明的目的是针对现有麦草畏微生物降解机制研究的缺乏，提供3,6-二氯龙胆酸脱氯酶基因，该基因参与麦草畏下游降解路径，麦草畏脱甲基生成3,6-二氯水杨酸(3,6-DCSA)，然后3,6-DCSA 5-羟基化生成3,6-二氯龙胆酸(3,6-DCGA)，本发明脱氯酶DsmH2能将3,6-二氯龙胆酸6号位置的氯离子脱去生成3-氯龙胆酸，代谢途径见图1。本发明的另一目的是提供该3,6-二氯龙胆酸脱氯酶基因编码的蛋白DsmH2。DsmH2是首次发现参与麦草畏下游代谢路径的依赖GSH的谷胱甘肽硫转移酶类型的还原脱氯酶。本发明的又一目的是提供基因dsmH2及其编码蛋白的应用。
发明内容
本发明的目的可通过以下技术方案实现：
一种3,6-二氯龙胆酸脱氯酶基因dsmH2，其核苷酸序列为SEQ ID NO.1。
3,6-二氯龙胆酸脱氯酶基因dsmH2核苷酸序列所编码的3,6-二氯龙胆酸脱氯酶DsmH2，其氨基酸序列为SEQ ID NO.2。
含有所述的还原脱氯酶基因dsmH2的重组蛋白表达载体pET-24b-dsmH2。
所述的蛋白表达载体，优选将还原脱氯酶基因dsmH2插入pET-24b(+)的酶切位点NdeI和XhoI之间所得，所述的还原脱氯酶基因dsmH2是以保藏号为CCTCC NO:M 2014550的菌株Ndbn-20基因组DNA为模板，SEQID NO.3和SEQID NO.4所示的引物进行PCR扩增得到。
含有所述的还原脱氯酶基因dsmH2的高效表达菌株，所述的表达菌株优选Ecoli.BL21(DE3)。
所述3,6-二氯龙胆酸脱氯酶基因dsmH2在构建麦草畏下游降解产物3,6-二氯龙胆酸脱氯反应转基因作物中的应用。
所述3,6-二氯龙胆酸脱氯酶基因dsmH2在3,6-二氯龙胆酸脱氯反应中的应用。
所述3,6-二氯龙胆酸脱氯酶DsmH2在降解3,6-二氯龙胆酸中的应用。
所述3,6-二氯龙胆酸脱氯酶DsmH2在去除土壤、水体中3,6-二氯龙胆酸的应用。
有益效果：
本发明首次公开了除草剂麦草畏微生物代谢过程中的一个脱氯酶DsmH2及其应用。麦草畏中间产物3,6-二氯龙胆酸(3,6-DCGA)脱氯酶基因dsmH2其核苷酸及氨基酸序列分别为：SEQ ID NO.1，SEQ ID NO.2，编码246个氨基酸。3,6-二氯龙胆酸(3,6-DCGA)6-脱氯酶基因dsmH2是首次发现参与麦草畏下游代谢路径的依赖GSH的谷胱甘肽硫转移酶类型还原脱氯酶的编码基因，DsmH2能高效快速降解3,6-二氯龙胆酸，因此DsmH2在降解麦草畏的过程中起着重要的作用，在构建降解麦草畏转基因作物中应用潜能巨大。
附图说明：
图1麦草畏降解菌Rhizorhabdus dicambivorans Ndbn-20麦草畏降解途径推测
图2Ndbn-20粗酶脱氯酶功能检测
A.Ndbn-20粗酶液+GSH降解3,6-DCGA的HPLC图谱；B.5.40min处产物峰的MS图谱
图3脱氯酶DsmH2SDS-PAGE电泳图，
M：蛋白Marker；1：E.coli BL21(Pet-24b-dsmH2)粗酶；2：钴柱纯化后的脱氯酶DsmH2
图4脱氯酶DsmH2降解3,6-二氯龙胆酸的紫外扫描图，
290nm处为3,6-DCGA的峰，325nm处为产物的峰
图5脱氯酶DsmH2降解3,6-二氯龙胆酸的液质图谱，
A：i，CK 3,6-DCGA 0.1mM；ii，3,6-DCGA 0.1Mm加脱氯酶DsmH2反应2min；iii，3,6-DCGA0.1mM加脱氯酶DsmH2反应5min；B：5.39min处的产物MS图谱
图6脱氯产物核磁检测图谱
图7敲除菌株Ndbn-20ΔdsmH2降解3,6-DCGA和麦草畏的降解曲线
生物材料保藏信息:
Ndbn-20，分类命名为Sphingomonas sp.Ndbn-20，保藏于中国典型培养物保藏中心，保藏地址为中国武汉大学，保藏日期为2014年11月5日，保藏编号为CCTCC NO:M2014550。
具体实施方式
以下实施例中使用的微生物来源如下：大肠杆菌DH5α购自宝生物工程(大连)有限公司，大肠杆菌高表达载体pET-24b(+)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。
实施例1.3,6-二氯龙胆酸脱氯酶基因dsmH1,dsmH2的克隆
1.1代谢产物检测
1.1.1麦草畏降解菌株Ndbn-20粗酶3,6-DCGA脱氯酶活性检测
本实验的采用的菌株为由本实验室成员分离得到的麦草畏高效降解菌Rhizorhabdus dicambivorans Ndbn-20(以前命名为Sphingomonas sp.Ndbn-20)，将Ndbn-20在100ml 1/5LB液体培养基中培养至对数期，离心收集菌体，采用PBS缓冲液洗涤2次，10ml PBS缓冲液重悬，采用超声破碎(Auto Science,UH-650B ultrasonic processor,30％intensity)5-10分钟，12000rpm离心40min，收集上清，此上清液即为粗酶液。
酶活反应体系(1ml)：加有PBS(50mM,pH 7.0)，0.1mM 3,6-二氯龙胆酸，粗酶液100μl，分别加入不同的辅因子NADH,NADPH,GSH,ATP，30℃反应1h。将酶反应足够长的时间，采用HCl酸化，然后用乙酸乙酯萃取，取上层有机相，采用无水硫酸钠吸干水分，再采用氮气将溶剂乙酸乙酯吹干，用甲醇溶解，采用0.22μm的滤膜对样品进行过滤，进行HPLC检测发现加辅因子NADH,NADPH,ATP的酶反应中3,6-二氯龙胆酸的峰没有下降，而加有GSH的粗酶反应中3,6-二氯龙胆酸的峰有明显的下降，并且有一个产物峰产生，通过质谱分析，此产物为脱掉一个氯原子的3,6-二氯龙胆酸的分子量，见图2。粗酶降解试验表明催化3,6-二氯龙胆酸脱氯反应的脱氯酶是一个依赖GSH的还原脱氯酶，而且此还原脱氯实验可以在有氧条件下发生，因此极有可能为一种依赖还原型GSH谷胱甘肽硫转移酶类型的还原脱氯酶。
1.1.2查阅文献对麦草畏降解下游途径进行推测
通过粗酶的实验，我们确定了脱氯酶的类型，很快通过查阅文献找到来自Sphingobium chlorophenolicum ATCC 39723的谷胱甘肽硫转移酶类型的还原脱氯酶PcpC，通过将它的氨基酸序列与菌株Ndbn-20的基因组进行比较，检索到与PcpC氨基酸序列具有31％同源性的基因序列，命名为dsmH2，采用PCR将基因扩增出来，进行蛋白表达及功能验证。
实施例2脱氯酶基因在BL21(pET-24b(+))中的高效表达
2.1脱氯酶基因的PCR扩增
以正向引物：5’-TAAGAAGGAGATATACATATGACCCATCTGGACCTGTACAATTAC-3’(SEQ IDNO.3),反向引物：5’-TGGTGGTGGTGCTCGAGGATCCCACCCTTCCAATTGGGCATC-3’(SEQ ID NO.4)。用PCR从Ndbn-20基因组DNA中扩增出dsmH2。
扩增体系：
PCR扩增程序：
a.98℃变性3min；
b.98℃变性0.5min，58℃退火0.5min，72℃延伸1.0min，进行30个循环；
c.72℃延伸10min，冷却到室温。
2.2PCR产物和质粒的双酶切、产物纯化和酶连
PCR产物使用凝胶纯化回收试剂盒，具体方法参考试剂盒说明书。纯化后的PCR产物与质粒分别用相应的酶进行酶切，以序列两端分别引入Nde I和Xho I酶切位点，酶切体系如下：
10×M Buffer 5μL
Nde I(10U·μL-1)2.0μL
Xho I(10U·μL-1)2.0μL DNA(纯化后的PCR或质粒)30μL
ddH2O 11μL
37℃酶切30min，0.75％的琼脂糖核酸电泳检测酶切效果。然后使用凝胶纯化回收试剂盒纯化回收相应的DNA片段，然后用T4连接酶与同样双酶切的表达载体pET-24b(+)进行酶连，酶连体系如下：
10×T4ligase buffer 1.0μL
DNA(PCR双酶切)4.0μL
pBBR1MCS-2 2.0μL
T4ligase 0.5μL
ddH2O 2.5μL
16℃温浴12h。
2.3酶连产物转化和阳性转化子的筛选
从-70℃取一管E.coli BL21(DE3)感受态细胞(100μL)，握在掌心融化后，加入10μL的酶连产物(体积不超过感受态细胞的10％)，轻轻转动管体混匀，静置于冰上30min。轻轻将离心管放入42℃水浴锅热激60-90s，然后将离心管重新置于冰上，10min。加入500-800μLLB培养基，将离心管放到37℃摇床，150rpm，45-60min复苏，并且表达质粒编码的抗生素抗性基因。离心管5,000rpm，2min，弃部分上清，留约200μL，用枪吹打将菌体混匀，吸取100μL混合液均匀涂布到含有卡那霉素100mg/L的LB平板上，37℃过夜培养，挑取长出的单菌落，测序验证目的基因连接到载体上而且末端连有6个His-tag，将此转化子保存。
2.4DsmH2的表达、纯化
BL21(DsmH2)在LB中，37℃，150rpm摇床培养至OD 600nm为0.4到0.6之间，然后加0.05mM IPTG，16℃诱导8个小时，离心收集菌体，采用PBS(50mM,pH 7.0)将菌体洗两遍，用10ml的PBS缓冲液重悬菌体，超声破碎5-10分钟，12000rpm离心40min，收集上清，用钴离子亲和层析柱对DsmH2进行纯化，纯化后的酶进行蛋白质电泳，见图3。
2.5DsmH2活力测定
酶活反应体系(1ml)：加有PBS(50mM,pH 7.0)，0.1mM 3,6-二氯龙胆酸，反应酶量(2.4中纯化所得)10μl，30℃反应。每个反应以加入酶开始计时，通过紫外扫描进行定时检测。3,6-二氯龙胆酸的紫外吸收峰最高是300nm，经过反应后产生脱氯产物，此物质的紫外吸收峰为325nm，因此通过紫外扫描(200nm-400nm)可以看到紫外峰会发生偏移，降解情况见图4。将酶反应液通过酸化，萃取，吸干水分，吹干，甲醇溶解，过滤，进行HPLC检测发现3,6-二氯龙胆酸的峰下降，并产生了新峰，质谱检测为脱氯产物，见图5。酶降解试验表明纯化后的脱氯酶DsmH2能降解3,6-二氯龙胆酸。通过酶活检测DsmH2降解3,6-二氯龙胆酸的比酶活为23.24±2.73μM/min/mg protein。
2.6脱氯产物的鉴定
通过质谱鉴定得到，脱氯产物是3,6-二氯龙胆酸脱掉一个氯原子的分子量，但是3,6-二氯龙胆酸的分子结构中有两个氯原子，因此需要确定脱氯反应脱去的氯离子是哪个位置的氯，我们采用的方法是核磁鉴定。采用薄层层析的方法将酶反应液中的产物进行分离纯化，通过硅胶板预实验确定展开剂的配方，通过大量实验得到合适的展开剂为：乙酸乙酯：氯仿：甲酸＝10:6:1，将纯化好的脱氯产物经过液相和质谱的鉴定确定为单一的物质后进行H1NMR检测，检测结果见图6，通过分析，核磁结果，脱氯产物脱掉的氯是6号位置的氯，脱氯生成3-氯龙胆酸。
2.7DsmH2底物谱的测定
通过选用不同的氯代芳香族化合物，优选3,6-二氯龙胆酸的类似物，主要有：3-氯龙胆酸，4-氯龙胆酸，6-氯龙胆酸，3-氯水杨酸，4-氯水杨酸，5-氯水杨酸，6-氯水杨酸，对硝基氯化苄，四氯氢醌，2,5-二氯对苯二酚，3-氯-4-羟基苯甲酸，4-氯苯甲酸，2-氯对苯二酚，3,5-二氯-4-羟基苯甲酸，2,4-二硝基氯苯。通过酶反应得出结果：DsmH2只能降解6-氯龙胆酸，因此DsmH2可能是专门参与麦草畏代谢中3,6-二氯龙胆酸的降解的。
实施例3脱氯酶在菌株Ndbn-20中的生理功能的验证
为验证脱氯酶在菌株Ndbn-20中的生理功能，本发明构建了dsmH2插入突变载体，是将还原脱氯酶基因中间的大约509bp的dsmH2’的片段，插入pJQ200SK的PstI和BamHI酶切位点之间所得。
以正向引物：5’-CTTGATATCGAATTCCTGCAGTTGATGAAGCTGGAGCATACGCGGC-3’(SEQID NO.5),反向引物：5’-GCTCTAGAACTAGTGGATCCGTCGTCCCATAGCCGGGCAAAATTC-3’(SEQ IDNO.6)，扩增出dsmH2’。
将插入突变的载体pJQ200SK采用PstI和BamHI进行双酶切，构建载体的方法见实施例2表达载体的构建。将构建好的载体转化进入DH5α中。
3.2插入突变
将供体菌DH5α(pJQdsmH2)、受体菌Ndbn-20、辅助菌PRK600分别接入含有相应抗生素的LB液体培养基中，培养至对数期，离心后用无菌水重悬菌体按1:2:1的体积比在离心管中混合均匀。将无菌滤膜放置在无抗性的LB平板上，随后吸取混合菌液200μL加至滤膜上，30℃静置培养2天后，用液体LB洗下滤膜上的菌体，震荡混匀，取100μL菌液涂布于选择性抗性LB平板(Str和Gm)上。培养4天挑取转入质粒的接合子在Gm和Sm双抗平板上划线纯化以备后续的降解功能验证试验。
通过PCR验证，得到插入突变成功的突变菌株Ndbn-20ΔdsmH2，进行降解3,6-DCGA和麦草畏的实验，实验结果证明Ndbn-20ΔdsmH2不能降解3,6-DCGA，而且Ndbn-20ΔdsmH2降解麦草畏的速率下降，并且有3,6-DCGA积累(见图7)。
序列表
<110> 南京农业大学
北京大北农生物技术有限公司
<120> 麦草畏中间产物3,6-二氯龙胆酸脱氯酶DsmH2及其编码基因的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 741
<212> DNA
<213> Ndbn-20菌株(Sphingomonas sp. Ndbn-20)
<400> 1
atgctcgaac tctatcatga ctggcggtcc ttctgctcga tcaaggtgag gctttgcctc 60
gccgaaaaac agctcccttg ggaaagccgg ttcgtcgatt tgatgaagct ggagcatacg 120
cggccggaat atacccggct caatcccaat ggggtcgtgc ccaccctggt gcacaatggt 180
gttccgatca tcgaatccac gatcatcaac gaatatctgg aggaggtatt cccggagata 240
tcgctggtcc ccagcgatcc ggtcgagcgt gcgagaatgc gcgcctgggt gaagttcgag 300
gatgacgtcc tgcatccgtc gatacgcccg gcaaccttca cgctgatgat gagtcaggaa 360
ctggcaaaat tgtcggatgt cgaactggat gagcaactgg caaaacatcc caaccagcag 420
cgcgcggaag aatatcggat tgctgcacgc tcgcccgtcg atcatgctgc ggtcgaggag 480
gcgaaggtca agatgagcaa ggcgctggat cggctggaaa agcagctcga caccaccccc 540
tatctcgccg gtgacagcta ttcgctcgcc gatgttgcgg cagcgccctt tgtcgatcgg 600
ctggaagagt tgaattttgc ccggctatgg gacgaccggc ccagcctgag cgcctggatc 660
gctcggttga agtcgcgtcc ttctttcagc gaggcggtgc cccgcagaga ccagcgcttc 720
gctgcggccg tcatcgcctg a 741
<210> 2
<211> 246
<212> PRT
<213> Ndbn-20菌株(Sphingomonas sp. Ndbn-20)
<400> 2
Met Leu Glu Leu Tyr His Asp Trp Arg Ser Phe Cys Ser Ile Lys Val
1 5 10 15
Arg Leu Cys Leu Ala Glu Lys Gln Leu Pro Trp Glu Ser Arg Phe Val
20 25 30
Asp Leu Met Lys Leu Glu His Thr Arg Pro Glu Tyr Thr Arg Leu Asn
35 40 45
Pro Asn Gly Val Val Pro Thr Leu Val His Asn Gly Val Pro Ile Ile
50 55 60
Glu Ser Thr Ile Ile Asn Glu Tyr Leu Glu Glu Val Phe Pro Glu Ile
65 70 75 80
Ser Leu Val Pro Ser Asp Pro Val Glu Arg Ala Arg Met Arg Ala Trp
85 90 95
Val Lys Phe Glu Asp Asp Val Leu His Pro Ser Ile Arg Pro Ala Thr
100 105 110
Phe Thr Leu Met Met Ser Gln Glu Leu Ala Lys Leu Ser Asp Val Glu
115 120 125
Leu Asp Glu Gln Leu Ala Lys His Pro Asn Gln Gln Arg Ala Glu Glu
130 135 140
Tyr Arg Ile Ala Ala Arg Ser Pro Val Asp His Ala Ala Val Glu Glu
145 150 155 160
Ala Lys Val Lys Met Ser Lys Ala Leu Asp Arg Leu Glu Lys Gln Leu
165 170 175
Asp Thr Thr Pro Tyr Leu Ala Gly Asp Ser Tyr Ser Leu Ala Asp Val
180 185 190
Ala Ala Ala Pro Phe Val Asp Arg Leu Glu Glu Leu Asn Phe Ala Arg
195 200 205
Leu Trp Asp Asp Arg Pro Ser Leu Ser Ala Trp Ile Ala Arg Leu Lys
210 215 220
Ser Arg Pro Ser Phe Ser Glu Ala Val Pro Arg Arg Asp Gln Arg Phe
225 230 235 240
Ala Ala Ala Val Ile Ala
245
<210> 3
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taagaaggag atatacatat gacccatctg gacctgtaca attac 45
<210> 4
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tggtggtggt gctcgaggat cccacccttc caattgggca tc 42
<210> 5
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cttgatatcg aattcctgca gttgatgaag ctggagcata cgcggc 46
<210> 6
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gctctagaac tagtggatcc gtcgtcccat agccgggcaa aattc 45
Claims (8)
1.一种3,6-二氯龙胆酸脱氯酶基因dsmH2，其核苷酸序列为SEQ ID NO.1。
2.权利要求1所述的3,6-二氯龙胆酸脱氯酶基因dsmH2所编码的3,6-二氯龙胆酸脱氯酶DsmH2，其氨基酸序列为 SEQ ID NO.2。
3.含有权利要求1所述的3,6-二氯龙胆酸脱氯酶基因dsmH2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3,6-二氯龙胆酸脱氯酶基因dsmH2分别插入pET-24b(+)的NdeI和XhoI位点之间所得。
5.含有权利要求1所述的3,6-二氯龙胆酸脱氯酶基因dsmH2的基因工程菌。
6.根据权利要求5所述的基因工程菌，其特征在于所述的基因工程菌的表达菌株为大肠杆菌BL21(DE3)。
7.权利要求2所述3,6-二氯龙胆酸脱氯酶DsmH2在降解3,6-二氯龙胆酸中的应用。
8.权利要求2所述3,6-二氯龙胆酸脱氯酶DsmH2在去除土壤、水体中3,6-二氯龙胆酸的应用。
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