【作者】 岳建建；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2010， 硕士

【摘要】 在长期重金属污染的环境中,许多微生物产生了对多种重金属及其复合污染物较高的抗性,产生许多由重金属诱导作用而产生的抗性基因及蛋白质。重金属基因可以激活和编码金属硫蛋白、操纵子、金属运输酶和透性酶等,通过这些物质结合重金属,形成失活晶体或促进重金属排出体外等机制对重金属进行解毒。PcoR基因的合成根据GenBank中Klebsiella pneumoniae （GeneID:NP₉43472）基因,设计并合成了1对引物（由TaKaRa公司合成）,用于扩增PcoR基因。PCR反应程序为:95℃5min; 94℃1min 30 s; 58℃1min 30 s; 72℃3 min; 34个循环;最后72℃延伸10min。对扩增产物进行电泳鉴定,最后将PCR产物经胶回收试剂盒纯化,以备酶切。按照DNA凝胶回收试剂盒的操作说明,回收纯化PCR产物。将回收的PCR产物插入pGEM-Teasy载体,构建重组质粒pGEM-PcoR,转化DH5α感受态细胞,37℃培养12～16 h。挑取白色菌落接种于含有Amp的LB肉汤中,37℃振荡培养12～16 h,提取重组质粒进行PCR和酶切鉴定。用EcoR I和Hind III酶切pET-41a载体及PcoR基因片段,酶切后进行产物回收,在T4 DNA连接酶的作用下做定向连接。连接产物转化至BL21感受态细胞,涂板、挑选单克隆进行菌落PCR。菌落PCR、酶切验证均为阳性的克隆即为含有目的重组pET-41a-PcoR的阳性克隆。选取一个阳性克隆送至南京金思特科技有限公司进行测序。以pET-41a空质粒转化菌为对照,将测序鉴定后的重组质粒转入BL21 RIL感受态细胞中,在含100 mg/L氨苄青霉素（Amp）的LB培养板上,37℃下培养过夜。次日,从LB培养板上挑取含有重组质粒的转化菌及空质粒转化菌各一个,分别接种到5 mL LB培养液中（含100 mg/L Amp）,37℃摇床培养。待菌液OD600值=0.5时,按照IPTG终浓度为1.0 mmol/L诱导不同时间（1、2、3、4和5h）,或不同IPTG浓度（0.1、0.25、0.5、0.75和1.0mmol/L）下诱导5h,比较重组蛋白的产量,确定最佳诱导条件。抗铜基因的BLASTn分析,苜蓿中华根瘤菌pcoR与大肠杆菌（E. coli）、阪崎肠杆菌（E. sakazakii）、克雷伯杆菌（K. pneumoniae）和粘质沙雷氏菌（S.marcescens）的质粒或基因组上的铜抗性基因的同源性达到99%。抗铜蛋白的BLASTp同源性搜索与分析,发现抗铜蛋白PcoR与大肠杆菌（E. coli）、克雷伯杆菌（K. pneumoniae）和克雷伯杆菌（K. pneumoniae subsp.）的转录调控蛋白PcoR、粘质沙雷氏菌（S. marcescens）的双组分抗铜应答调控子的同源性达到100%,与布克氏菌（Burkholderia multivorans）、罗尔斯顿菌（Ralstonia pickettii）和假单胞菌（Pseudomonas putida）等的重金属应答调控子的同源性在57%以上。采用PCR法可以成功合成长度为687 bp的苜蓿中华根瘤菌PcoR基因。重组子pET-41a-PcoR在大肠杆菌表达株系BL21 （RIL）中、28℃200 r/min经1mmol/L IPTG诱导5 h,可以大量表达可溶性的GST—PcoR融合蛋白。更多还原

【Abstract】 In long-term heavy metal contaminated environment, many microbes produce high resistance to a wide range of heavy metals and composites, resulting in induction of many heavy metal resistance genes and proteins. Metal genes can activate and coding metallothionein, operon,metal transport enzymes and permease, heavy metals through the combination of these substances to form crystals or inactivation mechanisms such as the promotion of excretion of heavy metals detoxification of heavy metals.PcoR gene synthesis according to GenBank, Klebsiella pneumoniae （GeneID: NP₉43472） gene, were designed and synthesized one pair of primers （synthesized by the TaKaRa Company） for the amplification PcoR gene. PCR reaction program was: 95℃5min; 94℃1min 30 s; 58℃1min 30 s; 72℃3 min; 34 cycles; final extension at 72℃10min. Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion. According to the instructions of DNA gel extraction kit, recover purified PCR product. The recovered PCR products were inserted into pGEM-Teasy vector, the recombinant plasmid pGEM-PcoR, transformed into DH5αcompetent cells, 37℃train 12 16 h. Picked white colonies were inoculated in LB broth containing Amp, 37℃shaking culture 12 16 h, extraction of recombinant plasmid PCR and restriction enzyme digestion. With EcoR I and Hind III digested pET-41a vector and PcoR gene fragment was recovered after digestion, in the role of T4 DNA ligase to do under the direction connection. The products were transformed into BL21 competent cells, coated plates, select monoclonal colony PCR. Colony PCR, enzyme restriction shall contain both positive recombinant clone pET-41a-PcoR positive clones. Choose a clone sent for sequencing Technology Co., Ltd. Nanjing Jin Site. With pET-41a as the control empty plasmid was transformed into bacteria, the DNA sequencing after plasmid into BL21 RIL competent cells, containing 100 mg / L ampicillin （Amp） of the LB culture plate, 37℃incubated overnight. The next day, picked from the LB culture plate containing the recombinant plasmid, empty vector transformed E. coli bacteria and one each were inoculated into 5 mL LB medium （containing 100 mg / L Amp）, 37℃shaking culture. When bacteria OD600 value = 0.5, in accordance with IPTG final concentration of 1.0 mmol / L induced at different times （1,2,3,4 and 5h）, or different IPTG concentrations （0.1,0.25,0.5,0.75 and 1.0mmol / L ） under the induction of 5h, more recombinant protein production, to determine the optimal induction conditions.BLASTn analysis of copper resistance genes showed the homology of the copper resistance gene on plasmid or the genome of Sinorhizobium pcoR and E. coli, E. sukiyaki, K. pneumonia, and S. marcescens is 99%. Anti-copper protein BLASTp homology search and analysis showed the homology of anti-copper protein pcoR and E. coli, K. pneumonia and transcriptional regulation protein pcoR of K. pneumonia subsp, two-component response regulators in anti-copper of S. marcescens is 100%, homology with Booker’s bacteria, Ralston bacteria and Pseudomonas and other heavy metal response regulator is more than 57%.The length of 687 bp of Sinorhizobium pcoR gene can be successfully synthesized by PCR. Recombinant pET-41a-PcoR in E. coli strain BL21 （RIL） in 28℃, 200 r / min by 1mmol / L IPTG induction 5 h, express a large number of soluble fusion protein GST-PcoR.更多还原