【作者】 鲁芸芸；

【导师】 孙雅量；

【作者基本信息】 华中科技大学 ， 无机化学， 2009， 硕士

【摘要】 在自然环境中,含镉的化合物具有较高的脂溶性和生物富集性,毒性强、危害大。传统的物理化学方法并不能正确反映出镉化合物对生物体引发的生物毒性效应,而生物方法可以直接检测镉化合物对生物体的毒理作用,更能够体现镉化合物在环境中的实际污染程度。本文以全套细菌荧光素酶基因( luxCDABE )为报告基因,以含有特异性镉启动子PcadA的质粒(pPcadA-gfp)为载体,构建由Cd2+诱导的含报告基因luxCDABE的重组质粒(pPcadA-luxCDABE-gfp),感受态转化法将其导入枯草杆菌B.subtilis受体菌,构建特异性检测Cd2+的工程菌,并应用于环境中痕量重金属镉的检测。研究方法如下:(1)鉴定实验所需的两个菌种:含有质粒pMD20-T-luxCDABE的大肠杆菌E.coli HB101和含有质粒pPcadA-gfp的枯草杆菌B.subtilis 1A751。E.coli HB101采用蓝白斑筛选法鉴定。将B.subtilis 1A751菌悬液制成载玻片,在荧光显微镜下观察菌体特征,提取质粒pPcadA-gfp,BamHI/HindIII和BamHI/EcoRI双酶切后,用琼脂糖凝胶电泳分析鉴定;(2)构建重组质粒(pPcadA-luxCDABE-gfp):利用基因工程技术,用BamHI单酶切获得目的基因luxCDABE及载体质粒pPcadA-gfp;在T4连接酶的作用下,将luxCDABE插入到PcadA的下游,构建重组质粒pPcadA-luxCDABE-gfp。将重组质粒(pPcadA-luxCDABE-gfp)导入E.coli进行增殖,通过Cm抗性平板筛选获得克隆并进行酶切鉴定。筛选的重组质粒导入耐受重金属镉的B.subtilis,做部分研究。实验结果:通过蓝白斑筛选E.coli HB101菌株,Amp抗性平板上生长白色菌落表示菌体中含有质粒pMD20-T-luxCDABE;B.subtilis 1A751菌悬液在蓝光激发下发出绿色荧光,说明菌体中含有质粒pPcadA-gfp,并能表达出GFP。从宿主菌中提取重组质粒pPcadA-luxCDABE-gfp,经酶切初步鉴定,pPcadA-luxCDABE-gfp构建成功,为进一步研究Cd2+是否诱导luxCDABE的表达及获得监测Cd2+的工程菌株打下基础。更多还原

【Abstract】 In the nature, cadmium compounds have a high liposolubility and bio-concentration, it’s very harmful and toxic. Traditional physical and chemical methods cannot correctly reply the biological toxic effects that cadmium compounds caused to organisms. But biological methods can directly detect the toxicological effect of cadmium compounds on the organisms, more reflect the actual pollution level. In my paper,a full set of bacterial luciferase gene(luxCDABE) serves as reporter gene, and the pPcadA-gfp plasmid containing the cadmium-specific promoter(PcadA) acts as vector to construct the recombinant DNA(pPcadA-luxCDABE-gfp) whose expression is induced by Cd2+. B.subtilis was employed as the host bacteria, and the recombinant DNA was transformed into B.subtilis in order to obtain the specific engineering strains which can detect trace cadmium in the environment. Research methods are as follows:(1)Identification of two laboratory keeping strains: E.coli HB101 containing pMD20-T-luxCDABE and B.subtilis 1A751 containing plasmid pPcadA-gfp. Blue-write spot screening was used to identify E.coli HB101.Making B.subtilis 1A751 solution into a glass slide, and observe the cell characteristics under the fluorescence microscope, Plasmid pPcadA-gfp was extracted and identified by BamHI/HindIII and BamHI/EcoRI double enzyme digestion and agarose gel electrophoresis analysis;(2)Construction of recombinant pPcadA-luxCDABE-gfp: Through the genetic engineering methods,BamHI was used for isolating the target gene luxCDABE and circular vector plasmid pPcadA-gfp, and then transformation and screening was performed. T4 ligase was applied to inserting luxCDABE into the downstream of PcadA to generate recombinant plasmid DNA pPcadA-luxCDABE-gfp. Recombinant plasmid DNA was induced into E.coli for growth, clones was screened by Cm resistance plate and the plasmid DNA was extracted and identified by restriction enzyme digestion. Screened recombinant DNA was induced into B.subtilis tolerancing the heavy metal cadmium, did some research.Results: E.coli HB101 strains containing plasmid pMD20-T-luxCDABE was screened by blue-white screening method, if white colonies growed on the Amp resistance plate, the phenomenon shows the successful transformation of the plasmid pMD20-T-luxCDABE; B.subtilis 1A751 bacteria solution in glass slide emited green fluorescent under blue light excitation, indicated the successful plasmid transformation and the expression of GFP. The plamid pPcadA-luxCDABE-gfp was extracted from the engineering strains, preliminary study has identified that the target gene had been inserted into the vector DNA. It provide the basis for further study on the luxCDABE expression induced by Cd2+ and obtianing the engineered stains for monitoring Cd2+ existing in the environment .更多还原