pUCD-recA重组发光菌构建及对遗传毒性污染物响应作用  

Construction of the pUCD-recA Recombinate Luminescence Bacterium to Evaluate the Genetoxicty of Environment Pollutants

作　　者：黄新新[1] 何苗[1] 罗虹[1] 施汉昌[1] 蔡强[1] 

机构地区：[1]清华大学环境科学与工程系,水环境保护研究所,北京100084

出　　处：《环境科学》2009年第6期1722-1726,共5页Environmental Science

基　　金：“十一五”国家科技支撑计划重点项目(2006BAC19B06);浙江省重大科技专项社会发展项目(2007C13010);浙江省嘉兴市项目(2006AY2059)

摘　　要：基因重组发光菌在水质毒性的评价中具有重要的作用,本研究从分析污染物毒性损伤的机制出发,构建新型pUCD-recA基因重组发光菌.用PCR法从大肠杆菌W3110中扩增recA基因,将其与pGEM-T easy载体连接后测序.测序正确的recA片段及pUCD615载体均用BamHⅠ、EcoRⅠ双酶切,连接后电转化导入宿主菌JM109.挑取克隆,提取质粒用PCR鉴定,阳性克隆再进行测序.将构建成功的pUCD-recA载体转化入大肠杆菌RFM443,加入相应的遗传毒性污染物,观察发光响应作用.结果表明,recA基因PCR扩增出的片段为293 bp,测序结果与GenBank中的recA序列进行BLAST比对,同源性为99%,表明扩增序列正确.与pUCD615载体连接后的测序结果表明,recA基因已正确地插入到pUCD615的多克隆位点,方向和读码框正确,重组发光菌载体构建成功.将构建好的重组载体转化入RFM443宿主菌,加入遗传毒性污染物观察响应效果.丝裂霉素C（MMC）对pUCD-recA重组发光菌诱导效果最好,0.01 mg/L即可有很好的响应曲线;N′-甲基-N′-硝基亚硝基胍（MNNG）则在50-100 mg/L时可发挥最佳响应作用.Recombinate luminescence bacteria have the important role in evaluating water toxicity. A recombinate luminescence bacteria vector pUCD-recA was constructed to investigate the general toxicity of pollutants. The gene of recA amplified by PCR from W3110 was cloned into pGEM-T easy vector and sequencing. The correct PCR product and pUCD615 vector were digested with BamH Ⅰ , EeoR Ⅰ , then be fused and imported into JM109 with Several clones were selected and identificated by PCR and sequencing. The result revealed that the length of the recA fragment was 293 bp. When it was sequenced and blasted with the recA in GenBank, the homology of the sequences reached 99% indicating the amplified result correct. The sequencing result of the fragment fused with pUCD615 revealed that the gene of recA had been inserted into the multiple clone site correctly, and the insert direction and reading frame were also exactly. Transforming the pUCD- recA vector to E. coli RFM443, then added the genetoxic pollutants and observed the responding effect. The effect of MMC inducing the pUCD-recA strain was best with the dose only of 0.01 mg/L, while need the dosage of MNNG reached 50-100 mg/L.

关 键 词：重组发光菌 遗传毒性 pUCD-recA载体 响应效应 

分 类 号：X171.5[环境科学与工程—环境科学]