Hg^(2+)诱导发光报告基因系统的构建及其检测红壤中残汞的研究  

Construction of Hg^(2+)-induced Luminescent Reporter Gene System and Its Application to Detect Mercury in Red Soil

作　　者：何伟[1,2] 韩诚[3] 钟文辉[3] 林先贵[1] 

机构地区：[1]中国科学院南京土壤研究所土壤与农业可持续发展国家重点实验室,南京210008 [2]南京师范大学生命科学学院,南京210046 [3]南京师范大学环境科学与工程系,南京210046

出　　处：《环境科学》2011年第10期3045-3052,共8页Environmental Science

基　　金：土壤与农业可持续发展国家重点实验室基金项目(0751010017);南京师范大学"211工程"重点学科建设项目(1843203623);江苏省普通高校自然科学研究计划资助项目(10KJD610001)

摘　　要：将汞诱导型启动子PmerT和其调控基因merR与egfp基因融合构建发光报告基因系统.通过mini-Tn5系统把merR-egfp插入到Pseudomonas putida染色体DNA上,构建成为在Hg2+诱导下能特异性表达绿色荧光蛋白的工程菌,并将其应用于检测江西红壤中汞污染.当土壤中汞含量在0.04～50 mg.kg-1范围内时,采用流式细胞仪测定工程菌表达的荧光强度,发现荧光强度与汞污染的含量呈正相关,且最低检测含量为0.04 mg.kg-1.土壤背景中一定含量的Cr2+、Zn2+、Co2+、Cu2+、Pb2+、Ag+对汞污染测定均无显著影响.正交试验结果表明,孵育温度是影响荧光测定的主要因素,其最佳范围为30～35℃.将初始含量为0.1 mg.kg-1的Hg2+添加入江西红壤并孵育15和30d后,运用原子吸收和工程菌分别对残汞进行检测,结果显示工程菌对可溶性残汞、生物质残汞、有机残汞和剩余残汞均有响应,有效检出35%～64%,其有效性限与传统分析方法一致.A luminescent reporter gene system was constructed by fusing the mercury-inducible promoter,PmerT,and its regulatory gene,merR with a promoterless reporter gene EGFP.A stable whole-cell reporter was created by mini-Tn5 and introducing the merR-egfp system cassette into the chromosome of Pseudomonas putida strain,then applied it for mercury detection in the red soil of Jiangxi province.the fluorescence density of the sensor strain was confirmed in soil extraction and fluorescence intensity was quantified by flow cytometry.The results showed positive correlation with the mercury pollutant in the concentration range of 0.04-50 mg·kg-1.The background heavy metal irons such as Cr2＋,Zn2＋,Co2＋,Cu2＋,Pb2＋,Ag＋ at certain level did not interfere with the measurement.The key factor for detecting the fluorescence density was the induction time and the optimal temperature for EGFP expression was 30-35℃.Spiked with 0.1 mg·kg-1 Hg2＋ and after 15 and 30 days incubation,red soil samples were extracted and evaluated water soluble,bioavailable,organic matter bound and residual fractions of mercury by both sensor strain and analytical way.The sensor strain appeared to have a detection range similar to that of atomic absorption spectroscopy（AAS） method and the effective detection ratio was 35%-64%.

关 键 词：发光 汞 报告基因 红壤 构建 

分 类 号：X172[环境科学与工程—环境科学]