一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达  

Isolation and identification of a bacterial strain KHg2 with high resistance to mercury and cloning and expression of its merA gene

作　　者：曾艳[1,2] 陈强[2] 王敏[1] 孙建光[3] 高俊莲[1] 

机构地区：[1]北京市农林科学院北京农业生物技术研究中心,北京100097 [2]四川农业大学资源环境学院微生物学系,雅安625014 [3]中国农业科学院农业资源与农业区划研究所,农业部作物营养与施肥重点实验室,北京100081

出　　处：《微生物学报》2009年第12期1628-1633,共6页Acta Microbiologica Sinica

基　　金：北京市自然科学基金(5062010);国家"863计划"(2006AA06Z386)~~

摘　　要：【目的】本研究旨在从重金属汞抗性细菌中分离鉴定汞抗性基因【方法】从北京凉水河河床底泥中分离抗汞细菌,采用16SrRNA基因序列分析结合生理生化特征对菌株进行鉴定。根据GenBank中已发表的多种抗汞细菌的merA基因序列设计引物,以抗性细菌基因组DNA为模板,扩增merA基因,并在大肠杆菌(Escherichia coli)BL21(DE3)表达。同时对表达菌株的重金属汞抗性进行测定。【结果】分离得到一株能在含HgCl2为70mg/L的平板上生长良好的高抗汞细菌,编号为KHg2。16S rRNA基因序列分析结果表明,菌株KHg2与Bacillus silvestris的模式菌株DSM12223T有96%的同源性,其形态特征及生理生化特性与文献报道的Bacillus silvestris一致。从菌株KHg2中扩增得到1687bp DNA片断,其序列与Pseudomonas putida的merA基因序列同源性达到99%,将该DNA片断克隆于pET-30a(+)得到重组质粒pZY2,转化宿主菌获得表达菌株E.coliBL21(DE3).pZY2,经IPTG诱导后产生相对分子量约为33KD的蛋白。重金属汞抗性测定结果表明,表达菌株E.coliBL21(DE3).pZY2可以在含HgCl2为20mg/L的培养基中生长,而转入空载体pET-30a(+)的阴性对照菌株E.coliBL21(DE3)pET-30a(+)则不能在含HgCl2为20mg/L的培养基中生长。【结论】分离的抗汞菌株KHg2与Bacillus silvestris关系密切;从菌株KHg2中克隆到汞抗性基因merA,并在大肠杆菌中得到了成功表达;表达merA基因的大肠杆菌具有抗重金属汞的特性。[Objective] The aim of our study was to isolate and identify merA gene from a bacterial strain with high resistance to mercury. [nethodsl A bacterial strain with resistance to mercury was isolated from the river sediment of Liangshui river in Beijing,The strain was identified according to the sequence analysis of 16S rRNA gene and its physiological and biochemical properties. A pair of PCR primers was designed according to the merA gene sequences of some bacteria published in the GeneBank to amplify the complete merA gene using the genomic DNA of KHg2 as a template. The PCR-amplified DNA fragment was expressed in Escherichia coli BL21（DE3）. Mercury resistance of the expression strain was tested. [Results] A bacterial strain which could grew on LB medium plate containing 70 mg/L HgCl2 was isolated,it was named as KHg2. The strain KHg2 shares 96% sequence identity with the type strain DSM12223T of Bacillus silvestris. The morphological characteristics and the results of physiological and biochemical properties of the strain were agreement with that of Bacillus silvestris. One PCR fragment of 1680 bp was obtained from the strain, which shares 99% sequence identity with merA gene of Pseudomonas putida. The PCR- amplified DNA fragment was cloned into a pET-30a（ ＋ ） vector to obtain a expression plasmid pZY2. The expression plasmid was transformed into E. coil host strain to obtain a expression strain E. coli BL21 （DE3）·pZY2, a protein of 33 kDa was expressed by the expression strain after induced with isopropyl-b-D-thiogalactoside （IPTG）. The result of heavy metal resistance test showed that the expression strain could grew on LB medium containing 20 mg/L HgCl2 , meanwhile a negative control, E. coli BL21 （DE3）, harbouring pET-30a （ ＋ ）, could not grew on LB medium containing 20 mg/L HgCl2. [ Conclusion] The isolated strain KHg2 was closely related to Bacillus silvestris. A merA gene was cloned from the strain KHg2 and was expressed successfully in E. coll. The expression strain E. coli BL

关 键 词：重金属污染土壤 抗汞细菌 分离鉴定 16S RRNA基因序列分析 merA基因克隆与表达 

分 类 号：Q93[生物学—微生物学]