一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达

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16S rRNA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70 mg/L的平板上生长良好的高抗汞细菌，编号为KHg2。16S rRNA

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

Objective]The aim of our study was to isolate and identify merA gene from a bacterial strain with high resistance to mercury.Methods]A bacterial strain with resistance to mercury was isolated from the river sediment of Iiangshui 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 HgCl_2 was isolated, it was named as KHg2. The strain KHg2 shares 96% sequence identity with the type strain DSM12223~T 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 HgCl_2, 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. Coli. The expression strain E. Coli BL2l(DE3) ·pZY2 has resistance to heavy metal mercury.