芽孢杆菌BC4铬抗性相关基因chrA的克隆表达

Clonal Expression of Bacillus CereusBC4 Chromium resistance-associated gene chrA

以芽孢杆菌BC4菌株为对象,通过对其铬抗性相关基因chr A的克隆表达来测定其编码蛋白ChrA还原Cr(Ⅵ)的能力以及该蛋白在芽孢杆菌Cr(Ⅵ)去除中的作用,可帮助进一步阐明芽孢杆菌与Cr(Ⅵ)的作用机理,并为芽孢杆菌应用于铬污染治理奠定理论基础。PCR扩增出芽孢杆菌BC4菌株铬抗性相关基因chr A,PCR产物经克隆与测序,得到chr A完整的DNA序列。该序列大小为1182bp,共编码393个氨基酸,预测编码蛋白分子量为43kDa。根据利用NCBI所进行的BLAST序列比对结果,判断该基因为铬转运蛋白编码基因。将chr A基因PCR产物双酶切后连接到表达载体pET28b并转化进大肠杆菌BL21中,对其表达产物进行分析。结果表明,chr A基因在大肠杆菌BL21中表达约43kDa的蛋白,与预期结果相符;重组菌株对Cr(Ⅵ)的耐受能力高于对照菌株,说明ChrA蛋白在芽孢杆菌BC4的Cr(Ⅵ)抗性机制中起重要作用,且重组菌株对Cr(Ⅵ)具备较强的抗性。

In this paper,Bacillussp.BC4 strain was used as the target,and its chromium resistance-related gene chrAwas cloned and expressed to determine the ability of its encoded protein ChrA to reduce Cr(VI)and the role of thisprotein in the removal of Cr(VI)by Bacillus,which can help to further elucidate the mechanism of Bacillus and Cr(VI)and lay the theoretical foundation for theapplication of Bacillus to chromium pollution treatment.The PCR amplified the chromium resistance-related gene chrAof BacillusstrainBC4,and the PCR product was cloned and sequenced to obtain the complete DNA sequence of chrA.The sequence size was 1182bp,encoding 393 amino acids,and the predicted molecular weight of the encoded protein was 43kDa.The expression product was analyzed by double digestion of the chrAgene PCR product and ligated into the expression vector pET28b and transformed into E.coliBL21.The results showed that the chrAgene expressed about 43kDa protein in E.coli BL21,which was consistent with the expected results;the tolerance ability of the recombinant strain to Cr(VI)was higher than that of the control strain,indicating that the ChrA protein played an important role in the Cr(VI)resistance mechanism of BacillusBC4,and the recombinant strain possessed a strong resistance to Cr(VI).