一株高效抗砷喜温硫杆菌工程菌的构建

利用DNA体外重组技术，将大肠杆菌质粒载体pUM3上的抗砷基因簇片段亚克隆到含有强启动子（tac启动子）并具有广泛寄主范围特性的IncQ族质粒pMMB24上，删除调节基因片段，构建了含有强启动子、可在tra基因诱动下转移的组成型表达的抗砷质粒pSDRA4。通过接合转移的方式将其导入专性自养极端嗜酸性喜温硫杆菌Acidithiobacillus caldus中，构建了冶金工程菌Acidithiobacillus caldus (pSDRA4)，接合转移频率为（1.444±0.797）×10-4。表明在大肠杆菌和喜温硫杆菌之间成功地建立了一个遗传转移系统。经检测，重组质粒在喜温硫杆菌中具有较好的稳定性，在无选择压力条件下传代50次基本保持稳定（重组质粒保留76% 以上）。经抗砷性能检测，与野生菌相比，构建的喜温硫杆菌工程菌抗砷能力明显提高，从10mmol/L提高到45mmol/L。

Construction of an engineered Acidithiobacillus caldus with high-efficiency arsenic resistance

Using the recombinant technique in vitro, a new arsenic resistance plasmid pSDRA4 was constructed by subcloning the arsenic resistance genes from plasmid pUM3 into the wide-host-range IncQ plasmid pMMB24 with the hybrid trp-lac ( tac ) promoter, and followed by deleting the regulative gene of the promoter, the lacIQ gene. Then plasmid pSDRA4 was introduced from E. coli into extremely acidophilic obligately chemolithotrophic Acidithiobacillus caldus by conjugative transfer with a frequency of( 1.444 +/- 0.797) x 10(-4), and the engineered strain of Acidithiobacillus caldus (pSDRA4) for biomining was constructed. The successful transfer demonstrates the development of a conjugational system between strains of E. coli and A. caldus. The recombinant plasmid pSDRA4 is stable in A. caldus. Compared with wild type A. caldus, the level of the arsenic resistance of A. caldus (pSDRA4) is greatly raised from 10mmol/L to 45mmol/L.