低温菌穿梭质粒的构建及转化方法研究

Construction of shuttle vector for a cold-adapted bacterium, Acinetobacter sp.DWC6

摘要: 由于低温微生物在细胞结构上的特殊性,使得对它们进行遗传操作受到很大的限制.以分离自冻土的低温菌Acinetobacter sp.DWC6为宿主菌,构建了一套外源DNA导入系统.通过在质粒pBR322和pUC118中插入一段Acinetobacter属特异性的Ori片段,成功构建了一系列穿梭质粒,并建立了稳定的转化方法,所有重组质粒均可在Escherichia coli和Acinetobacter sp.DWC6中正常复制.通过优化转化方法,使质粒在低温菌Acinetobacter sp.DWC6中的转化率达3×106转化子/μg DNA.

Abstract: Genetic manipulation of cold-adapted microorganismshas been limited so far.A host-vector system comprised of a cold-adapted bacterium Acinetobacter sp.DWC6 was shuttle plasmids were obtained by inserting an Acinetobacter-specific Ori region into pBR322 and pUC118,respectively.All vectors can been replicated both in Escherichia coli and Acinetobacter sp.DWC6,and present as a low-copy-number plasmid in Acinetobacter sp.DWC6.These plasmids were introduced into Acinetobacter sp.DWC6 by electroporation with the maximal transformation efficiency of 3×106 transformants/μg DNA.