【摘要】 通过生物信息学分析发现,pprM基因含有冷休克DNA结合域。利用Overlap PCR方法获得pprM基因DNA结合域缺失的基因片段pprMΔCSD后,酶切扩增的片段和自杀质粒空载体pk18mobsacB,并将目的基因片段与自杀质粒载体连接构建重组载体pk18mobsacB-pprMΔCSD,将重组子转化大肠杆菌JM109感受态,筛选出阳性克隆,酶切及测序鉴定。成功构建pprM DNA结合域基因敲除重组体,且生物信息学分析预测结果显示pprM DNA结合域与耐辐射奇球菌抗辐射功能相关。更多还原

【Abstract】 Bioinformatics analysis showed that pprM gene contained a Cold-shock DNA-binding domain. The pprM△CSD was obtained by Overlap-PCR technology. And then it was connected with the suicide vector pk18mobsacB. Before ligation, amplified fragment and the empty vector were digested by the restriction enzyme EcoR? and BamH?. The ligation products were transformed into E.coil JM109. Positive clones were selected and followed by restriction enzyme digestion and sequencing analysis results. The pprM gene DNA binding domain knockout vector recombinants were successfully constructed. The bioinformatics analysis shows that pprM gene DNA binding domain is associated with the radiation resistance function in Deinococcus radiodurans.更多还原