Pseudomonas plecoglossicida NyZ12基因无痕敲除方法的建立

A Method for Constructing Unmarked Deletion Mutants of Pseudomonas plecoglossicida NyZ12

旨在建立一种适合环己胺降解菌NyZ12基因无痕敲除的可靠方法。通过overlapping PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pEX18km上,将重组质粒转化到大肠杆菌S17pir中,再通过接合转移到假单胞菌NyZ12菌株内,经pEX18km质粒上sacB基因的反向筛选得到突变株并通过PCR方法和测序鉴定。结果显示,成功构建了假单胞菌NyZ12菌株orf4637的基因突变株(NyZ12Δ4637)。通过自杀载体同源重组可以成功获得敲除的无痕突变株,且突变株基因组上没有任何抗性筛选标记残留,为环己胺降解菌NyZ12基因功能研究提供了可靠的基因敲除技术。

This study aims to establish a practical method for an unmarked gene knockout of Pseudomonas plecoglossicida NyZ12 that degrades cyclohexylamine, which is significant for further studying the molecular mechanism of it degrading cyclohexylamine. The upstream and downstream flanking DNA fragments of the target gene were fused by overlapping PCR, and then cloned into the suicide vector pExl 8km. The recombinant plasmid was introduced into Escherichia coli strain S17 pit and then transferred into NyZ12 by conjugation. The mutants by inverse screening sacB gene in pExl8km was identified by PCR and sequencing. The mutant NyZ12A4637 from NyZ12 strain orf4637 were successfully constructed. In conclusion, the unmarked gene-knockout mutant was acquired using homologous recombination of suicide vector, and there was no resistance residual of screening marker on the genome of the mutant. This study provides a reliable gene-knockout technology for investigating the genetic functions of NyZ 12.