论文部分内容阅读
目的构建奇异变形菌(PMI)聚磷酸盐激酶1(polyphosphate kinase 1,PPK1)基因缺失株,为研究ppk1基因在PMI致尿路感染中的作用及其机制奠定基础。方法通过融合PCR将目的基因上游及下游同源臂连接成一个片段,并克隆入自杀质粒pCVD442,将重组质粒转化入大肠埃希菌SM10λpir中,再与PMI受体菌进行接合试验,通过氨苄西林、蔗糖筛选得到ppk1基因缺失株(△pk1),进行PCR和DNA测序鉴定;体外比较野生株和缺失株在低营养条件下的生存能力。结果利用融合PCR将目的基因上、下游片段连接成一个重组片段,并连接至pCVD442自杀质粒;重组自杀质粒进入PMI后出现了同源重组事件,同源片段发生替换,ppk1基因被敲除,通过PCR及测序分析证实基因组目的基因已缺失;经体外测定MOPS培养基中的生长曲线,发现敲除株与野生株相比,在低营养环境中生存能力明显降低。结论自杀质粒同源重组方法可用于PMI基因敲除株的构建,是研究PMI基因功能的重要手段,ppk1基因与PMI在低营养环境中的生存能力有关。
Objective To construct a deletion mutant of PMI polyphosphate kinase 1 (PPK1) gene and lay a foundation for studying the role and mechanism of ppk1 gene in urinary tract infection caused by PMI. Methods The homologous arms upstream and downstream of the target gene were ligated into a fragment by fusion PCR and cloned into suicide plasmid pCVD442. The recombinant plasmids were transformed into Escherichia coli SM10λpir, and then joined with PMI receptor bacteria. (△ pk1) was screened by sucrose. PCR and DNA sequencing were used to identify the surviving ability of wild-type and mutant strains in low-nutrition condition. Results The upstream and downstream fragments of the target gene were ligated into a recombination fragment by fusion PCR and ligated into pCVD442 suicide plasmid. The homologous recombination event occurred after recombinant suicide plasmid was introduced into PMI, the homologous fragment was replaced, the ppk1 gene was knocked out and passed PCR and sequencing analysis confirmed that the target gene of the genome was deleted. The growth curve of MOPS medium was determined in vitro and found that the survivability of the knockout strain in the low nutrient environment was significantly lower than that in the wild strain. Conclusion The method of suicide plasmid homologous recombination can be used to construct PMI knockout strain, which is an important means to study the function of PMI gene. The relationship between ppk1 gene and the viability of PMI in low nutrition environment.