铜绿假单胞菌clpP基因缺陷株的构建

目的 构建铜绿假单胞菌clpP基因缺陷株。方法 分别以质粒pUCGM和铜绿假单胞菌PAO1基因组为模板PCR扩增庆大霉素抗性基因(GM)和铜绿假单胞菌clpP基因及其3'、5'侧翼序列,将clpP基因及其3'、5'侧翼序列克隆至pMD19T载体, EcoRV切除clpP基因37 bp~453 bp片段后,引入庆大霉素抗性基因,构建重组质粒pCKR2;EcoRⅠ/HindⅢ双酶切重组质粒pCKR2,回收FclpP-GM-clpPR片段,与自杀质粒pEX18Tc连接,得到clpP基因缺陷的同源重组载体pCKR3;将pCKR3质粒转化大肠埃希菌SM10,与铜绿假单胞菌PAO1双亲杂交,庆大霉素筛选得到铜绿假单胞菌clpP基因缺陷株。结果 经酶切鉴定同源重组载体pCKR3构建正确;PCR和DNA测序鉴定铜绿假单胞菌clpP基因缺陷株构建成功。结论 本研究成功敲除了铜绿假单胞菌clpP基因,为进一步研究clpP基因的生物学功能奠定了基础。

Construction of the clpP mutant strain of Pseudomonas aeruginosa

To delete clpP gene of Pseudomonas aeruginosa PAO1, the clpP gene with 3' and 5' flanking sequences was amplified by PCR using the primers clpP-F1and clpP-R1, and then the PCR product was cloned into pMD19T for construction of pCKR1. A gentamicin resistance cassette amplified from pUCGM was inserted into EcoRV sites of pCKR1 for construction of pCKR2. Then the FclpP-GM-clpPR fragment was obtained by cutting pCKR2 with EcoRⅠ/HindⅢ and gel purification, and inserted it into EcoRⅠ/HindⅢ sites of pEX18Tc to construct the homologous recombinant vector pCKR3. The P. aeruginosa clpP mutant strain was obtained by conjugations between P. aeruginosa PAO1 and E. coli SM10 transformed with pCKR3. The homologous recombinant vector pCKR3 was verified by enzymatic digestion and the final P. aeruginosa clpP mutant strain was verified by PCR analysis and DNA sequencing. The clpP gene of P. aeruginosa was successfully deleted, which laid a foundation for further study of its biological function.