大肠杆菌pqqL基因敲除与功能分析

Knockout and function analysis of pqqL gene in Escherichia coli

【目的】通过构建大肠杆菌pqqL基因缺陷突变株,研究大肠杆菌pqqL基因的功能。【方法】首先通过PCR扩增得到pqqL基因和kan抗性基因,在体外构建线性打靶片段pqqL-kan-pqqL。然后通过Red同源重组敲除大肠杆菌的pqqL基因,构建大肠杆菌缺失突变体DH5αΔpqqL。在此基础上通过DCIP法检测山梨糖脱氢酶活性来比较大肠杆菌突变株与亲本株中PQQ合成的情况。【结果】成功敲除了大肠杆菌的pqqL基因,DCIP法检测结果显示大肠杆菌pBCP162/DH5αΔpqqL和pMD19T Simple-pqqABCDE/DH5α能够合成PQQ,而大肠杆菌pMD19T Simple-pqqABCDE/DH5αΔpqqL不能合成PQQ。【结论】大肠杆菌pqqL基因和pqqF基因具有同样的功能。

[ Objective ]To confirm the involvement of pqqL gene of Escherichia coli in PQQ biosynthesis, a pqqL deletion mutant of E. coli DH5α was constructed and investigated. [ nethods] pqqL and kan gene were cloned and a linear targeting fragment pqqL-kan-pqqL was constructed in vitro. Then pqqL gene was knocked out and DH5α△pqqL mutant was constructed by Red homologous recombination. The PQQ biosynthesis was compared between the mutant and its parential strain by detection of bio-activity of sorbose dehydrogenase with DCIP method. [ Results] The DH5α△pqqL deletion mutant was successfully constructed, and the result indicated that PQQ would be synthesized in pBCP162/DH5α△pqqL and pMD19T Simple-pqqABCDE/DH5α, but not in pMD19T Simple-pqqABCDE/DH5α△pqqL. [Conclusion] The function of pqqL gene in Escherichia coli is the same to that of pqqF gene.