大肠杆菌pqqL基因敲除与功能分析

Knockout and function analysis of pqqL gene in Escherichia coli

摘要:

摘要：【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DH5α?pqqL。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/DH5α?pqqL和pMD19T Simple-pqqABCDE/DH5α能够合成PQQ，而大肠杆菌pMD19T Simple-pqqABCDE /DH5α?pqqL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。

Abstract:

Abstract: [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. [Methods] 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.