检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

Construction and application of a dual fluorescence reporter plasmid for measuring c-di-GMP levels in Escherichia coli

摘要:

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法，为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物，通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP (pACVcE)，然后构建c-di-GMP代谢基因过表达菌株和缺失菌株，利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】Overlap PCR扩增产物与目的靶序列一致，测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高，而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结论】本研究构建了基于核糖开关的双荧光素报告质粒，可方便、快速检测大肠杆菌胞内c-di-GMP水平。

Abstract:

Bacteria modulate intracellular concentrations of the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP), which facilitates the adaptation to environments, survival, and infection. [Objective] To establish an effective method for measuring the c-di-GMP levels in Escherichia coli. [Methods] We designed the primers for the dual fluorescence reporter plasmid based on the regulation pattern of the riboswitch receptor by c-di-GMP and the fluorescence reporter genes. The dual fluorescence reporter plasmid pAmCherry-Vc2EGFP (pACVcE) was constructed by overlap polymerase chain reaction (overlap PCR) and homologous recombination. Then, we constructed the mutants overexpressing and lacking the genes involved in c-di-GMP metabolism, and used pACVcE to measure the c-di-GMP levels in Escherichia coli. [Results] The targeted genes were successfully amplified with correct sequences. The intracellular c-di-GMP levels in Escherichia coli expressing the c-di-GMP synthase DgcZ were significantly increased, while the intracellular c-di-GMP levels expressing the c-di-GMP phosphodiesterase PdeK were significantly decreased. The deletion of the pdeK gene encoding c-di-GMP phosphodiesterase elevated the level of c-di-GMP in avian pathogenic Escherichia coli. [Conclusion] We constructed a dual fluorescence reporter plasmid based on riboswitch, which can facilitate the rapid determination of c-di-GMP levels in Escherichia coli.