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

作　　者：张贝贝 胡剑刚 王欣宇 郭伟奇 姚澜 王芷洋 王帝 祁晶晶[1] 田明星 鲍衍清[1] 王少辉[1] ZHANG Beibei;HU Jiangang;WANG Xinyu;GUO Weiqi;YAO Lan;WANG Zhiyang;WANG Di;QI Jingjing;TIAN Mingxing;BAO Yanqing;WANG Shaohui(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《微生物学报》2023年第12期4814-4822,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32172856,31972654);上海市自然科学基金(22ZR1476100,23ZR1476600);中央级公益性科研院所基本科研业务费专项资金(2023JB02);中国农业科学院科技创新工程(SHVRI-ASTIP-2014-8)。

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。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.

关 键 词：环二鸟苷酸 核糖开关 双荧光素报告质粒 检测 

分 类 号：R446.5[医药卫生—诊断学]