脱水四环素诱导的大肠杆菌Targetron基因打靶系统构建  

Construction of Anhydrotetracycline-inducible Targetron system in Escherichia coli

作　　者：崔古贞 花登雄 陈相好 陈峥宏 管玉祝 谷俊莹 CUI Gu-zhen;HUA Deng-xiong;CHEN Xiang-hao;CHEN Zheng-hong;GUAN Yu-zhu;GU Jun-ying(Department of Microbiology,School of Basic Medicine,Guizhou Medical University,Guiyang,Guizhou 550025,China;不详)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]遵义市第一人民医院(遵义医科大学第三附属医院),贵州遵义563002 [3]贵州医科大学医学检验学院,贵州贵阳550004

出　　处：《现代预防医学》2021年第16期3005-3010,共6页Modern Preventive Medicine

基　　金：国家自然科学基金(31760318);贵州省科技计划项目([2019]1441,[2018]5779-17和[2020]1Z067)。

摘　　要：目的在大肠杆菌中构建脱水四环素诱导的Targetron基因打靶系统,为微生物遗传改造提供可控的新型Targetron基因打靶工具。方法首先,分析大肠杆菌对脱水四环素的敏感性,筛选合适的诱导剂浓度。其次,以绿色荧光蛋白为报告基因,构建脱水四环素诱导的荧光蛋白报告系统,检测其诱导效果。然后,将上述验证的脱水四环素诱导系统与Ⅱ型内含子打靶元件组合,构建脱水四环素诱导型Targetron系统。最后,以大肠杆菌pyrF79a、lacZ1063a和lacZ635s三个基因位点为例,分析其在大肠杆菌中的打靶效率。结果脱水四环素诱导系统能够在大肠杆菌中诱导Targetron元件表达,使用0.2μg/ml脱水四环素诱导3 h该诱导型Targetron系统在pyrF79a、lacZ1063a和lacZ635s三个位点的打靶效率分别为(31.9%±5.2%)、(56.3%±10.8%)和(8.2%±3.6%)。结论在大肠杆菌中成功构建脱水四环素诱导的Targetron基因打靶系统,为微生物基因工程改造提供了良好的遗传工具。Objective To construct an anhydrotetracycline-inducible Targetron system in E.coli,and to provide a new controllable Targetron tool for microbial genetic transformation.Methods Firstly,we determined the appropriate inducer concentration by testing the sensitivity of E.coli to anhydrotetracycline and then constructed an anhydrotetracycline-induced green fluorescent protein(EGFP)report system to test the induction effect.Secondly,we constructed an anhydrotetracyclineinduced Targetron system by combining the GroupⅡintron element with the inducible operon.Finally,we used the pyrF79a,lacZ1063a and lacZ635s three sites of E.coli as examples and determined the targeting efficiency of anhydrotetracycline-inducible Targetron system through blue white spot screening and colony PCR.Results The anhydrotetracycline-inducible system could induce the expression of Targetron in E.coli,and the targeting efficiency at sites of pyrF79a,lacZ1063a and lacZ635s were(31.9%±5.2%),(56.3%±10.8%)and(8.2%±3.6%)respectively after 3 hours of induction using 0.2μg/ml anhydrotetracycline.Conclusion The anhydrotetracycline-inducible Targetron system is successfully constructed in E.coli,which provides a good tool for microbial genetic engineering.

关 键 词：大肠杆菌 脱水四环素诱导系统 EGFP Targetron Ⅱ型内含子 

分 类 号：Q812[生物学—生物工程]