嗜热基因打靶系统Thermotargetron构建及其打靶效率检测  

Construction of thermophilic gene targeting system Thermotargetron and detection of targeting efficiency

作　　者：花登雄 管玉祝 鲍江舰 洪伟 陈峥宏 张馨月 王鑫鑫 郑建国 谷俊莹 崔古贞 HUA Deng-xiong;GUAN Yu-zhu;BAO Jiang-jian;HONG Wei;CHEN Zheng-hong;ZHANG Xin-yue;WANG Xin-xin;ZHENG Jian-guo;GU Jun-ying;CUI Gu-zhen(School of Clinical Laboratory Sciences,Guizhou Medical University,Guiyang 550004,China;Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou,School of Basic Medical Science,Guizhou Medical University,Guiyang 550025,China;Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education&Key Laboratory of Medical Molecular Biology of Guizhou Province,Guiyang 550004,China;Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学医学检验学院,贵州贵阳550004 [2]贵州医科大学基础医学院病原生物学重点实验室,贵州贵阳550025 [3]地方病与少数民族疾病教育部重点实验室&贵州省医学分子生物学重点实验室,贵州贵阳550004 [4]贵州医科大学附属医院,贵州贵阳550004

出　　处：《生物技术》2022年第3期276-283,共8页Biotechnology

基　　金：国家自然科学基金项目(31760318);贵州省科技计划项目([2019]1441,[2020]1Z067);贵州医科大学培育项目(20NSP0004);大学生创新训练项目(S202010660052)。

摘　　要：[目的]在大肠杆菌中构建基于嗜热Ⅱ型内含子的高效Thermotargetron基因打靶系统,并验证其温度敏感性及打靶效率,为嗜热Ⅱ型内含子功能分析建立快速遗传检测平台。[方法]首先,以大肠杆菌lacZ基因为靶基因,根据嗜热Ⅱ型内含子碱基识别规律设计基因打靶引物,构建IPTG诱导型Thermotargetron打靶载体。然后,利用蓝白斑筛选及菌落PCR方法检测其打靶效率,并通过优化温度、IPTG浓度及诱导时间,确定Thermotargetron在大肠杆菌中的最佳打靶条件。[结果]在大肠杆菌正常生理条件下,Thermotargetron不具有基因打靶功能,而在45℃及以上条件下可获得较好的打靶效率,且在48℃、1.0 mmol/L IPTG诱导5 h时在lacZ-369a、lacZ-60a、lacZ-515a三个位点的打靶效率分别达到98.05%±1.67%、91.76%±7.08%和69.77%±5.86%。[结论]在短暂耐高温的大肠杆菌中建立了IPTG诱导的Thermotargetron基因打靶系统,该系统在任意选择的3个靶位点的打靶效率平均为86.5%±12.1%,为嗜热Ⅱ型内含子的功能机制研究奠定了平台基础。[Objective]To construct an efficient Thermotargetron gene targeting system based on thermophilic groupⅡintrons in Escherichia coli,and verify its temperature sensitivity and targeting efficiency,and finally to establish a rapid genetic detection platform for functional analysis of thermophilic groupⅡintrons.[Method]First,using the lacZ gene of E.coli as the target gene,the gene targeting primers and IPTG inducible Thermotargetron targeting vector were designed according to the base recognition rules of thermophilic groupⅡintrons.Then,blue-white spot screening and colony PCR methods were used to detect its targeting efficiency,and the best targeting conditions of thermotargetron in E.coli were determined by optimizing the temperture,IPTG concentration and induction time.[Result]Thermotargetron did not have gene targeting function under normal physiological conditions of E.coli,but could get high efficiency at 45℃and above,and the highest targeting efficiency at the three sites of lacZ-369a,lacZ-60a and lacZ-515a reached 98.05±1.67%,91.76±7.08%and 69.76±5.86%,respectively,when induced 5 h with 1.0 mmol/L IPTG at 48℃.[Conclusion]IPTG-induced Thermotargetron gene targeting platform was successfully established in transient high temperature resistant E.coli,and the targeting efficiency of this system was 86.5±12.1%on average at three randomly selected target sites,which provides a platform foundation for the study of functional mechanism of thermophilic groupⅡintrons.

关 键 词：大肠杆菌 Thermotargetron 嗜热Ⅱ型内含子 LACZ基因 遗传检测平台 

分 类 号：Q939.93[生物学—微生物学]