高效严谨型大肠杆菌Targetron基因打靶系统的构建

doi:10.13560/j.cnki.biotech.bull.1985.2018-0883

生物技术通报 ›› 2019, Vol. 35 ›› Issue (6): 213-220.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0883

高效严谨型大肠杆菌Targetron基因打靶系统的构建

陈相好^1,2, 刘芳², 王彩霞², 陈峥宏^2,3, 洪伟⁴, 蔡梦迪², 张峥嵘^2,3, 綦廷娜^2,3, 廖永慧², 谷俊莹^1,5, 崔古贞^1,2,3

1. 贵州医科大学医学检验学院,贵阳 550004;
2. 贵州医科大学基础医学院,贵阳 550025;
3. 贵州省普通高等学校病原生物学特色重点实验室,贵阳 550025;
4. 贵州医科大学分子生物学重点实验室,贵阳 550004;
5. 贵州医科大学附属医院,贵阳 550004

收稿日期:2018-10-16 出版日期:2019-06-26 发布日期:2019-07-08
作者简介:陈相好,男,硕士研究生,研究方向：微生物学;E-mail：1176184604@qq.com
基金资助:
国家自然科学基金项目（31500078,31560318,31601012,31760318）,贵州省科技计划项目（黔科合基础[2018]1132）,贵州省教育厅自然科学研究项目（黔教合KY字[2014]216）,贵州省研究生科研基金立项项目（11348）,贵州省大学生创新创业训练计划项目（201710660022）

Construction of Highly Efficient and Rigorous Targetron System in Escherichia coli

CHEN Xiang-hao^1,2, LIU Fang², WANG Cai-xia², CHEN Zheng-hong^2,3, HONG Wei⁴, CAI Meng-di², ZHANG Zheng-rong^2,3, QI Ting-na^2,3, LIAO Yong-hui², GU Jun-ying^1,5, CUI Gu-zhen^1,2,3

1. School of Clinical Laboratory Sciences,Guizhou Medical University,Guiyang 550004;
2. School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025;
3. Key Laboratory of Medical Microbiology and Parasitology in Higher Education Department of Guizhou,Guiyang 550025;
4. Key Laboratory of Molecular Biology,Guizhou Medical University,Guiyang 550004;
5. Affiliated Hospital of Guizhou Medical University,Guiyang 550004

Received:2018-10-16 Published:2019-06-26 Online:2019-07-08

摘要/Abstract

摘要： 构建高效严谨型大肠杆菌Targetron基因打靶系统。将来源于pET28a的T7-lac操纵子与来源于pSY6的II型内含子组装构建大肠杆菌IPTG诱导型Targetron质粒系统。以lacZ基因为例,选择lacZ-635s和lacZ-1063a两个位点为靶位点,利用构建的IPTG诱导型Targetron系统进行基因打靶,通过分析诱导前和诱导后II型内含子在靶位点的插入效率,验证大肠杆菌IPTG诱导型Targetron系统严谨性和打靶效率。最后,通过优化诱导剂浓度及诱导时间,建立高效严谨的诱导型大肠杆菌Targetron基因打靶系统。在没有IPTG诱导时,II型内含子在两个位点均不能插入,打靶效率均为0;当加入0.5 mmol/L IPTG诱导45 min时,其在lacZ-635s位点的打靶效率提高到90.8±5.5%,在lacZ-1063a位点的打靶效率提高到92.6±2.4%。成功建立高效严谨型大肠杆菌Targetron基因打靶系统,旨为II型内含子的机理研究及应用奠定基础。

关键词: Targetron, II型内含子, 诱导系统, 大肠杆菌, lacZ

Abstract: The objective is to construct a highly efficient and rigorous Targetron gene targeting system in Escherichia coli. Firstly,the T7-lac operon from pET28a and the group II intron from pSY6 were assembled to construct an IPTG-inducible Targetron plasmid system in E. coli. Then,taking the lacZ gene as an example,the efficiency and stringency of the IPTG-inducible Targetron targeting system were verified by analyzing the insertion efficiency of the group II introns before and after induction. Finally,a highly efficient and rigorous inducible Targetron gene targeting system in E. coli was obtained after optimizing the IPTG concentration and induction time. As results,group II introns were unable to be inserted into 2 target sites at all in the absence of IPTG induction,the targeting efficiency was 0. When induced for 45 min after adding 0.5 mmol/L IPTG,the targeting efficiency at lacZ-635s site increased to 90.8±5.5%,and the targeting efficiency at lacZ-1063a site increased to 92.6±2.4%. In conclusion,the IPTG-inducible Targetron gene targeting system in E. coli is successfully established,laying a foundation for the research and application of the group II intron.

Key words: Targetron, group II intron, inducible system, Escherichia coli, lacZ

陈相好, 刘芳, 王彩霞, 陈峥宏, 洪伟, 蔡梦迪, 张峥嵘, 綦廷娜, 廖永慧, 谷俊莹, 崔古贞. 高效严谨型大肠杆菌Targetron基因打靶系统的构建[J]. 生物技术通报, 2019, 35(6): 213-220.

CHEN Xiang-hao, LIU Fang, WANG Cai-xia, CHEN Zheng-hong, HONG Wei, CAI Meng-di, ZHANG Zheng-rong, QI Ting-na, LIAO Yong-hui, GU Jun-ying, CUI Gu-zhen. Construction of Highly Efficient and Rigorous Targetron System in Escherichia coli[J]. Biotechnology Bulletin, 2019, 35(6): 213-220.

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高效严谨型大肠杆菌Targetron基因打靶系统的构建

Construction of Highly Efficient and Rigorous Targetron System in Escherichia coli

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