内含子编码蛋白Mg2+结合位点功能分析及验证  

Functional Analysis and Validation of Mg2+Binding Sites of Intron-encoded Protein

作　　者：崔古贞 陈相好 洪伟 张峥嵘 綦廷娜 陈峥宏 CUI Gu-zhen;CHEN Xiang-hao;HONG Wei;ZHANG Zheng-rong;QI Ting-na;CHEN Zheng-hong(School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025;The Third Affiliated Hospital of Zunyi Medical Univesity(The First People’s Hospital of Zunyi),Zunyi 563002;Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou,Guiyang 550025;Key Laboratory of Molecular Biology of Guizhou,Guizhou Medical University,Guiyang 550004)

机构地区：[1]贵州医科大学基础医学院,贵阳550025 [2]遵义医科大学第三附属医院(遵义市第一人民医院),遵义563002 [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]贵州省分子生物学重点实验室,贵阳550004

出　　处：《生物技术通报》2020年第10期165-172,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31760318,31601012);贵州省科技计划项目(黔科合基础[2018]1132,[2019]1441);贵州医科大学培育项目(黔科合平台人才[2018]5779-17)。

摘　　要：旨为筛选并构建II型内含子编码蛋白Mg2+结合位点突变体,验证该位点突变对II型内含子“归巢”效率的影响。利用生物信息学技术筛选关键位点,利用定点突变技术构建突变体,利用Targetron及蓝白斑计数法验证其“归巢”效率。结果显示,筛选到D308和D309两个位点是II型内含子编码蛋白Mg2+结合的核心催化位点,并成功构建该位点的三种突变体,包括两个单点突变体(D308A和D309A)和一个双点突变体(D308A/D309A),大肠杆菌体内实验结果表明,三种突变体均完全失活了II型内含子的“归巢”功能。证实了II型内含子编码蛋白Mg2+结合位点是其发挥功能的核心催化位点。This work aims to screen and construct the mutants of having intron-encoded protein Mg2+binding site,and to verify its effect on the“Retrohoming”efficiency of group Ⅱ intron.Bioinformatics technology was used to screen key sites,site-directed mutation technology to construct mutants,and Targetron and blue-and-white spot counting methods to verify its“Retrohoming”efficiency.Results showed that 2 sites,D308 and D309,were identified as the core catalytic sites for group II intron-encoded protein Mg2+binding,and three mutants of these sites were successfully constructed,including two single-site mutants(D308A and D309A)and a double-site mutant(D308A/D309A).The experimental results in Escherichia coli showed that all three mutants completely inactivated the“Retrohoming”function of group II intron.In conclusion,this work confirms that the Mg2+binding sites of group Ⅱ intron-encoded protein is the core catalytic site of it playing function.

关 键 词：Ⅱ型内含子 内含子编码蛋白 Mg2+结合位点 Targetron 反转录结构域 

分 类 号：Q78[生物学—分子生物学]