嗜热Ⅱ型内含子Tel3c/4c-RT结构域活性位点分析  

Analysis of active sites from the structural domain of thermophilic group Ⅱ intron Tel3c/4c-RT

作　　者：花登雄 王鑫鑫 陈峥宏 洪伟 汪依航 俞青凤 谷俊莹 崔古贞 HUA Dengxiong;WANG Xinxin;CHEN Zhenghong;HONG Wei;WANG Yihang;YU Qingfeng;GU Junying;CUI Guzhen(School of Clinical Laboratory Science,Guizhou Medical University,Guiyang 550004,Guizhou,China;Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education,Key Laboratory of Medical Molecular Biology of Guizhou Province,Guizhou Medical University,Guiyang 550004,Guizhou,China;The Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,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：《微生物学通报》2022年第8期3049-3061,共13页Microbiology China

基　　金：国家自然科学基金(31760318,32160015);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：【背景】嗜热Ⅱ型内含子是一类由内含子RNA和内含子编码蛋白(intron encoded protein,IEP)组成且在高温条件下能够在染色体上高频移动的反转录转座子,目前已被开发为高效基因打靶工具Thermotargetron,阐明其活性催化位点,对深入研究其“归巢”机制及开发新型遗传工具具有重要意义。【目的】筛选嗜热Ⅱ型内含子Tel3c/4c-RT结构域关键活性位点,并获得失活反转录功能的内含子编码蛋白突变体。【方法】先利用生物信息学技术分析并筛选可能影响Tel3c/4c-RT反转录功能的关键氨基酸位点;然后对筛选到的关键氨基酸位点进行定点突变,并以Thermotargetron质粒为基础构建失活反转录功能的突变型嗜热Ⅱ型内含子打靶系统;最后以大肠杆菌lacZ基因为例,通过蓝白斑计数分析突变型Thermotargetron系统的打靶效率,体内验证Tel3c/4c-RT结构域关键活性位点突变对嗜热Ⅱ型内含子打靶效率的影响。【结果】共筛选到15个可能影响反转录活性的氨基酸位点,包括D194、I195、S196、G197、C198、F199、Q241、G242、R274、Y275、A276、D277、D278、L324和G325。其中,当G242和R274这2个位点突变后,嗜热Ⅱ型内含子Tel3c/4c的“归巢”效率几乎完全丧失,表明G242和R274是影响嗜热Ⅱ型内含子Tel3c/4c-RT结构域反转录功能的最核心催化位点。【结论】筛选并获得影响嗜热Ⅱ型内含子Tel3c/4c-RT结构域反转录功能的关键催化位点,为深入研究嗜热Ⅱ型内含子的“归巢”机制及其功能应用开发奠定了良好的基础。[Background]Thermophilic group Ⅱ introns are a class of retrotransposons that are composed of intron RNA and intron-encoded protein(IEP)and can move on chromosomes at high frequency under high temperature,and nowadays,they have been used to develop Themortargetron,an efficient gene-targeting tool.Therefore,it is highly important to elucidate their active catalytic sites for studying the“retrohoming”mechanism and developing new genetic tools.[Objective]To screen the key active sites from the domain of thermophilic group Ⅱ intron Tel3c/4c-RT and obtain IEP mutants with inactivated reverse transcription function.[Methods]Firstly,bioinformatics method was used to analyze and screen the key amino acid sites that might affect the reverse transcription function of Tel3c/4c-RT.Then,site-directed mutations were performed on the selected key amino acid sites,and Thermotargetron plasmids were used to construct a targeting system of thermophilic group Ⅱ intron with inactivated reverse transcription function.Finally,taking the lacZ gene of Escherichia coli as example,the targeting efficiency of the Thermotargetron mutant system was analyzed by blue-white screening,and the effect of the key active site of the Tel3c/4c-RT on the targeting efficiency of the thermophilic group Ⅱ intron was verified in vivo.[Results]A total of 15 amino acid sites that may affect reverse transcriptional activity were screened out:D194,I195,S196,G197,C198,F199,Q241,G242,R274,Y275,A276,D277,D278,L324 and G325.The“retrohoming”of Tel3c/4c was almost completely lost when the G242 and R274 were mutated,suggesting that the G242 and R274 were the core catalytic sites affecting the reverse transcription function of Tel3c/4c-RT.[Conclusion]The core catalytic sites affecting the reverse transcription function of Tel3c/4c-RT were screened out,which laid a good foundation for in-depth study of the“retrohoming”mechanism of the thermophilic group Ⅱ introns and the further development.

关 键 词：大肠杆菌 嗜热Ⅱ型内含子 Thermotargetron Tel3c/4c 内含子编码蛋白 反转录结构域 

分 类 号：Q75[生物学—分子生物学]