| 嗜热II型内含子Tel3c/4c-RT结构域活性位点分析 |
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| 引用本文： | 花登雄,王鑫鑫,陈峥宏,洪伟,汪依航,俞青凤,谷俊莹,崔古贞.嗜热II型内含子Tel3c/4c-RT结构域活性位点分析[J].微生物学通报,2022,49(8):3049-3061. |
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| 作者姓名： | 花登雄 王鑫鑫 陈峥宏 洪伟 汪依航 俞青凤 谷俊莹 崔古贞 |
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| 作者单位： | 贵州医科大学医学检验学院, 贵州 贵阳 550004;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;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;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;贵州医科大学医学检验学院, 贵州 贵阳 550004;贵州医科大学附属医院, 贵州 贵阳 550004;贵州医科大学医学检验学院, 贵州 贵阳 550004;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;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 |
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| 基金项目： | 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) |
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| 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、A2...
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| 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 |
| 收稿时间： | 2021/11/26 0:00:00 |
Analysis of active sites from the structural domain of thermophilic group II intron Tel3c/4c-RT |
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| HUA Dengxiong,WANG Xinxin,CHEN Zhenghong,HONG Wei,WANG Yihang,YU Qingfeng,GU Junying,CUI Guzhen.Analysis of active sites from the structural domain of thermophilic group II intron Tel3c/4c-RT[J].Microbiology,2022,49(8):3049-3061. |
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| Authors: | HUA Dengxiong WANG Xinxin CHEN Zhenghong HONG Wei WANG Yihang YU Qingfeng GU Junying CUI Guzhen |
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| Institution: | 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 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;School of Clinical Laboratory Science, Guizhou Medical University, Guiyang 550004, Guizhou, China;The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou, China; 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 |
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