基于MS2蛋白系统的可移动性RNA鉴定方法

doi:10.7668/hbnxb.20192131

华北农学报 ›› 2021, Vol. 36 ›› Issue (4): 31-36. doi: 10.7668/hbnxb.20192131

• 作物遗传育种·种质资源·生物技术 • 上一篇下一篇

基于MS2蛋白系统的可移动性RNA鉴定方法

李陆萍^1,2, 邓竹英^1,2, 汪志鹏^1,2, 梁大成^1,2

1. 湖北省主要粮食作物产业化协同创新中心, 湖北荆州 434025;
2. 长江大学湿地生态与农业利用教育部工程研究中心, 湖北省涝渍灾害与湿地农业重点实验室, 湖北荆州 434025

收稿日期:2021-04-06 出版日期:2021-08-28
通讯作者: 梁大成(1979-),男,河南信阳人,教授,博士,硕士生导师,主要从事活性氧及生物大分子长距离运输研究。
作者简介:李陆萍(1996-),女,湖北随州人,硕士,主要从事作物遗传育种研究。
基金资助:
国家自然科学基金项目（31671257）

Identification of Mobile RNA Based on the MS2 Protein System

LI Luping^1,2, DENG Zhuying^1,2, WANG Zhipeng^1,2, LIANG Dacheng^1,2

1. Hubei Collaborative Innovation Center for Grain Industry, Jingzhou 434025, China;
2. Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, Hubei Key Laboratory of Waterlogging Disaster and Wetland Agriculture, Yangtze University, Jingzhou 434025, China

Received:2021-04-06 Published:2021-08-28

摘要/Abstract

摘要： 为了方便快速地鉴定RNA的移动性，研究RNA参与植物生长发育以及生理响应过程的胞间运输机制，减少试验成本，将含有24个茎环串联重复的RNA结构（SL24）连入双元载体pUbiK成功构建了表达载体pSL24UbiK，再利用同源重组的方法将可移动性RNA FT和不可移动性RNA GUS片段分别连入表达载体pSL24UbiK成功构建了重组载体pSL24UbiK-FT、pSL24UbiK-GUS。利用农杆菌介导法，将pSL24UbiK-FT、pSL24UbiK-GUS分别与p1390-35S-MS2_FD-GFP共同在烟草叶片中瞬时表达。在Ubiquitin启动子驱动下，SL24和目标RNA形成融合片段。MS2/SL24系统的另一组分MS2蛋白与绿色荧光蛋白融合形成MS2-GFP，噬菌体外壳蛋白MS2可识别SL24 RNA结构，在共聚焦显微镜下观察烟草叶片中GFP的细胞定位情况。结果显示，可移动RNA FT在细胞质附近显示出点状信号，不可移动RNA GUS在细胞质附近没有检测到荧光信号，只在细胞核位置有荧光信号的出现。结果表明，利用该系统将目标RNA连接到表达载体pSL24UbiK上，通过与p1390-35S-MS2_FD-GFP共侵染烟草叶片，在共聚焦显微镜下观察GFP的细胞定位情况就能够确定RNA在细胞内是否可以移动，为研究RNA的移动性提供了新的技术方法。

关键词: 可移动性RNA, SL24, MS2, MS2-GFP

Abstract: In order to conveniently and quickly characterize RNA mobility, explore the intercellular transport mechanism of RNA involved in plant development and physiological response and reduce the experimental cost, this study successfully constructed the expression vector pSL24Ubik by adding the 24 tandem stem-loop repeats(SL24) to the binary vector pUbiK.The recombinant vector pSL24Ubik-FT and pSL24Ubik-GUS were sequentially constructed by homologous recombination of mobile RNA FT and non-mobile RNA GUS fragments into expression vector pSL24Ubik.pSL24UbiK-FT and p1390-35S-MS2_FD-GFP, pSL24UbiK-GUS and p1390-35S-MS2_FD-GFP were collectively transient expressed in tobacco leaves by Agrobacterium-infiltration method.MS2/SL24 system in which 24 tandem stem-loop repeats fused with target RNA were driven by ubiquitin promoter, and phage coat protein MS2 was fused with nucleus-localized GFP. Since MS2 could specifically recognize SL24 RNA sequence, the localization of fusion RNA could be revealed by the MS2-GFP fluorescent signal under the confocal scanning microscope. The result showed that mobile RNA FT had a spotty signal near the cytoplasm, while non-mobile RNA GUS had no fluorescence signal near the cytoplasm, but rather in the nucleus. The result indicated that the target RNA was linked to the expression vector pSL24UbiK. By co-infecting tobacco leaves with p1390-35S-MS2_FD-GFP, the cell localization of GFP could be observed under confocal microscope to determine whether the RNA could move in the cell. This study provides a new technical method for the future study of RNA migration.

Key words: RNA mobility, SL24, MS2, MS2-GFP

中图分类号:

Q78
Q943.2

李陆萍, 邓竹英, 汪志鹏, 梁大成. 基于MS2蛋白系统的可移动性RNA鉴定方法[J]. 华北农学报, 2021, 36(4): 31-36. doi: 10.7668/hbnxb.20192131.

LI Luping, DENG Zhuying, WANG Zhipeng, LIANG Dacheng. Identification of Mobile RNA Based on the MS2 Protein System[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(4): 31-36. doi: 10.7668/hbnxb.20192131.

http://www.hbnxb.net/CN/Y2021/V36/I4/31

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