基于高通量测序鉴定细菌RNA加工位点方法的开发  

Development of a Method to Identify Bacterial RNA Processing Sites Based on High-throughput Sequencing

作　　者：王娜 尤梦成 任振兴[2] 许成钢 WANG Na;YOU Meng-Cheng;REN Zhen-Xing;XU Cheng-Gang(Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology,Shanxi University,Taiyuan 030006,China; Institute of Applied Chemistry,Shanxi University,Taiyuan 030006,China)

机构地区：[1]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,太原030006 [2]山西大学应用化学研究所,太原030006

出　　处：《中国生物化学与分子生物学报》2020年第6期715-723,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by National Natural Science Foundation of China (No. 31571282 and 31871252);Program for the Innovative Talents of Higher Education Institutions of Shanxi。

摘　　要：简便的RNA剪切加工位点鉴定方法对于细菌核糖核酸内切酶功能和转录后水平调控机制的研究至关重要。本研究基于第二代测序技术,开发了一种能够精确鉴定RNA剪切加工位点及其剪切效率的高通量测序方法。在该方法中,首先将各种潜在RNA剪切加工的DNA片段分别克隆至报告系统进行转录,然后利用其下游特异引物进行反转录,直接构建约500 bp的双端测序cDNA文库,并在Illumina MiSeq平台对该文库进行测序。最后通过对reads 5′末端的比对定位,精确测定发生RNA剪切的位点。利用该方法,成功鉴定了来自Ruminiclostridium Cellulolyticum的cip-cel mRNA中的3个RNA剪切加工位点。与传统引物延伸和5′RACE等方法相比,该方法不仅具有更高的安全性和通量,同时还可像Northern印迹鉴定RNA的剪切效率。Conventional methods for identification of RNA processing sites are essential to research on bacterial endoribonuclease function and regulation at post-transcriptional level.Here,we developed a high-throughput sequencing method to determine the processed sites of RNA and their cleaving efficiency employing the Next Generation Sequencing technology.In this approach,various putative RNA-processing fragments were firstly cloned into a reporter system.A cDNA library containing about 500-bp cDNA fragments harboring all candidates were constructed by reverse transcription using their downstream special primers,and then paired-end sequenced on the Illumina MiSeq platform.Finally,the processing sites were identified by location of 5'end of sequencing reads.Using this new method,we successfully identified three processed sites of the cip-cel mRNA from Ruminiclostridium cellulolyticum.It is not only a safer and higher throughput than traditional methods,but also a determination method of the RNA processing efficiency like Northern blotting.

关 键 词：高通量测序 CDNA文库 RNA剪切位点 RNA剪切效率 

分 类 号：Q74[生物学—分子生物学]