基于小RNA深度测序和RT-PCR检测侵染大豆的病毒种类  

Small RNA sequencing and RT-PCR detection of virus species infecting soybeans

作　　者：王秋实 闫哲 李玮瑜 杨爱珍[1] 任俊达 尚巧霞[1] Wang Qiushi;Yan Zhe;Li Weiyu;Yang Aizhen;Ren Junda;Shang Qiaoxia(College of Bioscience and Resource Environment/Key Laboratory of Urban Agriculture(North China),Ministry of Agri-culture and Rural Affairs,Beijing University of Agriculture,Beijing 102206,China;College of Plant Science and Technol-ogy/Beijing Key Laboratory of New Technology in Agricultural Application/National Demonstration Center for Experimen-tal Plant Production Education,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]北京农学院,生物与资源环境学院/农业农村部华北都市农业重点实验室,北京102206 [2]北京农学院,植物科学技术学院/农业应用新技术北京市重点实验室/植物生产国家级实验教学示范中心,北京102206

出　　处：《山西农业大学学报（自然科学版）》2021年第4期25-32,共8页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：北京市科学技术协会2020-2022年度青年人才托举工程项目();北京农学院青年科学基金(QNKJ202001)。

摘　　要：[目的]大豆病毒病的发生影响大豆的产量和品质。本研究应用小RNA深度测序技术检测山西省太谷县侵染大豆的病毒种类,研究结果可以为防控大豆病毒病提供依据。[方法]2019年在山西省晋中市太谷县大豆田调查大豆病毒病发生情况,并采集36份样品进行小RNA深度测序,经RT-PCR检测和序列测定分析结果明确山西省侵染大豆的病毒种类。[结果]太谷县所调查大豆的主要症状是叶片变色、畸形和植株矮化等。采集有典型症状的36份样品经小RNA深度测序共获得32716152条小RNA序列。将拼接获得的contigs与NCBI数据库进行比对,最终获得377个被注释的contigs,包括黄瓜花叶病毒(cucumber mosaic virus,CMV)105条contigs、大豆花叶病毒(soybean mosaic vi‐rus,SMV)267条contigs、蚕豆萎蔫病毒2号(broad bean wilt virus 2,BBWV-2)5条contigs。选用以上病毒的特异性引物进行RT-PCR检测,选取病毒阳性样品进行序列测定和分析,验证了小RNA深度测序分析的结果。获得了CMV、SMV和BBMV2山西太谷大豆分离物(STS)的部分序列(GenBank登录号为MW556479,MW556478和MW556484),与来自NCBI数据库中不同地区的病毒分离物序列分别进行了比较,同源一致性为93.59%~99.59%。在采集的大豆样品中可以检测到CMV、SMV和BBWV-2三种病毒,检出率分别为97.2%、94.4%和13.9%。其中34株样品均为不同病毒的复合侵染,复合侵染率为97.1%,同时被CMV和SMV侵染的样品有29株,同时被3种病毒侵染的样品有5株。[结论]危害山西省太谷县大豆的病毒有CMV、SMV和BBWV-2,其中CMV、SMV为优势病毒,应控制毒源和传毒媒介来降低该地区大豆病毒病发生危害。[Objective]The occurrence of soybean virus disease affects the yield and quality of soybean.In this study,small RNA deep sequencing technology was used to detect viruses infecting soybean planted in Taigu County Shanxi Province.The results of the study can provide a research basis for the prevention and control of soybean virus diseases.[Methods]In 2019,we investigated the occurrence of virus diseases and collected 36 samples for small RNA deep sequencing in soybean fields in Taigu County,Jinzhong City,Shanxi Province.RT-PCR detection and sequence analysis,viruses infecting soybeans in Shanxi Province were identified.[Results]The results showed that the main symptoms of soybeans investigated in Taigu County were leaf discoloration,deformity,and plant dwarfing.And 32,716,152 small RNA sequences were obtained through deep sequencing of small RNAs from 36 samples collected with typical symptom.Obtained contigs were compared with sequences from the NCBI database and finally 377 annotated contigs were obtained including 105 contigs of cucumber mosaic virus(CMV),267 contigs of soybean mosaic virus(SMV)and 5 contigs of broad bean wilt virus 2(BBWV-2).The specific primers of the above viruses were selected for RT-PCR detection,and virus-positive samples were selected for sequence determination and analysis,which verified the results of small RNA deep sequencing.Partial sequences of CMV,SMV and BBMV2 Shanxi Taikoo Soybean Isolate(STS)were obtained(GenBank numbers are MW556479,MW556478 and MW556484),Compared with the sequences of virus isolates from different regions in the NCBI database,the homology is 93.59%~99.59%.The detection rates of CMV,SMV and BBWV-2 from collected soybean samples were 97.2%,94.4%and 13.9%,respectively.Furthermore 34 samples were detected by coinfection with 2 or 3 viruses with the complex infection rate of 97.1%in which 29 samples infected by CMV and SMV and 5 samples infected by three viruses at the same time.[Conclusion]The viruses that harm soybeans in Taigu County,Shanxi Province are CMV,SMV a

关 键 词：大豆病毒病 小RNA深度测序 RT-PCR检测 病毒检测 

分 类 号：S432.41[农业科学—植物病理学]