进境玉米种子中小麦花叶病毒的检测及其RNA3序列全长扩增分析  

Detection of wheat mosaic virus in imported maize seeds and sequence analysis of its full-length genome of RNA3

作　　者：田沂民 俞禄珍 易建平 王书平 印丽萍 于翠 Tian Yimin;Yu Luzhen;Yi Jianping;Wang shuping;Yin Liping;Yu Cui(Technical Center for Animal,Plant and Food Inspection and Quarantine of Shanghai Customs District,Shanghai 200135,China)

机构地区：[1]上海海关动植物与食品检验检疫技术中心,上海200135

出　　处：《植物检疫》2022年第1期34-38,共5页Plant Quarantine

基　　金：上海市科技兴农项目(2019-02-08-00-08-F01150)。

摘　　要：小麦花叶病毒(wheat mosaic virus,WMoV)是严重危害小麦、玉米等禾本科作物的种传病毒,在我国尚无发生,具有重要检疫意义。本研究利用引物WMoV-283F/R全国首次检测出玉米种子中的WMoV,成功扩增出了两个不同典型毒株NP序列,并分析其序列特征。根据WMoV-RNA3、WMoV-RNA3A和WMoV-RNA3B序列特点设计特异性引物,经RT-PCR扩增玉米种子样品2211-2和8044-10的RNA,扩增产物测序得到283 bp基因序列,分别与WMoV分离物GG1-RNA3B(GenBank登录号KT988872.1)、WMoV分离物K1-RNA 3(GenBank登录号KT988889.1)同源性为96.11%、96.47%。设计引物分别扩增玉米种子样品2211-2和8044-10的RNA核衣壳全长序列,得到WMoV-Germany RNA3、WMoV-Malaysia RNA3A和WMoV-Malaysia RNA3B全长序列。使用MEGA6软件采用Maximum likelihood法建立系统发育树,它们分别属于3个同源性差异明显的簇。通过DNAMAN序列比对分析三者的关系,RNA3序列在标尺碱基位点220~506区域序列差异性最大,而序列700~892、972~1102区段则相对保守。这为检测引物的设计提供了重要参考。WMoV的首次截获并鉴定为该病毒的检测鉴定和致病机理研究提供依据。Wheat mosaic virus(WMoV)is seed-borne virus that seriously endangers cereal plants,such as wheat and corn.It has no occurrence in China and therefore has important quarantine significance.WMoV was detected for the first time in China by using primers WMOV-283 F/R,and NP sequences of two different typical strains were successfully amplified,and the sequences characteristics were analyzed.The specific primers were used to detect WMoV in maize seeds,and its homology was analyzed.Specific primers were designed according to the sequence characteristics of WMoV-RNA3,WMoV-RNA3 A and WMo V-RNA3 B.RT-PCR was used to amplify the RNA of 2211-2 and 8044-10 samples of maize seeds,and 283 bp gene sequences were obtained by sequencing of the amplification products.The sequences had 96.11%and 96.47%similarity to WMoV isolate GG1-RNA3 B(GenBank login number KT988872.1)and K1-RNA3(GenBank login number KT988889.1)respectively.The full-length sequences of WMoV-Germany RNA3,WMo V-Malaysia RNA3 A and WMoV-Malaysia RNA3 B from samples of corn seed 2211-2 and 8044-10 were amplified.Maximum likelihood method was used to establish phylogenetic tree,which belong to 3 clusters with obvious homology difference.Through DNAMAN sequence alignment to analyze the relationship among the three,the RNA3 sequence showed the largest sequence diversity in the region of scale label sequence 220-506.The sections of scale label sequences 700-892 and 972-1102 were relatively conservative.This provided an important reference for the design of detection primers.The first time interception and identification of WMoV at imported port provided reference for the of detection and pathogenic mechanism research of the virus.

关 键 词：小麦花叶病毒 进境玉米种子 RT-PCR检测 序列分析 系统发育树 

分 类 号：S41[农业科学—植物保护] S685