基于小RNA深度测序技术的苜蓿病毒病鉴定与分析  

Identification and analysis of alfalfa virus disease based on sRNA deep sequencing technology

作　　者：杜江[1] 马振男 王晨燕 张丽[2] 王德富[1] 牛颜冰[1] DU Jiang;MA Zhen-nan;WANG Chen-yan;ZHANG Li;WANG De-fu;NIU Yan-bing(College of Life Sciences,Shanxi Agricultural University,Taigu 030801,China;College of Grassland Sciences,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学生命科学学院,山西太谷030801 [2]山西农业大学草业学院,山西太谷030801

出　　处：《草业学报》2023年第12期115-125,共11页Acta Prataculturae Sinica

基　　金：山西省基础研究计划(自由探索类)青年项目(20210302124676);山西农业大学科技创新基金(2020BQ51);山西省高等学校科技创新项目(2021L172);国家现代农业产业技术体系项目(CARS-21)资助。

摘　　要：紫花苜蓿是一种重要的牧草,然而紫花苜蓿病毒病的侵染严重影响其品质。本研究通过小RNA深度测序技术和RT-PCR/PCR的方法对采自山西农业大学植物园中表现花叶、皱缩、卷曲的紫花苜蓿样品进行病毒病原的鉴定,结果表明病样被苜蓿花叶病毒(AMV)、豌豆线条病毒(PeSV)、苜蓿矮化病毒(ADV)和苜蓿卷叶病毒(ALCV)4种病毒复合侵染。PCR扩增获得了ALCV山西太谷紫花苜蓿分离物SXTG(OP748371)的全基因组序列,分析表明该分离物与ALCV阿根廷紫花苜蓿分离物Colonia Dora(MG792026)的序列相似性最高,达到97.34%。对扩增获得的AMV CP基因(OP748369)核苷酸序列及其编码的氨基酸序列进行序列比对分析显示,AMV山西太谷紫花苜蓿分离物SX与阿根廷紫花苜蓿AMV分离物ACat(MW835977)相似性最高,核苷酸和氨基酸相似性分别为99.24%和99.54%。同样将扩增获得的ADV CP基因序列上传至GenBank获得登录号OP957285,命名为ADV山西太谷紫花苜蓿分离物(SXJZ),通过序列分析表明该分离物与ADV分离物N1(MZ221810)亲缘关系最近,氨基酸序列相似性达到99.43%。进一步对扩增获得的PeSV CP基因编码的氨基酸(OQ108501)进行序列比对分析显示,PeSV山西紫花苜蓿分离物(SXJZ)与其他PeSV分离物CP基因编码的氨基酸相似性达到100%。这是首次从种植于山西的紫花苜蓿上检测到ALCV、ADV、AMV和PeSV的复合侵染,该研究结果有助于深入了解侵染紫花苜蓿的病毒病分子进化,为病毒病的防控提供一定的理论依据。Medicago sativa is an important forage,but the infection with alfalfa virus disease seriously affects the yield and nutritional quality of alfalfa.In this study,the virus pathogens of alfalfa samples,which exhibited mosaic,wrinkled,and curled leaves,collected from Botanical Garden of Shanxi Agricultural University were identified by small RNA deep sequencing technology and RT-PCR/PCR methods.It was found that the diseased leaf samples were co-infected by alfalfa mosaic virus(AMV),pea streak virus(PeSV),alfalfa dwarf virus(ADV)and alfalfa leaf curl virus(ALCV).The whole genome sequence of ALCV isolate SXTG(OP748371)was obtained by PCR amplification.Sequence analysis showed that the complete sequence of ALCV isolate SXTG shared the highest similarity with the ALCV isolate Colonia Dora(MG792026),namely 97.34%.Sequence alignment analysis of the AMV CP gene(OP748369)nucleoside sequence and its encoded amino acid sequences showed that the AMV isolate SX had the highest similarity with the AMV isolate ACat(MW835977),with nucleotide and amino acid similarities of 99.24%and 99.54%,respectively.Similarly,the amplified ADV CP gene sequence was uploaded to GenBank,the registration number OP957285 was obtained and the isolate was named ADV Shanxi Taigu alfalfa isolate(SXJZ).Sequence analysis showed that the isolate SXJZ had the closest genetic relationship with ADV isolate N1(MZ221810),with an amino acid sequence similarity of 99.43%.Further sequence alignment analysis of the amino acid(OQ108501)encoded by PeSV CP gene showed that the similarity of the amino acid encoded by the coat protein of the Shanxi alfalfa isolate(SXJZ)and other PeSV isolates was 100%.This is the first time that the compound infection of ALCV,ADV,AMV and PeSV has been detected in alfalfa plants in Shanxi Province.These results will help to understand the molecular evolution of the virus disease infecting alfalfa and provide a theoretical basis for the prevention and control of the virus disease.

关 键 词：紫花苜蓿 苜蓿花叶病毒 苜蓿卷叶病毒 苜蓿矮缩病毒 豌豆线条病毒 序列分析 

分 类 号：S435.4[农业科学—农业昆虫与害虫防治]