基于小RNA深度测序和RT-PCR鉴定鹅绒藤花叶病的病毒病原  

Small RNA sequencing and RT-PCR detection of viral pathogens infecting Cynanchum chinense

作　　者：杜江[1] 马振男 王德富[1] 牛颜冰[1] DU Jiang;MA Zhennan;WANG Defu;NIU Yanbing(College of Life Sciences,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China)

机构地区：[1]山西农业大学生命科学学院,山西晋中030801

出　　处：《草业科学》2024年第10期2229-2239,共11页Pratacultural Science

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

摘　　要：鹅绒藤(Cynanchum chinense)是一种传统中药,为明确造成鹅绒藤叶片花叶症状的原因,本研究采用小RNA深度测序技术结合RT-PCR的方法鉴定引起山西太谷鹅绒藤病毒花叶症状的病原,并通过生物信息学的手段对病毒序列进行分析。结果表明,发病鹅绒藤样品经小RNA深度测序技术共获得15039334个原始序列,将拼接contigs与NCBI中的病毒数据库进行比对注释,结果显示病原为苜蓿花叶病毒(AMV)和黄瓜花叶病毒(CMV)。利用特异引物克隆了AMV和CMV的外壳蛋白(CP)和移动蛋白(MP)基因全序列,分别命名为AMV-BR和CMV-BR。通过序列比对分析,发现AMV-BR CP氨基酸序列和核苷酸序列与AMV分离物AMV-Gyn(MH332899)、Dich-rep(MW835989)和VIC-320(MF075254)的相似性均达到100%;MP氨基酸序列和核苷酸序列与AMV分离物AMV-Gyn(MH332899)的相似性最高,均为99.3%。CMV-BR CP氨基酸序列和核苷酸序列与CMV亚组ⅠA中CMV分离物YA17(MH119159)的相似性最高,分别为100%和99.1%;MP氨基酸序列和核苷酸序列与CMV亚组Ⅰ分离物PV-0185(ON013887)的相似性最高,分别为98.2%和96.4%。MP和CP氨基酸序列系统进化分析表明,CMV-BR属于CMV亚组Ⅰ中的成员。这是首次在山西鹅绒藤上检测到AMV和CMV,该研究有助于深入了解AMV、CMV的分子进化,也为鹅绒藤病毒病的防治提供一定的理论依据。Cynanchum chinense is used in traditional Chinese medicine.To identify the causes of mosaic symptoms in the leaves of C.chinense,sRNA deep sequencing technology with RT-PCR was used to identify pathogens causing mosaic symptoms in Taigu,Shanxi,and the resulting viral sequence was analyzed using bioinformatics.The results showed that a total of 15039334 original sequences were obtained from the C.chinense samples by sRNA deep sequencing technology.The contigs were compared and annotated with the virus database in NCBI,and the results showed that they were alfalfa mosaic virus(AMV)and cucumber mosaic virus(CMV).The complete sequences of the coat protein(CP)and movement protein(MP)genes of AMV and CMV were cloned using specific primers and named AMV-BR and CMV-BR,respectively.Through sequence alignment analysis,it was found that the amino acid sequence and nucleotide sequence of AMV-BR CP were 100%higher than those of AMV isolates AMV-Gyn(MH332899),Dich-rep(MW835989),and VIC-320(MF075254),respectively.The amino acid sequence and nucleotide sequence of MP have the highest similarity with AMV isolate AMVGyn(MH332899)at 99.3%.The amino acid and nucleotide sequences of CMV-BR CP had the highest similarity with the CMV isolate YA17(MH119159)in CMV subgroup IA(100%and 99.1%,respectively),and the MP amino acid and nucleotide sequences had the highest similarity with the CMV subgroup I isolate PV-0185(ON013887)(98.2%and 96.4%,respectively).Phylogenetic analysis of the MP and CP amino acid sequences showed that CMV-BR belongs to the CMV subgroup I.This is the first time that AMV and CMV have been detected in C.chinense in Shanxi Province.This study will help to better understand the molecular evolution of AMV and CMV and provide a theoretical basis for preventing and treating C.chinense viral diseases.

关 键 词：鹅绒藤 黄瓜花叶病毒 苜蓿花叶病毒 小RNA深度测序 

分 类 号：S435.672[农业科学—农业昆虫与害虫防治]