葡萄卷叶伴随3型病毒和葡萄A病毒的多重检测及其系统进化分析

Optimizing Multiple Detection and Phylogenetic Studies on Grapevine leafroll associated virus-3 and Grapevine virus A

葡萄卷叶伴随3型病毒(Grapevine leafroll associated virus-3,GLRaV-3)和葡萄A病毒(Grapevine virus A,GVA)常常复合侵染部分主栽欧美杂交葡萄品种。以半定量RT-PCR与qRT-PCR方法研究了‘希姆劳特’葡萄(Vitis vinifera L.×V.labrusca L.‘Himrod’)中不同组织内病毒的相对积累量,结果表明叶脉和韧皮部内的病毒相对积累量显著高于其他组织。以成熟枝条韧皮部组织的cDNA为模板,优化了RT-PCR多重扩增体系,可以同时扩增待测样本中两种病毒的目标基因。从一株复合感染GLRaV-3与GVA的‘藤稔’葡萄(Vitis vinifera L.×V.labrusca L.‘Fujiminori’)中,分别克隆了GLRaV-3外壳蛋白(coat protein,CP)基因全长序列和GVA部分基因组区域序列,后者包括CP基因的部分序列与病毒RNA沉默抑制子(viral suppressor of RNA silencing,VSR)基因全长序列。病毒核酸同源性分析与系统进化研究表明,不同GLRaV-3分离物的CP高度保守;而‘藤稔’葡萄的GVA分离物包含多种变异株,具有准种病毒的特点。

Co-infection of Grapevine leafroll associated virus-3（GLRaV-3）and Grapevine virus A（GVA）was a common phenomenon on some cultivars of Vitis vinifera L.× Vitis labrusca L.in Sichuan Province.Semi-quantitative RT-PCR and qRT-PCR were used to investigate the relative accumulations of the aforementioned two viruses in different tissues of infected grape trees（Vitis vinifera L.× V.labrusca L.‘Himrod’）.The results suggested the relative accumulations of viruses in petiole and phloem were higher than that of others tissues.The multiple RT-PCR was developed to amplify the target genes of GLRaV-3 and GVA in phloem tissues of mature shoots（Vitis vinifera L.× V.labrusca L.‘Fujiminori’） simultaneously.The full length of coat protein gene（CP）of GLRaV-3 and partial genome region of GVA（including partial sequence of CP and full length of viral suppressor of RNAsilencing gene,VSR）were cloned from V.vinifera L.× V.labrusca L.‘Fujiminori’separately.The nucleotide homology analysis and phylogenetic studies on different isolates of each virus were possibly revealed that CP from different GLRaV-3 isolates was highly conserved,whereas GVA Sichuan isolate contained very divergent variants and thus it could be quasispecies.