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园艺学报 ›› 2011, Vol. 38 ›› Issue (12): 2401-2410.

• 新技术与新方法 • 上一篇    下一篇

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

王建辉1,2,3,刘建军4,陈克玲2,李洪雯2,席德慧1,林宏辉1,*   

  1. (1生物资源和生态环境教育部重点实验室,四川大学生命科学学院,成都 610064;2四川省农业科学院园艺研究所,成都 610066;3农业部西南地区园艺作物生物学与种质创制重点实验室,成都 610066;4四川省农业科学院,成都 610066)
  • 收稿日期:2011-08-09 修回日期:2011-10-24 出版日期:2011-12-25 发布日期:2011-12-25
  • 通讯作者: 林宏辉

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

WANG Jian-hui1,2,3,LIU Jian-jun4,CHEN Ke-ling2,LI Hong-wen2,XI De-hui1,and LIN Hong-hui1,*   

  1. (1Key Laboratory of Bio-resources and Eco-environment,Ministry of Education,College of Life Science,Sichuan University,Chengdu 610064,China;2Horticulture Institute,Sichuan Academy of Agricultural Sciences,Chengdu 610066,China;3Key laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest Regions,Ministry of Agriculture,Chengdu 610066,China;4Sichuan Academy of Agricultural Sciences,Chengdu 610066,China)
  • Received:2011-08-09 Revised:2011-10-24 Online:2011-12-25 Published:2011-12-25
  • Contact: LIN Hong-hui

摘要: 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。

关键词: 葡萄, 葡萄卷叶伴随3型病毒, 葡萄A病毒, 病毒外壳蛋白基因, 病毒RNA沉默抑制子基因, RT-PCR多重检测, 病毒变异株

Abstract: 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 RNA silencing 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.

Key words: grape, Grapevine leafroll associated virus-3, Grapevine virus A, coat protein, virus silencing suppressor, multiple RT-PCR, variant

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