三种葡萄病毒的RT-PCR检测和系统进化分析  

RT-PCR detections and phylogenetic studies on three viruses from grapevine

作　　者：王建辉[1,2] 刘建军[2,3] 陈克玲[1,2] 李洪雯[1,2] 何建[1,2] 关斌[1,2] 

机构地区：[1]四川省农业科学院园艺研究所,成都610066 [2]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 [3]四川省农业科学院,成都610066

出　　处：《果树学报》2013年第2期197-201,共5页Journal of Fruit Science

基　　金："十二五"四川省财政基因工程:果树生物技术研究与创新(2011JYGC05-018)

摘　　要：【目的】为了研究不同葡萄病毒病快速检测方法和四川分离株系统进化,【方法】比较3种葡萄总RNA提取方法,还优化了葡萄病毒病的RT-PCR检测方法。分别克隆了3种病毒四川分离株的部分基因组区域,并开展了系统进化分析。【结果】结果表明,改进硅胶颗粒吸附法快速从葡萄枝条的韧皮组织中提取了总RNA,并以葡萄18SRNA为内参基因,RT-PCR分别成功扩增出3种病毒的目的片段。把16个地理起源的GLRaV-2分离株的外壳蛋白基因(Coat protein,CP)分成5个系统进化组。而7个地理起源的GVB分离株CP同源性为79.1%～98.7%。此外,5个GVA四川分离株分别位于系统进化树的4个聚类群。【结论】GLRaV-2、GVB和GVA不同地理起源的分离株在核酸水平上具有变异性,其中GVA四川分离株之间具有显著的遗传差异,可能包括4种株系。[Objective]The objective of the study is to develop a sensitive detection technique on grapevine viruses and understand the phylogenetic relationships of Sichuan isolates with other geographic origins. [Method]Comparison with three different RNA extraction protocols, the Reverse Transcription-Poly- merase Chain Reaction （RT-PCR） were optimized to detect three different grapevine viruses. Partial genomes of three different viruses isolated from Sichuan were cloned and the phylogenetic studies of each isolate were conducted, respectively, [Result]The results indicated high quality RNA was obtained quickly from the mature phloem of shoots by the modified silica particles extraction only： RT-PCR was successfully used to amplify partial fragments of the GLRaV-2, GVB and GVA with 18S RNA of grapevine as internal control, respectively. In addition, phylogenetic analysis and nucleotide identities studies clearly clustered the 16 CP genes of GLRaV-2 isolates collected from different geographic origins into five phylogenetic groups. The nucleotide identities of GVB CP genes between 7 isolates of distinct ge- ographic origins were calculated to 79.1%-98.7%. Five isolates of GVA from Sichuan were clustered into 4 clades based on the phylogenetic structure, so it was suggested that the virus populations of GVA from Sichuan consisted of genetic diversity of variants. [Conclusion]The studies suggested that GLRaV-2, GVA and GVB had genetic variability at level of nu^leotide acids. Furthermore, GVA populations in Sichuan had significant genetic diversity and included 4 different strains.

关 键 词：葡萄卷叶伴随2型病毒 葡萄B病毒 葡萄A病毒 病毒外壳蛋白基因 病毒沉默抑制子基因 

分 类 号：S663.1[农业科学—果树学]