我国部分地区沙地葡萄茎痘相关病毒分离物外壳蛋白序列变异分析

Genetic variation analysis of the coat protein of Grapevine rupestris stem pitting associated virus isolates from China

为明确我国葡萄中沙地葡萄茎痘相关病毒(GRSPaV)的感染情况及病毒外壳蛋白(coat protein,CP)基因的变异特点,从而为其致病性、病害的防治以及抗病毒基因工程等研究提供依据,本研究对采自我国16个省市自治区的65个葡萄品种305株葡萄样品中的GRSPaV进行RT-PCR检测,根据地区与品种差异选取了24个阳性样品进行cp基因克隆与测序分析,并对不同RNA提取方法进行了比较。结果显示,114株样品被GRSPaV侵染,平均带毒株率为37.4%;分离物间及同一分离物不同克隆间的序列差异较大,从24个分离物克隆获得的37条cp基因序列与来源于不同国家的12个GRSPaV分离物的核苷酸序列同源性为80.5%~99.7%,氨基酸序列同源性为88.8%~100%;各个分离物的遗传距离无明显地域差异;SiO2吸附法比SDS法和CTAB法更适宜葡萄样品RNA的提取。

To determine the infection situation and coat protein gene variant of Grapevine rupestris stem pitting associated virus( GRSPaV) in China,and provide the basis for the research of pathogenicity, disease control and anti-virus genetic engineering,GRSPaV in 305 grapevine samples of 65 varieties from 16 regions were detected by RT-PCR,and 24 positive samples for coat protein gene sequences analysis were selected by origins and varieties,and different RNA extraction methods were compared. The detection results of RT-PCR showed that 114 samples were infected with GRSPaV,with an average infection rate of 37. 4%. Sequence alignment showed that the sequence variations among different isolates and different clones from the same isolate were very high. Thirty-seven clones from 24 isolates of GRSPaV cp gene shared 80. 5% ~ 99. 7% nucleotide sequence identity and 88. 8% ~ 100% amino acid sequence identity with 12 isolates from different countries. Phylogenetic analysis demonstrated that the genetic distance among all isolates had no significant regional differences. Comparatively,the silica-capturebased extraction method was more suitable for RNA extraction from grapevine than SDS and CTAB methods.