草莓镶脉病毒侵染性克隆的构建

 利用草莓镶脉病毒(Strawberry vein banding virus, SVBV)全长克隆pSVBV-E3构建SVBV侵染性克隆。pSVBV-E3经限制性内切酶酶切分别获得0.5-mer SVBV和1.0-mer SVBV,依次正向插入植物表达载体pBINPLUS,成功构建侵染性克隆重组质粒pBIN-1.5SVBV。pBIN-1.5SVBV转化农杆菌,分别接种森林草莓(Fragaria vesca)和4种烟草属植物(Nico-tiana spp.)验证其侵染性。结果表明,SVBV侵染性克隆接种森林草莓8周后发病,表现出典型的叶脉镶边黄化症状,PCR法可以从显症森林草莓中检测出SVBV cp基因,Southern blot法可以检测出SVBV基因组。而接种4种烟草属植物8周后未观察到发病症状,PCR法也未检测出SVBV cp基因。构建的SVBV侵染性克隆经接种验证能够侵染森林草莓,为进一步研究SVBV侵染森林草莓的致病机制奠定了基础。

Construction of infectious clone of Strawberry vein banding virus

Based upon the plasmid pSVBV-E3 containing the full-length Strawberry vein banding virus (SVBV) genomes, infectious clone of SVBV was constructed. Fragments of 0.5-mer SVBV and 1.0-mer SVBV were obtained by digesting plasmid pSVBV-E3 with restriction enzymes, respectively, and then sequentially ligated to the plant expression vector pBINPLUS to generate the infectious clone pBIN-1.5SVBV. Plasmid pBIN-1.5SVBV was transformed into Agrobacterium tumefaciens, and then innoculated onto Fragaria vesca and 4 species of Nicotiana plants to validate its infectivity, respectively. The result showed that typical vein banding and yellowing symptoms appeared on F. vesca plants inoculated with SVBV infectious clone 8 weeks later, and cp gene of SVBV could be detected from symptomatic F. vesca plant by PCR, and the presence of SVBV genome was further confirmed by Southern blot. In contrast, no symptoms could be observed on 4 species of Nicotiana plants inoculated with SVBV infectious clone 8 weeks later, and cp gene of SVBV could not be detected by PCR, either. Taken together, successful establishment of infection of SVBV in F. vesca through agroinoculation of the infectious clone would provide a basis for further study the pathogenic mechanism of SVBV in F. vesca.