大豆花叶病毒半夏分离物侵染性克隆构建及鉴定

Construction and characterization of an infectious clone of Soybean mosaic virus isolate from Pinellia ternata

大豆花叶病毒(Soybean mosaic virus,SMV)作为半夏(Pinellia ternata(Thunb.)Breit.)主要病毒病害之一,已对其产量和品质造成严重影响。构建病毒侵染性克隆是反向遗传学研究病毒基因功能、病毒与宿主相互作用的有力工具,为明确SMV侵染半夏的分子机制,开展SMV全长cDNA侵染性克隆的构建特别重要。因此文中利用Gibson体外重组系统对大豆花叶病毒山西半夏分离物(SMV-SXBX)侵染性克隆进行组装,通过农杆菌浸润法接种健康半夏;进一步通过机械传代、逆转录-聚合酶链式反应(RT-PCR)证实SMV-SXBX侵染性克隆3′末端含有poly(A)尾56 nt时具有稳定侵染性。该方法便捷、高效,且避免了SMV侵染性克隆在大肠杆菌中的不稳定问题。SMV全长侵染性cDNA克隆的构建,为进一步研究SMV复制和发病的分子机制奠定了基础。

Soybean mosaic virus(SMV),one of the major viral diseases of Pinellia ternata(Thunb.)Breit.,has had a serious impact on its yield and quality.The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host.To clarify the molecular mechanism of SMV infection in Pinellia ternata,it is particularly important to construct the SMV full-length cDNA infectious clone.Therefore,the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate(SMV-SXBX)was constructed in this study by Gibson in vitro recombination system,and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration,further through mechanical passage and RT-PCR,confirming that the 3′end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A)tail.This method is not only convenient and efficient,but also avoids the instability of SMV infectious clones in Escherichia coli.The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.