利用RNAi技术研究水稻瘤矮病毒P8蛋白在病毒装配中的作用

摘要

Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus in the family Reoviridae, which is transmitted by leafhopper Recilia dorsalis and Nephotettix cincticeps in a persistent-propagative manner. The viral genome of RGDV encodes 6 structural proteins and 6 nonstructural proteins. The structural protein P8 is a component of viral outer capsid protein, but the function of this protein in viral infection in insect vector is still unknown. Here, RNA interference (RNAi) induced by double-stranded RNA(dsRNA) synthesized in vitro was used to investigate the role of RGDV P8 protein in viral infection in the cultured cells derived from R. dorsalis. Immunofluorescence assay of RGDV infection in cultured cells after the treatment of dsRNAs from P8 gene (dsP8) howed that dsP8 did not only knock down the expression of P8, but also reduced the accumulation of RGDV in the insect vector cells. Then viral dsRNA genome and proteins were detected by SDS-PAGE and Western blot, the results indicated that the treatment of dsP8 significantly decreased the synthesis of viral dsRNA genome and the expression of P8 protein in insect vector cells. Thus, the results suggested that structural protein P8 of RGDV might play a role in viral assembly and multiplication in insect vector cells. This provides important theoretical basis for controling the spreading of rice gall dwarf disease by using RNAi technology.%水稻瘤矮病毒(Rice gall dwarf virus, RGDV)是呼肠孤病毒科(Reoviridae)植物呼肠孤病毒属(Phytoreovirus)的一个成员，由介体电光叶蝉(Recilia dorsalis)和黑尾叶蝉(Nephotettix cincticeps)以持久增殖型方式传播。其基因组编码6个结构蛋白和6个非结构蛋白。已知结构蛋白P8是病毒外层衣壳的主要成分，但其在病毒侵染介体细胞过程中的功能尚未弄清。本研究利用体外合成的dsRNA诱导的RNA干扰(RNA interference, RNAi)技术，研究RGDV P8蛋白在病毒侵染电光叶蝉培养细胞中的功能。通过免疫荧光标记技术对dsP8处理后的电光叶蝉培养细胞进行RGDV侵染观察，发现dsP8处理不仅抑制了P8在细胞内的正常表达，同时也抑制了病毒在细胞内的积累。随后通过SDS-PAGE凝胶电泳和Western blot分别检测病毒的dsRNA基因组和病毒蛋白水平的变化，发现dsP8处理显著降低了病毒双链RNA (double-stranded RNA, dsRNA)基因组的合成水平和病毒P8蛋白的表达量。由此推测RGDV P8蛋白参与了病毒粒体的组装过程和病毒在介体昆虫细胞内的有效增殖和侵染过程。本研究结果为利用RNAi技术控制水稻瘤矮病的传播提供了重要的理论依据。