水稻齿叶矮缩病毒非结构蛋白Pns7形成伸出昆虫细胞膜的纤维丝状结构.docVIP

  水稻齿叶矮缩病毒非结构蛋白 Pns7 形成伸 出昆虫细胞膜的纤维丝状结构# ** 5 10 15 20 25 30 35  摘要：水稻齿叶矮缩病毒（Rice ragged stunt virus, RRSV）引起的水稻齿叶矮缩病是我国水 稻生产中的一种重要病毒病害。RRSV 编码的非结构蛋白 Pns7 的功能未知。本研究构建了 含有 Pns7 基因的原核表达载体，通过诱导表达获得原核表达产物，免疫注射兔子获得该蛋 白的抗体。Western blot 检测表明制备的 Pns7 抗体可以特异性地识别水稻病株中 Pns7 的积 累。利用制备的 Pns7 抗体，通过免疫荧光实验发现 Pns7 在异源昆虫细胞 Sf9 内形成可以沿 着细胞膜表面肌动蛋白(actin)突起伸长的纤维丝状的结构。另外，Pns7 的表达促使 Sf9 细胞 膜上 actin 突起进一步聚集和伸长，从而改变细胞的形态。以上的研究表明 Pns7 可能参与了 RRSV 在寄主植物或介体昆虫中的扩散。 关键词：RRSV；Pns7 蛋白；抗体制备；Sf9 细胞；纤维丝状结构 中图分类号：S435.111.4+9 Nonstructural protein Pns7 of Rice ragged stunt virus induces the formation of filament structures growing from insect cell surface Guo Nianmei, Han Qingmei, Mao Qianzhuo, Jia Dongsheng, Wei Taiyun (Institute of Plant Virology, Fujian Province Key Laboratory of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002) Abstract: Rice ragged stunt virus (RRSV), a member of the genus Oryzavirus, is one of the most economically important pathogens of rice in China. At present, the function of viral nonstructural protein Pns7 of RRSV is unknown. In this study, the RT-PCR product of Pns7 gene was recombinated into the Gateway prokaryotic expression vector pDEST-17 and then transformed into component cell Escherichia coli Rosetta. The expression of Pns7 proteins were induced by IPTG treatment. We obtained antisera against Pns7 by injected the expressed Pns7 proteins into rabbit. Western blot analysis indicated that the antisera against Pns7 could specifically recognize Pns7 proteins in diseased plants. Expression of RRSV Pns7 in insect cell Sf9 by a recombinant baculovirus resulted in the formation of filamentous structures which could protrude from the cell surface, revealed by immunofluorescence microscopy with prepared Pns7 antibodies. Furthermore, the protrusion of filaments from cell surface induced by Pns7 was accompanied by actin-based cellular protrusion. Interestingly, the expression of Pns7 led to the extension of actin-based cellular protrusion, and thus, t