水稻齿叶矮缩病毒非结构蛋白Pns7在水稻原生质体内的表达动态  

Expression dynamics of Rice ragged stunt virus nonstructurai protein Pns7 in rice protoplast

作　　者：张洁[1] 陈晓敏[1] 宛柏杰 吴锦鸿[1] 林文武[1] 吴祖建[1] ZHANG Jie CHEN Xiao-min WAN Bai-jie WU Jin-hong LIN Wen-wu WU Zu-jian(Institute of Plant Virology, Fujian Agriculture and Forestry University, Key Laboratory of Plant Virology of Fujian Province, Fuzhou 350002, Chin)

机构地区：[1]福建农林大学植物病毒研究所,福建省植物病毒学重点实验室,福州350002

出　　处：《植物病理学报》2017年第1期61-67,共7页Acta Phytopathologica Sinica

基　　金：高等学校博士学科点专项科研基金资助项目(20133515120004);国家自然科学基金资助项目(31301640);福建省教育厅科技项目(JA13103)

摘　　要：水稻齿叶矮缩病毒(Rice ragged stunt virus,RRSV)属于呼肠孤病毒科(Reoviridae)水稻病毒属(Oryzavirus)。该病毒编码的非结构蛋白Pns7在昆虫细胞中可形成伸出细胞膜的纤维丝状结构。本研究利用水稻原生质体培养体系,对Pns7蛋白在水稻原生质体内的复制与表达情况进行了分析。本研究首先构建了Pns7蛋白的原核表达载体,通过IPTG诱导获得大量Pns7蛋白,免疫兔子获得抗血清。Western blot证明抗血清具有特异性,间接ELISA测得其效价为1∶2 500。利用实时荧光定量PCR技术,对病毒侵染水稻原生质体后的Pns7 RNA含量进行检测,结果表明:Pns7 RNA在8 h时开始积累,24h左右达到最大值,32 h后表达量维持在一个平台期;同时,以制备的抗血清为探针,通过Western blot检测到Pns7蛋白在病毒侵染原生质体后16 h开始表达,32 h左右达到最大值,60 h后开始下降,但仍保持在较高水平。Rice ragged stunt virus（RRSV） is a member of the genus Oryzavirus,family Reoviridae.Nonstructural protein Pns7 encoded by RRSV induces the formation of filament structures protruding from the surface of insect cells.In this study,based on a rice protoplast culture system,the expression dynamics of Pns7 protein in rice protoplasts were studied.Pns7 gene was cloned into the Gateway prokaryotic expression vector,and large amount of protein was obtained after IPTG induction.Polyclonal antiserum against Pns7 protein was prepared by immunizing the rabbit.The specificity of the antiserum was confirmed by Western blot analysis.The titer of the antiserum was measured by indirect ELISA and found to be 1：2 500.Real-time quantitative RT-PCR was used to analyze the amount of Pns7 RNA in rice protoplasts after virus infection.Results showed that,accumulation of Pns7 RNA arised at 8 h after virus inoculation,and reached a maximum at about 24 h.The level of Pns7 RNA declined to a platform stage after 32 h.Meanwhile,using the prepared antiserum as the probe,Western blot analysis indicated that Pns7 protein started to express at 16 h after the virus inoculation,and reached a maximum level at about 32 h.The expression of Pns7 protein began to decline after 60 h,but still held a high level.

关 键 词：水稻齿叶矮缩病毒 水稻原生质体 Pns7 RNA Pns7蛋白 

分 类 号：S432.41[农业科学—植物病理学]