水稻齿叶矮缩病毒Pns10蛋白在水稻原生质体内的表达

Expression of Pns10 Protein of Rice ragged stunt virus in Rice Protoplasts

【目的】水稻齿叶矮缩病毒(Rice ragged stunt virus,RRSV)Pns10蛋白在介体昆虫细胞内可形成类似病毒原质(viroplasm)的内含体,是RRSV侵染介体所必需。然而Pns10蛋白在水稻寄主中是否具有类似功能及其表达情况如何未见报道。【方法】利用大肠杆菌系统表达Pns10蛋白,免疫家兔制备多克隆抗体;通过水稻原生质体病毒侵染体系,利用免疫荧光技术分析Pns10蛋白在水稻原生质体内的分布情况,利用实时定量PCR技术和Western blot技术分别检测Pns10 RNA和Pns10蛋白在水稻原生质体内的积累情况。【结果】将Pns10基因克隆到Gateway系统原核表达载体p DEST17中,IPTG诱导表达成功后,制备融合蛋白抗血清。Western blot检测显示,该抗血清可检测感病水稻叶片中的Pns10蛋白。病毒侵染水稻原生质体后,Pns10蛋白可形成类似病毒原质的内含体;Pns10 RNA在病毒接种8 h后开始积累,24 h后达到最大值,随后开始下降;Pns10蛋白在24 h后开始表达,之后维持较高水平,60 h后略有下降。【结论】成功获得了Pns10抗血清;Pns10在水稻原生质体内成功表达,可形成类似病毒原质的内含体,并且Pns10 RNA的表达先于其蛋白的表达。

【Objective】Pns10of Rice ragged stunt virus(RRSV)forms viroplasm-like inclusion bodies which are essential for virus infection to vector insects.However,the expression pattern and function of Pns10in rice remain unknown.【Method】Polyclonal antibody against Pns10protein was prepared by immunizing rabbits with a bacterially expressed Pns10;the cellular distribution of Pns10protein in rice protoplasts was observed with an immunofluorescence microscopy;the accumulation of Pns10RNA in rice protoplasts infected by RRSV was quantified with real-time quantitative RT-PCR and that of the Pns10protein was investigated using Western blot.【Result】Pns10ORF was cloned into the Gateway prokaryotic expression vector pDEST17and its expression was induced by IPTG.Polyclonal antiserum against the Pns10was obtained and it can react with the Pns10protein from the naturally RRSV-infected rice by Western blot detecting.Pns10formed viroplasm-like inclusion bodies in protoplasts.The Pns10RNA was first detected at8h post RRSV inoculation.The accumulation of Pns10RNA peaked at24h post virus inoculation but began to decline thereafter.The Pns10protein was first detected at24h post virus inoculation.A high level of Pns10protein was maintained until60h post virus inoculation.【Conclusion】Antiserum against RRSV Pns10protein was obtained.Pns10was expressed and viroplasm-like inclusion bodies formed in rice protoplasts.In addition,the expression of Pns10RNA was earlier than that of the Pns10protein.