【作者】 张开玉；

【导师】 周益军；

【作者基本信息】 南京农业大学 ， 植物病理学， 2008， 硕士

【摘要】 水稻条纹病毒(Rice stripe virus,Rsv)是纤细病毒属(Tenuivirus)的典型成员,由灰飞虱以持久方式传播(Laodelphax striatellus Fallen,),曾分布于我国16个省市,至今仍在部分省市暴发流行,给我国水稻生产造成了严重损失。本文利用已经制备的多克隆抗体SV21和单克隆抗体389,应用多孔板间接ELISA、DIBA和Western blotting方法都能检测出单头灰飞虱体内水稻条纹病毒,检测灵敏度以多孔板间接ELISA最高,其次为DIBA,Western最低,用单克隆抗体检测灵敏度高于多克隆抗体。RT-PCR、IC-RT-PCR和DB-RT-PCR 3种方法中,RT-PCR对单头灰飞虱稀释到400倍都可以检测到病毒;IC-RT-PCR在多克隆抗体SV21稀释浓度大于500倍就检测不出RSV,单克隆抗体稀释浓度大于800倍也检测不到RSV;DB-RT-PCR结果显示单头灰飞虱在稀释400倍后都无阳性反应。检测灰飞虱体内由水稻条纹病毒(RSV)编码的外壳蛋白CP、病害特异性蛋白SP,以及非结构蛋白NS2、NS3和NSvc4 5种蛋白以及病毒粒子(CP)与SP、NS2、NS3、NSvc4的体外结合,为进一步研究水稻条纹病毒与其介体昆虫互作提供有用信息。本文提取了高带毒灰飞虱(Q家系)总蛋白,用免疫方法检测了昆虫体内CP、SP、NS2、NS3和NSvc45种重要蛋白的表达以及病毒粒子(CP)与SP、NS2、NS3、NSvc4的体外结合情况。结果表明在带毒灰飞虱体内以上5种蛋白都能检测到,而且CP含量最高,NSvc4、SP、NS3、NS2含量依次降低;用体外原核表达后纯化的蛋白进行了RSV粒子(CP)与SP、NS2、NS3、NSvc4的体外结合情况实验,实验结果表明病毒粒子(CP)与sP、NSvc4之间存在体外结合关系。检测结果为灰飞虱作为RSV的昆虫寄主并且可以在其体内增殖的结论提供了支持,表达差异表明病毒编码蛋白在介体灰飞虱体内可能存在着一定的协调关系;CP与SP的结合为前人关于CP与SP可能共同作用于叶绿体进而影响症状的严重度的报道又提供了一个新的证据,CP与NSvc4之间存在体外结合关系,也为NSvc4可能作为运动蛋白诱导病毒在胞间运动需要CP的辅助提供了佐证。为了获取灰飞虱体内RSV受体蛋白的情况。本文以Bio-Rad高通量双向电泳系统为技术平台,通过改进和优化灰飞虱蛋白的提取方法及电泳条件,建立了适合灰飞虱总蛋白的高通量、高分辨率和高重复性的双向电泳技术体系。在灰飞虱蛋白双向电泳图谱上能检测到至少54个蛋白点。应用病毒覆盖检测技术,我们从高亲和性无毒灰飞虱蛋白质中筛选出一个大小约为60KD,等电点约为6.7的蛋白,该蛋白可以与RSV在体外发生特异性结合。更多还原

【Abstract】 Rice stripe virus (RSV), a typical member of Tenuivirus, is one of important rice viruses in Easten Asian. It had inflicated damage over a huge hectarage from 2000 in Jiangsu province and its neighor areas. Polyclonal antibody SV21 and monoclonal antibody 3B9 were used in the paper. RSV in a single small brown plant hopper (Laodelphax striatellus Fallén, SBPH) could be detected with the ELISA, DIBA and Western blotting methods successfully. Results showed that the monoclonal antibody was more sensitive than polyclonal antibody and polywell-plate indirect-ELISA with polyclonal antibody or monoclonal antibody as testing antibody had a higher specificity and sensitivity than DIBA and Western blotting. Among the three molecular detection methods, RSV could be detected with 1:400 dilution of a single SBPH using RT-PCR, however, RSV could not be detected using IC-RT-PCR if the dilution of polyclonal antibody SV21 was less than 1:500 or the dilution of monoclonal antibody was less than 1:800. The results of DB-RT-PCR also showed that RSV could not be detected with less than 1:400 dilution of a single male or female SBPH.Detecting the five proteins encoded by RSV (CP, SP, NS2, NS3 and NSvc4) in SBPH, investigating the interactions between virion (CP) and the other four proteins of RSV in vitro, and providing useful data for further studying relationship between RSV and its insect vector. The total protein of virus infected SBPH was extracted, and hybrided with specific antibodies of the five RSV proteins. The gel overlay assay was performed. All the five proteins encoded by RSV presented in the SBPH. Further more, CP has a higher density among the five proteins. Besides, results indicated that there are interactions with virion (CP) and SP or virion (CP) and NSvc4 in vitro. Our study confirmed that RSV replicates in SBPH, and different concentration of the five proteins presented in SBPH body indicated the different roles in its replication. The interaction in vitro between CP and NSvc4 (the potential movement protein) indicates that the cell-to-cell movement of RSV may depend on this relationship between CP and NSvc4. Our results also showed the interaction between CP and SP, which has been reported previously on chloroplast of rice and influence to the severity of symptoms.In order to parse the case of receptor protein of RSV in SBPH . In this dissertation, through optimizing the protein of SBPH extraction methods and electrophoresis conditions, a large-scale, high- resolution and reproducible two-dimensional electrophoresis(2-DE) analysis system was established based on the technical platform of Bio-Rad large-scale 2- DE system. At least 54 protein pots were detected on the gels of SBPH.The protein of Mr= 60 KD, pI=6.7 was detected by virus overlay assay (VOA) from SBPH of non-infected and high-affinity. There is interaction with the protein and RSV in vitro, specially.更多还原