【作者】 李云琴；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 植物病理学， 2004， 硕士

【摘要】 烟草曲茎病毒（TbCSV）和中国番茄黄化曲叶病毒（TYLCCNV）在我国云南地区的烟草和番茄上引起严重危害。本论文对TbCSV AC4和Rep蛋白及与TYLCCNV伴随的卫星DNA的βC1蛋白进行了分子细胞学研究。 利用大肠杆菌表达系统和pET-30a载体对TbCSV Y35分离物的AC2和AC4基因进行了原核表达，用Ni²⁺-NTA树脂纯化了AC2和AC4的重组蛋白——6×His-AC2和6×His-AC4融合蛋白，并且制备了AC4蛋白的多克隆抗体。然后利用制备的AC4抗体通过胶体金标记技术对AC4蛋白在感病烟草中进行了亚细胞定位，结果发现，TbCSV的Y35AC4蛋白主要在寄主细胞质的叶绿体和线粒体中表达并积累。同时利用实验室已制备的Y35分离物的复制相关蛋白（Rep）多克隆抗体对Y35Rep蛋白在感病烟草中进行免疫胶体金标记定位并发现，Y35Rep蛋白主要在感病细胞的细胞核中表达和积累。 利用已制备的与TYLCCNV Y10分离物伴随的卫星DNA的βC1蛋白多克隆抗体对βC1蛋白在感病烟草和转基因烟草中进行亚细胞定位，结果发现，βC1蛋白在单独Y10DNA-A侵染的烟草中没有积累；在Y10DNA-A与DNA β共同侵染的烟草和转基因烟草中均主要在寄主细胞的细胞核中表达并积累，在叶绿体上也有少量积累。此外，对出现瘤样畸形生长物的转基因普通烟中的βC1蛋白也进行了免疫定位，发现βC1蛋白也是主要在细胞核中积累，而且在不同类型细胞中的表达量没有明显差异，叶绿体中也是只有少量积累。更多还原

【Abstract】 Tobacco curly shoot virus (TbCSV) and Tomato yellow curl leaf virus-China (TYLCCNV) caused serious diseases on tobacco and tomato in Yunnan province of China. In this paper the AC4 and Rep proteins encoded by TbCSV and PCI protein of satellite DNA associated with TYLCCNV were investigated by molecular cytological methods.The AC2 and AC4 genes of TbCSV Y35 isolate were expressed with pET-30a expression vector in Escherichia coll. The expressed fusion proteins (6xHis-AC2 and 6xHis-AC4) were purified with Ni2+-NTA resin and the polyclonal antibody of Y35AC4 was then prepared. Then immuno-gold labeling with the Y35AC4 antibody indicated that TbCSV AC4 protein accumulated in the chloroplast and mitochondria of infected tobacco cells. At the same time, immuno-gold labeling with the produced Rep polyconal antibody of TbCSV Y35 isolate indicated that Rep protein localized in the nuclear of the infected tobacco cells.The polyclonal antibody of C1 protein of satellite DNA of TYLCCNV Y10 isolate had been prepared by our laboratory. Immuno-gold labeling with this antibody found that TYLCCNV CI protein didn’t accumulated in tobacco cells when infected by Y10 DNA-A alone, but accumulated mainly in the nuclear of tobacco cells when infected by Y10 DNA-A and DNA together. Occasionally, the gold particles were found in the chloroplast of the infected tobacco cells. The localization of C1 protein in transgenic tobacco cells was same as that in the infected’tobacco cells. In interveinal tissue outgrowths of transgenic Nicoliana tabacum, we found that CI protein also mainly localized in the nuclear and no obvious difference was found among different cells. Minor accumulation was also found in the chloroplast.更多还原