【作者】 王辉；

【导师】 谢联辉；

【作者基本信息】 福建农林大学 ， 植物病理学， 2003， 硕士

【摘要】 水稻条纹病毒(Rice stripe virus，RSV)是纤细病毒属(Tenuivirus)的代表种，广泛分布于我国，给我国的水稻生产造成了严重损失。为了进一步研究RSV的分子变异及其基因组编码NS3蛋白的功能，本文通过RT-PCR及单链构象多态性分析(SSCP)并结合序列测定对我国水稻条纹病毒21个分离物NS3基因的分子变异进行了研究。 21个分离物的RNA3编码的NS3基因片断经RT-PCR扩增后均得到一段长约630bp左右的片断。通过测序结果以及这些分离物与已报道的云南Y、日本T、M的相比结果表明，这24个分离物长度均为636nt。根据核苷酸序列和氨基酸序列同源性，这些分离物可划分为两个组，第一组包括云南除昆明(KM)以外的所有分离物，包括云南保山(BSH)、沙坝(SHB)、大理(DL)、宜良(YL)、江川(JCH)、玉溪(YX)等共11个分离物，这11个分离物的NS3基因的核苷酸序列的同源性为95.2％—98.7％，其编码的氨基酸序列的同源率在95.3％—99.1％之间。其它的13个分离物可划分为第二组，主要包括来自福建、浙江、江苏、上海、北京、辽宁、河南的9个分离物，分别是三明(SM)、浙江(ZJ)、大丰(DF)、嘉定(JD)、北黑(BH)、大洼(DW)、开封(KF)、原阳(YY)、原阳2(YY2)，云南Y分离物和日本T、M分离物与第二组的关系较近，划入第二组，有趣的是云南的KM分离物也被划入这一组中去。这一组的核苷酸序列同源率在96.2％—99.4％之间，编码的氨基酸序列的同源率在93.9％—100％之间。而在两个组之间，NS3基因仅有92.6％—95.9％的同源率，编码的氨基酸的同源率在90.6％—97.2％之间。 对2个分离物经RT-PCR、克隆后的30个阳性克隆白斑进行SSCP分析，结果表明，只存在两种带型，93％的克隆的SSCP带型一致，而只有接近6％的克隆白斑的SSCP带型有异，这说明了每一个分离物都有一个主要的序列变异类型。通过21个分离物以及这两个分离物的SSCP分析结果初步表明了RSV病毒符合准种结构特征分布。 构建了含RSV NS3基因的融合蛋白原核表达载体，并在大肠杆菌(Escherichia coli)BL21中得到表达，SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)福建农林大学硕士学位论文测定融合蛋白的分子量为49kDa。认怂stem blot印迹结果显示，GST-Ns3与RSV抗血清有很强的免疫反应，表明GST-Ns3具有天然状态下病毒蛋白的抗原性。制备了GST-Ns3抗血清，并应用此抗血清建立了RSV间接EUSA检测技术，检测灵敏度为lmg鲜重的病株叶片。更多还原

【Abstract】 Rice stripe virus (RSV), a type member of Tenuivirus, it distributes abroad in China and has caused great decreases to rice yield. In order to further study the molecular variabiliby and the Function of NS3 protein, we detected molecular variations of NS3 gene among twenty-one isolates in china by reverse transcription-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis and clone sequencing.By RT-PCR, all the NS3 of twenty-one isolates were 630 bp in length. By sequence, these isolates and Y isolate of Yunnan and T, M isolates of Japan which have been previously reported were all 636nt .According to the sequences identity and characteristics of base variation of NS3 gene, these isolates could be divided into two groups. Besides KM isolate, eleven isolates which were all collected from Yunnan Province shared high homology and formed group I, including BSH^ SI^ DL, YLx JCH> YX and the others, nucleotide identities among these eleven isolates ranged from 95.2% to 98.7%, amio acid identities ranged from 95.3% to 99.1%. The other 23 isolates formed group II, including nine isolates collected from Fujian, Zhejiang, Jiangsu, Shanghai, Beijing, Liaoning, Henan, those are SM, ZJ, DF,JD,BH, DW, KF, YY, YY2, Y isolate of Yunnan and T, M isolates of Japan which have been reported belong to this group. It is interesting that KM of Yunnan belong to this group, too. Nucleotide identities among these isolates ranged from 96.2% to 99.4% and amio acid identities ranged from 93.9% to 100%. Between two groups, there were only 92.6-95.9% and 90.6-97.2% indentities in nucleotide and amino acid level, respectively.We also estimated the within-isolate population composition of SHE and KM isolates by SSCP analysis of 30 NS3 clones per isolate, more than 93% of the clones showed the same SSCP pattern as did the original RT-PCR product and 6% of the clones SSCP pattern are different. These support the hypothesis that each isolate had one predominant sequence variant and our results showed also that these RSV isolates had a qusispecies structure.The expression vector of RSV NS3 gene is constructed, a 49kDa fusion protein product is expressed in Escherichia coll BL21 successfully. The result ofwestern blot shows that GST-NS3 can reacts with the antiserum of RSV and GST-NS3 has the same immunocompetence as the natural protein of RSV. The antiserum of GST-NS3 is produced and is used in the detection of RSV by ELISA, the sensitivity is Img fresh leaf.更多还原