【作者】 魏太云；

【导师】 谢联辉； 林奇英；

【作者基本信息】 福建农林大学 ， 植物病理学， 2003， 博士

【摘要】 近年来，由水稻条纹病毒(Rice stripe virus，RSV)引起的水稻条纹叶枯病在我国的发生呈上升趋势，2000～2002年在江苏、河南等省市的一些地区大范围暴发，而在云南、山东、北京、辽宁、福建、浙江、上海、河北和四川等地则持续发生或流行。RSV及其介体的广泛分布以及广泛的寄主范围极可能导致其丰富的遗传多样性。本研究对采自有代表性的江苏、河南病害暴发区及云南、山东、北京、辽宁、福建、浙江、上海等病害常发区的RSV分离物RNA2片段上的非结构蛋白(non-sructure，NS2)基因、RNA3片段上的外壳蛋白(coat protein，CP)基因和RNA4片段上的病害特异蛋白(disease-specific protein，SP)基因的遗传多样性进行了比较分析，以期为抗病育种和分子流行病学以及进一步研究病毒的演化关系与基因功能奠定基础。 通过单链构象多态性(SSCP)和序列分析技术对我国9个省RSV在NS2、CP和SP 3个基因片段上的种群遗传多样性进行分析，结果表明，我国RSV自然种群遗传结构包括1-2种优势的序列变异类型和一些低频率的微小序列变异类型。分离物内遗传结构分析结果也表明，单一分离物也包括一种主要的序列变异类型和一些次要的序列变异类型。这些结果显示我国RSV自然种群遗传结构符合准种(quasispecies)遗传结构特征分布。这是首次有关负链RNA植物病毒遗传多样性的报道。进一步分析表明，在遗传多样性上，SP基因＞CP基因＞NS2基因；在保守性方面，NS2基因＞CP基因＞SP基因。这可能与这3个基因所编码的功能蛋白在病毒与植物寄主及介体昆虫互作过程中所受到的负选择压力不同有关。对我国RSV 3个基因遗传进化亲缘关系分析结果表明，我国RSV存在以云南和云南以外病区为代表的两个亚种群，而且两个亚种群间可能存在由介体昆虫迁入而引起的奠基者效应(founder effect)，即两个亚种群间发生了基因漂变。试验结果显示，各分离物的变异及亲缘关系与其地理分布位置有一定的关系。但是在不同寄主植物和不同抗性类型的水稻品种上产生的寄主适应性对病毒种群变异的影响并不明显。 首次测定了我国水稻条纹叶枯病常年流行区的云南楚雄(YCX1)及病害暴发区的江苏洪泽(JHZ)的RSV 2个分离物全长序列，其中YCX1分离物全长为17093nts，JHZ分离物全长为17150nts。与已报道的日本T分离物全福建农林大学博十学位论文长序列(17145 nts)比较结果表明，JHz分离物和T分离物相比仅在RNA3片段基因间隔区(IR)上有5个碱基的缺失。·3个分离物基因组结构组成一致，5‘端和3‘末端非编码区最为保守;7个编码区保守性次之;变异主要发生在IR上。同源性分析表明，JHZ和YCXI分离物基本上能够子弋表病毒自然利‘群中存在的两个差异较为明显的类群。 通过对一纤细病毒属病毒RNAI编码的依赖于RNA的RNA聚合酶(RdRp)氨基酸序列分析的结果表明，该蛋白除了具有RNA聚合酶特征的基元序列结构外，还存在mRNA的转录过程中所采取的加帽起始机制的保守性结构域位点，这表明，纤细病毒属病毒和布尼安病毒科病毒及甲型流感病毒一样，都是采取加帽起始机制进行转录的。 测定了来源于我国水稻条纹叶枯病常年流行区的辽宁盘锦(LPJI)，二、南昆明(YKMI)、YCXI及暴发区江苏JHZ RSV4个分离物RNA3全长序列。与己报道的RSV云南楚雄(YCXS)、日本T和M分离物RNA3序列比较的结果表明，这7个分离物可分为两组，其中，YKMI、YCXI分离物为一组，LPJI、JHZ、YCXS、T和M分离物为另一组。测定了来源于辽宁LPJI，云南YKMI、YCXI、保山(YBSI)、永仁(YYR)、大理(YDLI)及江苏JHZRSV7个分离物RNA4全长序列。与YCXS、T和M分离物RNA4序列比较的结果表明，这10个分离物根据SP基因可分为两组，其中，云南6个分离物为一组，其它分离物为另一组;云南分离物根据NSvc4基因又可以划分为两个不同组。RNA4 IR存在634 nts、654 nts与732 nts3种不同的长度类型。RNA4片段不同区域具有相差较为明显的起源关系的现象很可能是由病毒的同源重组(homologous reeombination)变异机制引起的。 结合本研究所分析的RSV基因组不同区域序列变异特征，云南部分分离物不同片段具有明显不同的来源可能是由病毒基因组重配(:eassortmellt)引起的。 测定了我国RSV 22个分离物RNA4 IR序列。序列比较结果表明3种不同类型的序列在云南省均有存在，而在其它省份仅存在654 nts类型的序列，云南省还存在不同片段类型序列在同一分离物中混合侵染的现象。序列内部具有两个重要的结构特征。一是具有插入序列，相对于634 nts类型分离物，654 nis类型分离物都有一段长19 nts的插入序列，7犯nis类型分离物都有-段长103 nis的插入序列，插入片段间序列高度保守，Blast分析表明19 nts序列中有一段和小麦cDNA文库的序列是一致的，1 03nt:序列中有1段和大福建农林大学博卜学位论文麦cDNA文库的序列是一致的，暗示可能发生了病毒与寄主间的重组，IR序列内存在典型的重组热点(reeombination hotsPot)特征则进一步证实了这个结论;二是有两处反向重复序列，可形成两个明显的发夹结构，其中一个序列比较保守，?更多还原

【Abstract】 Rice stripe disease caused by Rice stripe virus (RSV) has a trend of higher epidemiology in China in last two years. This disease has broken out in Jiangsu and Henan provinces, and also caused severe damage to rice production in Yunnan, Shandong, Beijing, Liaoning, Fujian, Zhejiang and Shanghai provinces. Broad distribution and broad host range of RSV resulted into likely rich virus genetic diversity. Here we analyzed the population genetic diversity of NS2, coat protein (CP) and disease-specific protein (SP) genes of RSV isolates from outbreak sites at Jiangsu and Henan provinces, as well as from endemic sites at Yunnan, Shandong, Beijing, Liaoning, Fujian, Zhejiang and Shanghai provinces, in order to further discuss the molecular variability and evolution mechanisms of RSV.The population structure and genetic variation of NS2, CP and SP genes encoded by RNA2, RNA 3 or RNA 4 of RSV within and between eighty isolates collected from 9 different provinces in China were estimated by single-strand conformation polymorphism (SSCP) and sequence analysis. The results showed that Chinese RSV isolates analyzed here were composed of populations each of which contained a majority haplotype and a few haplotypes in a very low frequency. The intra-isolate genetic diversity also consisted of one major variant and other sequence variants of lower frequencies. These results suggested that population structure and genetic diversity of Chinese RSV was predicted by the quasispecies structure for RNA viruses. From the data of the dN/ds ratio (0.1975 for NS2 gene, 0.2866 for CP gene and 0.3410 for NS3 gene) and genetic diversity (0.021 for NS2 gene, 0.033 for CP gene and 0.037 for SP gene), we can concluded that NS2 gene was the most conserved and lowest diversity compared to CP and SP genes, and SP gene was the most variable among the three genes. These results indicated that different negative selection constraints imposed by different functional regions might have maintained different conserved regions of these three genes. Sequence analysis of the three genes showed that these isolates and two Japanese isolates could be divided into two diverged groups. A part of isolates collected from Yunnan province formed one group and the other group contained the rest of the RSV isolates. Phylogenetic studies based on the sequences confirmed the geographical isolation of RSV virus population, but showed that gene flow might occur between populations. However, In terms of the effects of hosts on the population structure of RSV, the finding that different isolates from different rice varieties and three different host species of wheat, barely and cockspur grass had the same SSCP patterns suggested that different host plants did not contribute to differentiation of the virus population, as observed with other plant viruses. This was the first report about population genetic structure and genetic diversity of negative-sense RNA plant viruses.The complete nucleotide (nts) sequences of RNA 1-4 of two isolates of RSV,isolated from Chuxiong (YCX1), Yunnan Province, and Hongze (JHZ), Jiangsu Province, were determined. RNA1-4 of YCX1 isolate were 8970 nts (RNA1), 3506 nts (RNA2), 2489 nts (RNA3) and 2137 nts (RNA4) in length, respectively; RNA1-4 of JHZ isolate were 8970 (RNA1), 3514 nts (RNA2), 2509 nts (RNA3) and 2157 nts (RNA4) in length, respectively. Compared with the complete nucleotide sequences of RNA1-4 of T isolate from Japan, the numbers of nucleotides in the terminal untranslated regions (UTR), intergenic regions (IRs) and open reading frames (ORF) were identical between JHZ and T isolates except for a deletion of 5 nt in length in the RNA3 IR of JHZ isolate. The most conserved regions of RNA1-4 among these three isolates located in 5’ and 3’ UTR, suggesting that these regions played a very important role in transcription and replication of viral genome. The most variable regions of these three isolates located in the IRs of RNA2-4, sequence comparison showed that there were deletion and insert sequences in the IRs, computer-a更多还原