番茄斑萎病毒属病毒适应性进化研究

Tospovirus have caused severe damages on most field crops and flower plants. The complex climates, abundant hosts and effective trips in Yunnan Province have promoted spread of this disease. Therefore, Yunnan has become the most popular and severest area of the disease caused by Tospovirus. On the other side, this also provided great opportunities for evolution research. Due to the high dynamic evolution, continuous creation of new viruses, more hosts and wider areas of the disease, the research will utilize traditional biological methods, modern molecular technologies, and bioinformatics: ① To obtain viruses from different ecological conditions and different hosts through extensive field investigation, and furthermore to screen wild-type isolates with different phenotypes; ② To obtain reasserted and mutant isolates by mixed inoculations, screening, and domestication; ③ To obtain the whole genome sequences for wild-type, reasserted and mutant isolates by RT-PCR, clone and sequencing; ④ To differentiate the natural selection and directional selection by finding the polymorphism within species and difference between species using alignment of GenBank sequences and sequences from the research in the step 3. Based on the above results, the evolution mechanism and the causes for severe damages can be illustrated and solid theory can be provided for the practical prevention.

Tospovirus属病毒能侵染大多数农作物和花卉引起严重病毒病。云南因有立体的气候、丰富的植物寄主和大量的传毒蓟马，而成为我国爆发该类病害种类最多、发病最严重的区域，也为研究Tospovirus属病毒进化提供了最有利条件。针对目前该属病毒进化速度快、新病毒不断产生、寄主范围和分布区域逐级扩大的现状，本研究拟应用传统生物学研究法、现代分子生物学技术和生物信息学手段，开展①田间调查，获取不同生境和不同寄主的该属病毒，筛选表型差异明显的野生型株系；②人工混合接种、筛选和驯化，获取重排株系和突变株系；③RT-PCR、克隆和测序获得野生型株系、重排和突变株系的全序列；④与GenBank等发布的该类病毒序列进行比对分析，根据其种内遗传多态性和种间差异性，探明自然选择和定向选择的进化取向差异，进而揭示该类病毒适应性进化的机制，解析其日趋严重的原因，为制定有效防控策略提供可靠的理论依据和实践指导。

Tospovirus属病毒能侵染大多数农作物和花卉引起严重病毒病。针对目前该属病毒进化速度快、新病毒不断产生、寄主范围和分布区域逐级扩大的现状，本研究选取全球危害最严重、Tospovirus属代表种番茄斑萎病毒（TSWV）、在中国云南首次发现新种朱顶红褪绿环斑病毒（HCRV）和辣椒褪绿病毒（CaCV）为材料，应用传统生物学研究法和现代分子生物学技术从全国16个省、市、自治区采集采集300多份疑似Tospovirus样本中筛选获得9份TSWV、16份HCRV和 1份CaCV样品全基因组序列，通过Mega、RDP4、PAML软件分析等生物信息学手段，与NCBI GenBank数据库公布的全基因组序列进行比对分析研究，结果表明：(1) TSWV、HCRV和CaCV三种病毒的不同株系均与地理分布正相关与入侵寄主不相关，如TSWV主要分为欧洲和美洲株系，欧美株系明显聚为一支，亲缘关系近，而亚洲则聚为一支；进一步分析发现该类病毒地理扩张进化策略主要有3种，即基因突变、重组和重排；(2)在TSWV编码的NSs，NSm和GnGc的区域，HCRV病毒编码的NSs，NSm和GnGc以及CaCV编码的NSs，NSm，GnGc和RdRp上均检测出存在正选择位点，说明这些病毒经历了适应性进化过程；(3) 通过人工接种和复合侵染研究发现，复合侵染可以导致病毒寄主范围扩大。(4) 温度，光照等环境因子对HCRV进化研究表明，环境因子是导致病毒变异的主效因子。其次，本项目共培养博士研究生1名，硕士研究生9名，发表论文11篇，其中SCI刊源文章5篇，申请国家发明专利3项。