苜蓿花叶病毒群体对环境的适应性进化机制研究

Alfalfa is the main pasture of China, The previous working of collection and identity of AMV in Chinese different alfalfa ecological regions reflected alfalfa mosaic virus (AMV) disease occurred with high incidence and severe symptoms, and there is an indicated drift of year-on-year increase. How AMV adapts different varieties and environments to fitness? What is the molecular evolutionary success for the adaption? Understanding the causes and consequences of AMV evaluability has important implications for designing new and robust strategies for disease control. The proposed project has three aims. First, characterizing the genetic diversity of AMV natural populations from different cultivars and climatic regions across China; Second, by performing evolution experiments starting with cDNAs on resistant cultivar NO. 5 Gannong and susceptible cultivar NO.1 Zhongmu, and under controlled conditions (high, low and permissive temperatures as 37℃, 10℃ and 25℃ respectively) and we will identify targets of selection and adaptation in AMV genome, which also need to be depended on the data of RT-qPCR to assess the fitness of the cDNA infectious populations and analyzed by the informatics mathods. The adaptive value of mutations identified in natural and experimental populations will be evaluated by reconstructing them in the infectious cDNAs and measuring fitness as relative accumulation using RT-qPCR”. Third, comparing the different mutations between the natural population and cDNA clone population to sure the best fitness genome sequences, construct the cDNA clone of best adapted AMV genome sequence and infected on the alfalfa to test the best fitness contrarily. In summary, the expected results from this project will not only disentangle the basic mechanisms of adaptation and genome evolution for this agronomically important virus but also seed light into the design of new antiviral strategies aiming to control the spread of AMV into the Chinese agroecological landscape.

苜蓿是我国主要牧草，前期调查发现AMV引起的苜蓿花叶病在全国发生普遍，危害严重，并有逐年加重趋势。AMV是通过怎样的分子进化来适应品种抗性（寄主基因型）和环境压力的?其机制是什么?解析这些问题对AMV进化生物学和该病防控具有重要的理论和应用价值。本研究拟连续5年在不同生态区采集苜蓿AMV样本，进行基因组序列测定与分子群体遗传结构分析，获得AMV自然群体多样性和变异特点；利用AMV侵染性克隆在高温37℃、低温10℃和适温25℃下分别对抗病品种（甘农5号）和感病品种（中苜1号）进行连续继代（20代），利用RT-qPCR分析群体适应性变化，研究AMV基因组高变异区段与位点；综合分析自然群体和侵染性克隆群体AMV基因组变异区段或位点的差异，获得各种选择压下AMV适应性进化特点；筛选不同选择压力下病毒群体适合度最高的基因型，构建侵染性克隆突变体，接种验证其适应性，阐明AMV广泛适应性的进化机制。

苜蓿是我国主要牧草，我们连续五年在我国苜蓿主产区内蒙、新疆、河北等13省的21个采集点获得疑似标样516株，对它们进行9种常见病毒的分子鉴定，鉴定结果为：引起苜蓿病毒病害的主流种为AMV，此外还有ToMV、CPMV、TMV、BYMV、CMV。现除了四川甘孜，其他苜蓿产区均受到了AMV的侵染。AMV引起的苜蓿病毒病害有逐年增加的趋势。此外，在以下几个地区发现了其他病毒侵染苜蓿，在河北廊坊发现了BYMV侵染；在四川甘孜发现了CPMV侵染；在甘肃兰州发现了BYMV、TMV、CPMV、CMV、ToMV侵染；在青海民和发现了TMV侵染；在黑龙江齐齐哈尔发现了BYMV、TMV、SMV侵染。说明这几个地区导致苜蓿发生病毒病害的病原丰富，出现了多种病毒复合侵染的现象。此外在河北沧州和黑龙江齐齐哈尔发现了植原体感染。这是首次在全国范围内对我国各种苜蓿病毒病害分布的调查。. 对AMV群体进行分子遗传结构分析，发现苜蓿花叶病毒病害已经形成比较稳定的种群，该病毒的MP蛋白选择压力最大，意味着该蛋白在不断适应进化中。分析发现我国分离物多样性丰富，甘肃兰州的分离物在RNA1和RNA2系统进化树中有典型的地域性特点，河北沧州样品在RNA2系统进化树中都有地域性聚类特点，西班牙分离物与青海乌兰的分离物在RNA1、RNA2和RNA1+2+3系统进化树中聚类在一起，意大利的分离物与我国香格里拉的分离物在RNA2系统进化树中聚类在一起，说明我国西部和西南部分离物与欧洲分离物有相同的进化来源，亲缘关系较近。. 利用BEAST和BaTS等软件对我国、伊朗、西班牙以及全球AMV群体的cp基因进行了分子遗传结构分析。西班牙的AMV病害发生最早，其次是中国和伊朗，我国和西班牙的AMV-cp种群间差异显著，世界范围看，各国群体之间有极显著的差异，寄主植物在种或属的层面没有显示出显著差异。这是首次对各国的AMV-cp种群进行比较分析。. AMV的cDNA侵染性克隆群体在不同抗感品种中的适应性进化研究中发现，不断的继代接种12代，致使适应性增强，而在AMV-mp的选择压力上显示，mp基因的变异是适应进化的主要表现，这为进一步揭示AMV在不同抗感品种中为适应进化而进行的遗传变异位点的研究奠定了基础。