大豆花叶病毒抗病基因Rsc15的功能鉴定及抗病机制解析

Soybean mosaic virus is the most serious virus disease in soybean production in China, and SC15, the most popular and virulent strain in major soybean producing areas in China, can break the resistance of many varieties with broad spectrum resistance, potentially threating the soybean production. Based on the fine mapping of SC15 resistance gene Rsc15 (~100 kb) in former project, here we expect to narrow the interval through the creating of a nearly isogenic line (NIL) population, developing of SNP markers etc., haplotype analysis, and sequence and expression analysis of putative candidates, eventually define the resistance gene of Rsc15. Secondly we mean to validate the function of Rsc15 in resistance through transforming the vector of overexpressing and RNAi construct (CRISPR/Cas9) into soybean. Proteomics and phosphoproteomics method (with NILs) combining yeast two hybrid system would be employed, it will allow us to know the proteins involved on Rsc15 mediated resistance and interplay of signaling pathways in defense mechanism. For revealing its disease-resistant mechanism, the resistance related factors, such as salicylic acid level and callose deposition, among NILs treated with SMV and mock, would be measured; the process of infection and spreading of SMV infectious cloning (pSC15-GFP/GUS) would be observed. Briefly, by identification and understanding the defense mechanism of Rsc15, this project would provide helpful theoretical support and breeding materials of soybean breeding for SMV resistance.

大豆花叶病毒（Soybean Mosaic Virus, SMV）病是我国最主要的大豆病毒病害，SC15是我国大豆主产区的优势和强毒株系，能突破诸多广谱抗病品种的抗性，对大豆生产构成潜在威胁。本项目拟在前期对SC15抗病基因Rsc15精细定位（~100kb）基础上，创制近等基因系（NIL）、开发SNP等标记、进行单倍型分型以缩小定位区间，并通过对抗病候选基因的序列和表达分析，克隆并构建抗病候选基因功能互补和沉默载体（CRISPR/Cas9）转化大豆以验证其功能；以NILs进行蛋白质组学和磷酸化蛋白质组学分析，结合酵母双杂交系统探究其抗病通路（网络），观测SMV诱导后水杨酸、胼胝质等抗病相关物质的变化和SMV侵染性克隆（pSC15-GFP/GUS）的侵染、扩展规律，解析其抗病机制。本项目将通过Rsc15的功能鉴定和抗病机理解析，为选育SMV抗病品种提供重要理论指导和育种材料。

大豆花叶病毒（Soybean Mosaic Virus, SMV）病是我国最主要的大豆病毒病害，严重影响大豆产量和品质，我国目前存在22个SMV株系。其中SC15是我国大豆主产区的优势和强毒株系，能突破诸多广谱抗病品种的抗性，对大豆生产构成潜在威胁。本项目在前期对SC15株系精细定位基础上，开展了以下工作：基于21个SMV株系在12个大豆品种上的症状反应，进行聚类分析，发现21个SMV株系被分为4个亚组（A，B，C和D），强毒株系（SC7，SC10和SC15）被聚类成亚组D。对SC10（4050-3）、SC14（ZHZH132）、SC15（6768）和SC18（4273）四个不同致病力SMV株系进行了全基因组序列分析发现全基因组长度均为9529 nt，均包含1个开放阅读框（ORF）。解析了广谱抗病品种RN-9对4个不同致病力SMV株系SC10、SC14、SC15和SC18的抗性基因间关系，发现RN-9对SC10、SC14、SC15和SC18的抗性可能由不同的基因控制。对SC15株系的抗病候选基因进行了分析，探究其在大豆对SMV抗病过程中的作用。对抗病候选基因Glyma.06g175100、Glyma.06g182600进行了转基因功能验证，发现这2个候选基因不具有抗SMV株系SC15功能。对大豆真核翻译起始因子eIF4E进行功能验证及转基因后代的抗病性鉴定，发现eIF4Ei阳性转基因家系具有广谱抗性，对SC3、SC7、SC15、SC18、SMV-R及菜豆普通花叶病毒和西瓜花叶病毒均表现抗病。构建了SC15株系侵染性克隆载体pSC15，研究了SMV种传及SMV在大豆体内运动情况。利用与SMV株系SC15和SC18抗病基因Rsc15和Rsc18紧密连锁的5个分子标记（Satt246、Satt286和Satt634、BARCSOYSSR_02_0667和Satt266）对来自国内外的50份大豆种质以及杂交组合“科丰1号×RN-9”后代进行了针对SC15株系的抗病性评价和选择，发现Sat_246和Satt286对抗病基因Rsc15的选择平均符合率分别为63.89%和66.67%；表明这些分子标记可用于种质资源和杂交后代群体对SMV抗性的选择。本项目将通过Rsc15的功能鉴定和抗病机理解析，为选育SMV抗病品种提供重要理论指导和育种材料。