大豆对SMV抗性调控基因在不同阶段的鉴定及功能分析

Soybean mosaic virus (SMV) is one of the most broadly distributed soybean (Glycine max (L.) Merr.) diseases and causes severe yield loss and seed quality deficiency. Study of resistance genes and their functions will lay the foundation for clarifying mechanism of resistance to SMV in soybean. The objectives of this research are to develop Residual heterozygous line (RHL) using molecular markers closely linked to resistance genes on the basis of fine mapping of resistance genes and to obtain Near-Isogenic Line (NILs ) by screening resistance and susceptible lines from progeny of RHLs; To search the genes with sequence difference between resistance and susceptible lines after re-sequencing of NILs and to confirm the function of the genes with sequence difference by Virus Induced Gene Silencing (VIGS); To search resistance candidate genes and relating pathways by analysing the difference of the transcriptome RNA sequences and the difference of protein sequences of the genes between resistance and susceptible lines. To compare expression of the resistance candidate genes between the resistance and susceptible lines inoculated with SMV by RT-PCR to determine relation of the genes with resistance; To confirm relation of pathway with resistance by observing changes of symptoms of the resistance and susceptible lines inoculated with SMV after silencing key gene involved in the pathway.

大豆花叶病毒(SMV)病是我国大豆产区最重要的病害之一，严重影响大豆产量与品质。对大豆抗性基因及其功能研究将为阐明抗性机制奠定基础。本项目在精细定位基础上，利用与抗病基因紧密连锁的标记，构建高世代剩余杂合系（RHLs）。从剩余杂合系中筛选对SMV表现抗和感的近等基因系(NILs)。对近等基因系进行重测序，分析抗感近等基因系间存在序列差异的基因，筛选与抗性可能相关的基因进行功能验证；分析受SMV侵染后近等基因系在RNA水平和蛋白水平上存在显著差异的基因，筛选出可能与抗病调控有关的基因和参与的Pathway。通过沉默近等基因家系在精细定位区段内的候选基因，利用Real-Time PCR观察RNA和蛋白水平鉴定出的可能跟抗病相关的基因的表达，明确基因之间的关系。通过沉默Pathway中已知的关键基因后症状的变化，确定与抗病反应有关的Pathway，并明确候选基因在该Pathway中的作用。

大豆花叶病毒(SMV)病是我国大豆产区最重要的病害之一，严重影响大豆产量与品质。对大豆抗性基因及其功能研究将为阐明抗性机制奠定基础。本项目在把抗性基因定位在4条染色体基础上，利用与抗病基因紧密连锁的标记，构建高世代剩余杂合系（RHLs），精细定位和克隆抗性候选基因，另外利用构建的近等基因系进行转录组重测序，蛋白质组差异蛋白比较，筛选出多个抗性相关基因，对不同途径获得抗性候选基因如Gm26420、Gm26460、Gm40990、Gm39300以及NAC、NIK基因通过SMV诱导的表达差异Real-Time PCR、基因沉默（VIGS）、遗传转化以及基因敲除等技术进行了候选基因功能验证；通过候选基因与其它基因的互作分析，初步确定了候选基因可能参与的Pathway以及该候选基因在该Pathway中的作用。相关研究共发表标注论文25篇，其中SCI 论文12篇，申报专利1项，获省部级及社会奖3项，超额完成项目考核指标。