大豆花叶病毒P3蛋白与大豆互作蛋白的鉴定、功能分析及育种利用研究

Soybean Mosaic Virus (SMV) is one of the most destructive viruses that affects seed quality and yield of soybean. The SMV genome comprises a single ORF that produces 11 mature proteins. P3, one of 11 the proteins, affects SMV virulence. The objectives of this project are to screen soybean mosaic virus P3 interactors from soybean by yeast two-hybrid system; to confirm the interactions between P3 and the host proteins which have been screened using BiFc,Co-IP; to identify locations of the interactions between P3 and host proteins (candidate genes) using subcellular localization; to investigate response ways of the candidate genes to SMV infection at different time point by analysing their expression in different tissues; to research function characterizations of candidate genes by comparing the symptoms and SMV concentration level, ultrastructure and pathogenesis response genes expression level between the silenced and normal plants after inoculation with different SMV strains; to study specifity of interactions of the candidate genes with other pathogens such as TRSV and BYMV of Potyvirus and Psg strains; to identify the upstream and downstream genes of the candidate genes involved in the pathway by RNA–seq and proteomics; to verify the functions of the candidate genes through genetic transformation to soybean and to develop broad-spectrum transgenic resistance soybean germplasm.

大豆花叶病毒（SMV）严重危害大豆生产，其P3蛋白是决定SMV致病性的重要因素之一。本项目以P3为诱饵蛋白从大豆cDNA文库中筛选与其互作的蛋白，利用BiFC、免疫共沉淀等技术对重要互作蛋白进行体内、体外互作验证；利用组织表达和亚细胞定位揭示候选基因发挥功能的组织和亚细胞位置；利用VIGS沉默编码互作蛋白的（候选）基因，用Western blot及ELISA检测沉默材料接种SMV后的SMV含量及表型变化、对比不同时期的超微结构差异、分析病程相关蛋白表达变化，明确候选基因在SMV侵染大豆过程中的作用；鉴定沉默材料在马铃薯Y病毒属其它病毒及细菌侵染时的抗性变化，确定候选基因与病原互作的普遍性；沉默材料转录组测序及蛋白质组学分析，筛选与互作蛋白相关的基因，明确候选基因调控通路。转基因干扰关键候选基因功能，确认其在SMV侵染、复制和运动过程中的功能并选育广谱抗性转基因种质。

大豆花叶病毒（SMV）是危害大豆的最主要病害之一，严重已影响大豆产量和品质。SMV基因组编码的蛋白经切割加工后形成11个成熟蛋白，其中P3与SMV的毒性和寄主范围密切相关。筛选与P3互作的大豆蛋白对明确SMV侵染机制和大豆抗SMV机制都有重要意义。本项目以P3为诱饵蛋白从大豆cDNA文库中筛选与其互作的蛋白如GmEF1a、GmEF1b和GmVATPase等，并利用BiFC等技术对互作蛋白进行体内、体外互作验证；亚细胞定位把候选基因GmEF1a、GmEF1b定位在内质网上，少量在细胞核上，而GmVATPase在内质网上表达；利用VIGS沉默编码互作蛋白的（候选）基因，沉默后大豆抗性增强，研究发现候选基因GmEF1a参与内质网应激反应，从而抑制SMV的复制；转基因干扰技术GmEF1a的功能，发现其后代抗性增强，SMV引起的籽粒斑驳率下降。沉默GmVATPase材料接种SMV后症状明显减轻。Western和ELISA结果表明沉默材料的接种叶和上位叶病毒出现晚并且病毒的积累量明显降低，沉默GmVATPase影响SMV的复制和扩散，但对SMV的长距离运输没有影响。构建该基因的RNA干扰载体并进行遗传转化，转基因植株中大豆花叶病毒的含量都显著低于非转基因植株。另外，与SMV-P3互作的基因 CRISP沉默后，病毒含量上升，抗病品种出现感病症状，证明CRISP是抗病相关基因。