马铃薯Y病毒极端抗性基因Rychc的克隆与抗病机理解析

PVY侵染马铃薯后严重影响其产量和品质，开发利用寄主自身的抗性是解决病害问题的有效途径。项目前期已将S. chacoense的极端抗性基因Rychc定位于第9 号染色体末端834 kb的范围。在左侧筛选到8个连锁SNPs标记和1个基因标记M9，在右侧筛选到2个连锁的基因标记M32和M236。本研究在此基础上展开，筛选更多的重组单株并进行表型鉴定，开发定位区段内与抗性连锁的SNP标记，精细定位Rychc于100 kb以内的范围。分析候选基因在抗感池间的变异位点，并利用VIGS技术快速鉴定其功能。构建超量表达载体和抑制表达载体验证候选基因功能，以确认并克隆Rychc基因。进一步以Rychc为饵蛋白，采用Y2H、BiFC和Pull-down多种技术筛选与Rychc互作的无毒基因，明确寄主与病毒互作的分子机理。研究结果为马铃薯抗病毒育种提供基因资源和基因标记，为解析潜在的抗病机理奠定基础。

The damage caused by the Potato-Virus-Y (PVY) is a major yield-limiting and quality degradation factor in potato cultivation. The cultivation of PVY-resistant varieties is an effective strategy to address this problem. Previously, Rychc from S. chacoense was an extreme resistance gene to PVY had been mapped on the distal 834 kb region of chromosome 9. 8 SNP markers and 1gene marker (M9) showed linkages with Rychc on the left side. Gene marker M32 and M236 showed linkages with Rychc on the other side. In this study, more recombinant clones are screened by using linkage marker M9 and M32, and identification of phenotype to potato virus Y. Fine mapping will be carried out with recombinant plants, using new screened linkage SNP markers in the mapped region. The Rychc gene will be fine mapped to less than 100 kb region. The candidate genes were analyzed by comparing variation locus between resistant and susceptible pools to PVY and Rapid identification of their functions by Virus-induced gene silencing (VIGS) technology. The candidate genes were further confirmed by over expressing the gene in the susceptible parent and inhibiting expression in the resistant parent. The Rychc gene will be comfirmed and cloned. Further analysis of avirulence genes of PVY interacted with Rychc by Y2H, BiFC and Pull-down method using Rychc as bait protein. The research results will result in a molecular marker for PVY resistance, likely within the resistance gene. This will allow breeders to effectively select for PVY resistance in potato breeding. This research has the potential to make a major step toward understanding molecular interaction mechanism between the potato genome and its most significant viral pathogen.