马铃薯Y病毒极端抗性基因Rychc的克隆与抗病机理解析

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.

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。

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。利用左侧标记M4和右侧标记M32筛选1078个单株，得到12个重组单株。利用相同的方法，用标记M28和M50筛选2458个后代，又得到5个重组单株。重组单株的抗性鉴定结果表明5株为抗，12株为感。利用抗、感病亲本，抗、感池为模板，开发了8个与PVY抗性紧密连锁的标记。依据重组单株的表型和标记扩增的基因型，将Rychc基因精细定位于M28和M48-6之间约90 kb的范围。依据参考基因组DM1-3序列信息预测有8个候选基因。进一步分析候选基因在抗感单株间的变异位点，VIGS瞬时表达，病毒接种诱导基因表达量等，发现这些候选基因均不是目标基因。利用抗病亲本S. chacoense构建了BAC 文库，平均插入片段约130 kb，共挑选了74，880个BAC克隆，大约覆盖了基因组11.5倍。利用左翼标记M2526和M28筛选出71-24H和100-8O两个BAC片段，右翼标记M4950和M50筛选出74-24B。序列分析结果表明，71-24H和74-24B为Rychc所在的单链；100-8O为等位基因所在的另一条单链，而且与71-24H和74-24B均有重叠。但71-24H和74-24B无重叠。利用71-24H序列信息又开发了3个新的连锁标记，利用目标基因最左侧标记M71-28重新筛选文库，得到一个新的抗病基因所在的单链克隆64E14，将71-24H序列向右延伸了35 kb，与右翼74-24B仍有17.5 kb的缺口。应用PCR分段扩增弥补缺口，分段扩增片段至少有500 bp以上的重叠区域。至此将目标基因定位在M71-28和SNP48之间56959 bp的范围，利用抗病片段序列预测有8个可能的候选基因。构建了所有候选基因的超量表达载体同时转化感病烟草和马铃薯，获得4个载体的马铃薯转基因植株和2个载体的烟草转基因植株，已定植于生长室内等待抗性鉴定。剩余载体已全部完成转化，等待植株分化。转基因植株的抗性鉴定以克隆Rychc基因。构建了PVY病毒蛋白载体，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