黄瓜耐热关键基因的定位克隆及功能分析

Global warming and the frequent occurrence of extreme high temperature weather are seriously threatening the productivity of cucumber. It is urgent to accelerate the breeding process of new heat-tolerant varieties, and the precise and efficient molecular breeding can satisfy this demand. However, the molecular mechanism of heat tolerance is still unclear, which severely restricts the development of the molecular breeding of heat tolerance in cucumber. Recently, we found a spontaneous cucumber mutant, heat sensitive 1 (hs1), which is sensitive to high temperature. Genetic analysis showed that hs1 is controlled by a single recessive gene, indicating that hs1 is a precious material to study the molecular mechanism of heat tolerance in cucumber. In this project, HS1 will be isolated using map-based cloning, then transgenic complementation, gene knock-out, overexpression and silencing will be performed to confirm the biological function of HS1 involved in cucumber heat tolerance; meanwhile, the nearly isogenic line of hs1 will be constructed with marker-assisted selection for analyzing the influence of HS1 on the morphology, physiology and gene expression profile of cucumber plants under heat stress and non-heat stress conditions; the interacting proteins of HS1 will be screened and analyzed to reveal protein regulatory network of HS1 under heat stress. In summary, the molecular mechanism of heat tolerance controlled by HS1 might be illustrated, which will provide a theoretical basis and candidate target genes for molecular breeding of heat tolerance in cucumber.

全球气候变暖和由此引发的极端高温天气的频繁出现，正严重威胁着黄瓜生产，亟需加快耐热新品种的培育进程，而精准高效的分子育种能够满足这一需求。但是，黄瓜耐热分子调控机理仍不清楚，严重限制了黄瓜耐热分子育种的发展。本项目组分离到一个热敏感黄瓜自然突变体heat sensitive 1（hs1），遗传分析表明hs1为单基因隐性突变体，是研究黄瓜耐热分子机理的宝贵材料。本项目拟利用图位克隆技术分离HS1，并通过转基因互补、基因敲除、过表达及沉默，确证其在黄瓜耐热性中的生物学功能；同时，利用分子标记辅助选择构建hs1近等基因系，解析HS1在热胁迫及非热胁迫条件下，对黄瓜形态、生理及基因表达谱的影响；通过互作蛋白的筛选及分析，揭示HS1在黄瓜耐热性中的蛋白调控网络。总之，本项目拟阐明HS1调控黄瓜耐热性的分子机理，为黄瓜耐热分子育种提供理论依据及候选靶基因。

全球变暖引起极端高温天气的频繁发生，严重危害黄瓜的安全生产。挖掘耐热基因、解析耐热分子机理，进而培育耐热品种是应对高温胁迫的有效方式。本项目组获得一个黄瓜热敏感自然突变体heat sensitive 1（hs1），遗传分析表明，该突变体为单基因隐性突变体。利用图位克隆技术，我们获得了HS1的候选基因CsaV3_1G044470，该基因编码核黄素合成蛋白RibD。HS1和拟南芥的PHS1是同源基因，拟南芥phs1对强光敏感，我们发现黄瓜hs1也同样对强光敏感。我们利用病毒介导的基因沉默技术证明了CsaV3_1G044470为HS1。亚细胞定位表明，HS1在叶绿体中发挥作用，这与hs1突变体在高温、强光下叶片失绿表型一致。由于hs1为自然突变体，无法确定其亲本，所以我们利用9930为轮回亲本构建了hs1的近等基因系。在非胁迫条件下，hs1近等基因系植株无明显形态差异，但是种子胎萌严重，留种困难，无法进一步开展深入研究。为深入挖掘参与黄瓜热胁迫响应基因，我们用高温处理9930幼苗，进行转录组分析，发现受热诱导最显著的基因为CsHSFA2。利用黄瓜子叶瞬时表达系统和病毒介导的基因沉默技术，明确了CsHSFA2在黄瓜耐热中的关键作用。并且发现CsHSFA2不但可以直接结合耐热必需基因CsHSP70-1的启动子并激活其表达，而且还可以增强自身的表达。总之，本项目明确了CsHS1、CsHSFA2和CsHSP70-1等在黄瓜耐热中的重要作用，为黄瓜耐热育种提供了理论依据及基因资源。