黄瓜CsEIN2响应苗期低温胁迫的机制研究

Low temperature (LT) stress at seedling stage poses a severe threat to cucumber early maturity and high yield. With the increasing of the anti-seasonal cultivation area in northern China, improving the ability of cucumber LT tolerance has become more and more important. Study the mechanism of LT tolerance in cucumber seedlings and thus develop germplasm with good LT-tolerant performance is the fundamental way to solve the current problem. The project used CG104 (LT-tolerant inbred line) and CG37 (LT-sensitive inbred line) as parental lines for fine mapping of major QTL, and found a candidate gene CsEIN2, which is responsible for LT tolerance. Its homolog in Arabidopsis is EIN2, which is a key member in ethylene signaling transduction pathway. CsEIN2 was up-regulated by LT and its transcription was positively correlated with CsCBF1 which is a key transcription factor in cold stress signaling pathway. Based on this, the project will further verify the key role of CsEIN2 in LT stress response via genetic transformation, illustrate the molecular mechanism of CsEIN2-mediated LT stress tolerance and explore LT stress responsive signaling pathway in cucumber by means of hormone content determination, RNA-seq, and protein interaction analysis. The project aims to provide theoretical guidance for stress-tolerance molecular breeding and clarify the molecular mechanism of stress response in cucumber.

苗期低温胁迫严重制约着黄瓜的早熟丰产。随着北方反季节栽培面积的不断增加，提高黄瓜苗期耐低温能力成为亟待解决的问题，探究苗期耐低温的分子机制，选育低温耐受的优异黄瓜种质是解决当前问题的根本途径。本项目前期以耐低温的重组自交系CG104和低温敏感的重组自交系CG37为材料，通过苗期耐低温性的主效QTL位点的精细定位，挖掘到候选基因CsEIN2，该基因与拟南芥乙烯信号转导通路中关键因子EIN2同源性很高，受低温胁迫的诱导表达，并且正调控低温胁迫信号通路的关键转录因子CBF1的转录。基于此，本项目拟进一步通过转基因验证CsEIN2是调控低温胁迫响应的关键因子，然后综合运用激素含量测定、转录组测序、蛋白互作分析等技术手段，深入了解CsEIN2调控苗期低温胁迫响应的分子机理，探索黄瓜中响应低温胁迫的信号通路，为开展黄瓜抗逆分子育种与抗逆的分子机制研究提供理论指导。

苗期低温胁迫严重制约着黄瓜的早熟丰产。随着北方反季节栽培面积的不断增加，提高黄瓜幼苗耐低温能力成为亟待解决的问题，探究幼苗耐低温的分子机制，选育低温耐受的优异黄瓜种质是解决当前问题的根本途径。本项目克隆黄瓜幼苗耐低温候选基因CsEIN2，通过序列分析，亚细胞定位和拟南芥中乙烯三重反应对冷敏感材料中CsEIN2保守突变位点的进行功能分析，发现CsEIN2 N端编码区第257个碱基发生保守突变，天冬酰胺变成丝氨酸，从而使蛋白跨膜螺旋发生改变，此外野生型 CsEIN2 能部分恢复拟南芥 ein2-5 的表型而Csein2不能，证明该保守位点变异影响CsEIN2的功能。突变前后的CsEIN2均可以定位到细胞核，初步确定突变位点不影响CsEIN2的入核过程。利用CRISPR/Cas9技术获得两种CsEIN2纯合突变类型，低温表型鉴定验证了CsEIN2在调控黄瓜苗期低温胁迫响应中的作用，此外发现CsEIN2还调控黄瓜的生长发育、花性和耐盐耐高温等逆境胁迫。通过激素含量测定和转录组测序阐明了CsEIN2 与乙烯低温胁迫信号通路之间的关系。此外，针对CsEIN2基因开发了黄瓜幼苗耐低温SNP标记并申请专利。该研究对开展黄瓜抗逆分子机制研究及创制抗逆黄瓜育种具有重要意义。