赤霉素信号转导因子CsGAIP在黄瓜雄花发育中的功能分析与调控机制研究

Cucumber (Cucumis sativus L.) is a monoecious species with unisexual flowers, and the sex differentiation of reproduction organs directly affects the product yield and quality. Extensive studies have been performed previously on the molecular regulation of female flower development in cucumber, while the mechanism of male flower development has been neglected. Gibberellin (GA) can promote male flower formation in cucumber, but the molecular mechanism remains unknown. In our previous study, we found that a GA signal transduction factor CsGAIP may have a prominent role for male flower development of cucumber. Therefore, in this project we focus our study on CsGAIP, specifically, we will explore its expression pattern, then make the CsGAIP overexpression and RNAi constructs and introduce them into cucumber plants, analyze the biological function of CsGAIP through sexual phenotype, male flower development phenotype investigation and expression analyses of related genes in the transgenic plants. Further, ChIP-Seq and EMSA assays will be used to screen the downstream target genes of CsGAIP. Besides, we will clarify the responses of CsGAIP to GA and ethylene treatment and test whether there is a correlation between GA-CsGAIP signaling pathway and ethylene for regulating sex expression of cucumber through analyses of the promoter activity of CsGAIP and the content of GA and ethylene, the expressions of their biosynthetic genes in the transgenic cucumber plants. This project will shed light on the molecular mechanism of how CsGAIP regulate male flower development, and provide the basis for increasing product yield and improving quality of cucumber.

黄瓜属单性花植物，其性别分化直接影响产量和品质。前人已就黄瓜雌花发育分子调控开展了较多研究，而雄花发育分子机制则一直被忽视。赤霉素（GA）可促进黄瓜雄花形成，但作用机制尚不清楚。课题组前期研究表明，GA信号转导因子CsGAIP在黄瓜雄花发育中可能起着关键作用。本项目拟以黄瓜CsGAIP基因为对象，首先对其表达模式进行分析；其次，构建过表达和干扰表达载体，对黄瓜进行遗传转化，通过性别表现、雄花发育表型及相关基因表达等探讨其生物学功能；再者，利用染色质免疫共沉淀和凝胶迁移实验明确CsGAIP的下游靶基因，阐明其调控机制；此外，通过启动子活性分析，探讨CsGAIP对GA和乙烯处理的响应情况，结合转基因植株中GA和乙烯含量及合成基因表达，明确GA-CsGAIP信号途径与乙烯途径在调控黄瓜性别发育中是否存在关联。本研究旨在解析CsGAIP调控黄瓜雄花发育的分子机制，为黄瓜增产和品质改良提供理论依据。

黄瓜属单性花植物，其性别分化直接影响产量和品质。前人已就黄瓜雌花发育分子调控开展了较多研究，而雄花发育分子机制则一直被忽视。赤霉素（GA）可促进黄瓜雄花形成，但作用机制尚不清楚。本项目鉴定了一个GA信号转导因子CsGAIP，解析了其在黄瓜性别发育中的生物学功能，并探讨了GA和乙烯在黄瓜性别调控中的关系。黄瓜CsGAIP属于DELLA家族的同源基因，其N端含有DELLA和VHYNP两个保守域；时空表达分析表明，CsGAIP主要在黄瓜的茎和雄花芽处表达，而在雄花发育过程中，CsGAIP表达主要集中在两性期的萼片、花瓣、雄蕊、雌蕊原基以及后期的花粉母细胞、花粉囊壁、花粉粒中，且在两性期的表达量最高；亚细胞定位分析显示，CsGAIP蛋白定位于细胞核；基因功能验证发现，CsGAIP的过量表达下调了黄瓜植株中雄花节的比例，上调了雌花节的比例，表明CsGAIP可调节黄瓜的性别表达；此外，通过外源GA处理对黄瓜性别表达的影响及转录组分析揭示GA调控黄瓜性别发育的分子途径，结果表明，外源GA3可诱导黄瓜雄花形成，且抑制了茎尖中的乙烯合成，同时乙烯相关基因（包括乙烯合成基因、受体、乙烯响应因子）和花器官决定基因CAG2参与了GA调控的黄瓜性别分化，进一步分析得知，GA可以依赖于乙烯和不依赖于乙烯的两种方式调控黄瓜的性别表达。本研究结果丰富和补充了黄瓜性别分化的调控理论，可为黄瓜增产和品质改良提供理论依据。