MYB、NAC等转录因子响应相对低温调控扩展蛋白EXP控制桂花花开放的分子机制

桂花是我国传统名花，其花芽分化和花开放相对独立，花芽分化完成后花开放对温度有特殊需求，也正是因为这一需求精准控制其开花时间。扩张蛋白（EXP）在花开放中发挥最重要作用，通过分析关键EXP及其调控因子的功能将有助于解析温度控制桂花花开放的机制。前期已获得控制花开放的3个关键EXP基因以及共表达的关键MYB、NAC转录因子。本项目拟以‘堰虹桂’盆栽植株为材料，分离关键OfEXP基因启动子，分析关键基因及其启动子的功能和作用；通过启动子序列利用酵母单杂筛选上游转录因子，分析不同温度处理下的表达模式，利用Y1H、EMSA和Dual-LUC验证转录因子对关键OfEXP启动子的结合激活作用，进一步利用Y2H、BiFC结合Pull-down验证转录因子与其关键OfEXP蛋白互作关系，并通过转基因验证转录因子与OfEXP协同调控花开放的功能，构建相对低温调控花开放的分子调控网络，为花期预测和调控提供依据。

Osmanthus fragrans is very important traditional ornamental plants in China, which is of particular flowering process with relatively independent flower bud differentiation and flower opening. After flower bud differentiation, flower opening will occur only when they get enough cold accumulated temperatures. This accurately controls the flowering time. Expansins mediate cell wall loosening and play key roles in flower opening. By analyzing the function of key EXP genes and their regulation factors, we will be easy to understand the mechanism of relative low temperature contolling flower opening. At present, we have obtained all 12 EXP genes of O. fragrans, and confirm 3 EXP genes (OfEXPA2、OfEXPA4 and OfEXLA1) and some key transcription factors (MYB, NAC) directly related to flower opening through the analysis of gene expression levels. In this study, potted plants of ‘Yanhonggui’ as the materials, the promoter of key EXP genes will be cloned by genome walking then the functions of key EXP genes and their promoters will be investigated by use of Arabidopsis transformation. Meanwhile, the upstream transcription factors interaction with these key EXP genes were obtained by yeast one-hybrid (Y1H) from the sequence of their promoters. The expression patterns of these transcription factors during flower opening under the conditions of different temperature were also analyzed. Furthermore, the interactions of DNA-protein and protein-protein between key EXP genes and their transcription factors were studied by the molecular methods of Y1H, EMSA, Dual-LUC and yeast two-hybrid (Y2H), BiFC, Pull-down. And the function on flower opening of the interaction of the EXP with their transcription factors was confirmed by transformation. Then, the molecular regulation network between key EXP genes and their transcription factors will be constructed to understand the molecular mechanism of flower opening controlled by relative low temperature.