苹果MdWRI1转录因子介导糖信号在成花诱导中的功能及调控机制

Sugar play key role in regulating apple flower development. However, Very few reports have investigated how the sugar signaling transducts and regulates flower induction in apple. In addition, the key genes intergrated sugar signaling transduction and flowering pathway need to discover in apple as well as in model plant. So in this study, with ‘Changfu 2’and ‘Yanfu 6’ as expremental material, we take sucrose postive regulator MdWRI1 which were selected our previous study as the pointcut to identify and analyse the expression pattern of main responsive genes both in upstream and downstream. Combined with the promoter analysis of MADS-Box (MdAP1 and MdFUL) genes releted to flower induction, operating mode will be put up. Yeast one-hybird (Y1H) system and EMSA will be used to analyze the function pattern of MdNF-YB3 and MdWRI1 in post-transcriptional control, as well as will be used to analyze the function pattern of MdWRI1 and MADS-Box genes (MdAP1 and MdFUL). Transgenic systems of apple and Arabidopsis will be used to identify their functions. Through the above research, the molecular mechanism of MdWRI1 mediating sugar signal response to apple flower bud induction are hoped to clarify and to provide contribution in genetic improvement of apple flowering and development of flowering regulation techniques.

糖对苹果花芽孕育具有重要调控效应，但连接糖信号途径和成花调控途径关键结点基因有待挖掘。本项目拟以前期筛选关键MdWRI1转录因子为主要研究对象，以难成花‘长富2号’及其易成花芽变‘烟富6号’品种为材料，结合外源蔗糖喷施处理，分析MdWRI1及其上下游关键基因在花芽孕育过程中的表达模式，结合MADS-Box类成花基因(MdAP1和MdFUL)启动子序列分析，提出工作模型。利用Y1H和EMSA验证MdWRI1与其上游调控者MdNF-YB3转录调控关系；分析MdWRI1与成花途径MADS-Box基因(MdAP1和MdFUL)启动子顺式元件结合能力，解析其转录调控机制，并利用苹果和拟南芥转基因体系，验证其功能。通过以上研究，以期解析苹果MdWRI1响应糖信号在成花诱导中的功能及转录调控机制，为苹果成花特性的遗传改良和调控技术开发提供理论依据。

占我国苹果栽培面积72%以上‘富士’品种，其普遍存在花芽形成难，“大小年”结果严重等生产问题，严重制约了我国苹果生产的优质高效和可持续发展。碳水化合物（糖类物质）作为能量和信号分子在植物花芽诱导和形成中扮演重要作用。但连接糖信号途径和成花调控途径关键结点基因有待挖掘。本项目拟以前期筛选关健MdWRI1转录因子为主要研究对象，以难成花‘长富2号’及其易成花芽变‘烟富6号’品种为材料，结合外源蔗糖和葡萄糖喷施处理，发现外源蔗糖和葡萄糖喷施处理显著促进花‘长富2号’成花，且短枝顶芽糖信号途径候选MdWRI1及成花相关MdAP1和MdFUL基因显著上调表达。利用拟南芥原生质体系统，发现MdWRI1蛋白定位于细胞核。另外，发现MdNF-YB3转录因子不能直接结合在MdWRI1启动子区域诱导其表达，但是其可以直接结合在MdFT1启动子，诱导MdFT1表达，促进成花。此外，通过分析MdWRI1下游潜在靶基因MADS-Box基因(MdAP1和MdFUL)启动子顺式元件，结合酵母单杂交试验，证实其可以直接调控MdAP1, 促进其上调表达。最后，通过转基因AC番茄，发现过表达OE-MdWRI1较之野生型（WT）具有显著早花表型，且生长量（株高和茎粗等）显著增加。综上，本项目的实施为解析苹果MdWRI1响应糖信号在成花诱导中的功能及转录调控机制，为苹果成花特性的遗传改良和调控技术开发提供理论依据。