矮牵牛花衰老相关转录因子PhERF3互作蛋白的筛选、鉴定及功能研究

Ethylene is a gaseous molecule that regulates many aspects of plant development and physiology, including flower senescence. Ethylene signaling is mediated by a complex multi-component pathway. A few proteins that interacted with the main components were involved in the ethylene signaling transduction. The transcription factor ERF is the last component of ethylene signaling pathway. It is possible that the proteins interacted with ERF were also involved in ethylene signaling. We had found that PhERF3 gene, one of the PhERF family, was associated with flower senescence and that some proteins,such as F-box and WRKY, were interacted with PhERF3. On this foundation, we will identify the founction of these proteins. First, the false positive proteins that interacted with PhERF3 will be eliminated using back cross of yeast 2-hybrid and BiFC technology. Second, the full-length cDNA of the genes will be cloned. The effects of ethylene on expression of the genes will be analyzed and subcellular localization of the proteins will be on using fluorescence techniques. Last, the effect of these genes on flower senescence will be analyzed by transgenic technology. The results would be important for the regulation of flower senescence and ethylene signaling transduction mechanism.

乙烯是许多重要观赏植物花器官衰老的主要因子，乙烯信号路径中多个重要组分的互作蛋白都参与了乙烯信号转导的调控。ERF转录因子是乙烯信号转导路径的最后一个组分，本项目组前期已从矮牵牛ERF转录因子家族中筛选出PhERF3为调控花瓣衰老的重要成员，并推测ERF互作的蛋白也可能参与了花衰老相关的乙烯信号转导。本项目组已用PhERF3为诱饵与cDNA文库进行酵母双杂交，初步获得一些互作蛋白如F-Box等。本申请项目拟在此基础上，1.进一步用酵母回复杂交和荧光双分子互补技术验证PhERF3与所获蛋白的互作（排除假阳性）。2.分离互作蛋白编码基因cDNA全长；分析其时空表达以及对乙烯的应答,并对其进行亚细胞定位。3. 鉴定基因功能，通过转基因技术增加或减少互作蛋白基因的转录量，分析互作蛋白在花瓣衰老以及乙烯信号转导中的功能。该研究结果将对花衰老调控以及乙烯信号转导机制具有重要意义。

乙烯是调节花衰老的主要激素。本课题组前期通过转录分析筛选到花衰老相关的转录因子PhERF3。本研究进一步分析了PhERF3以及其上游组分PhEIL1和PhEIL2在花衰老中的功能，沉默PhERF3以及PhEIL1和PhEIL2都推迟了花衰老，并减少了乙烯释放，酵母双杂交分析显示PhEIL1和PhEIL2可以互作，该结果表明EIL家族可能通过异源二聚体的形式形式功能。PhERF3自激活区域在其C端，进一步用PhERF3的N端为诱饵与构建的矮牵牛酵母cDNA文库杂交，结果获得多个互作蛋白，其中包括PhWD1和PhEOL1。PhWD1、PhEOL1以及PhEOL2基因的全长得以分离。qPCR技术对PhWD1、PhEOL1以及PhEOL2的表达进行了分析，三者都显示出一定的时空差异。对三个基因的功能分析显示沉默PhWD1和PhEOL2对花衰老没有明显影响，而沉默PhEOL1则导致花提前1.6天衰老，并增加了乙烯释放。同时PhEOL1也可以与PhACS家族成员可以互作。这些结果表明PhEOL1可能在多个乙烯反应节点参与花衰老调节。