矮牵牛ODO1和EOBII基因启动子互作的ERFs在花香合成调节中的作用研究

Floral scent is one of the most important qualities of ornamental plant. In recent years, some research showed that ethylene is the important regulator of scent formation in petunia. Yet, it is not known how does ethylene regulate volatile production and emission and which components of ethylene signal transduction pathway are involved in the regulation. We have verified that ethylene treatment significantly reduced the expression of ODO1 and EOBII genes, whose promoters included ERE box motif, which is the binding domain of ethylene response factor (ERF). We further propose that ethylene reduces the scent by ERF regulating the expression of ODO1 and EOBII genes. In this application, we will identify the ERFs that interact with the promoters of ODO1 and EOBII genes and further analyze the spatial and temporal expression patterns for these genes. The effects of ethylene on expression of the genes will be analyzed and subcellular localization of the binding protein will be on by fluorescence techniques. The full long cDNA of the genes will be cloned and the effect of these genes on flower scent will be analyzed by VIGS and transgenic technology. The results will be important for the regulation of flower scent biosynthesis and the breeding of improving the floral scent.

花香是观赏植物的重要品质，近年来研究发现，矮牵牛等植物的花香合成受乙烯调节，但乙烯调节花香合成的分子机制还不清楚。本课题组前期研究发现，乙烯处理会显著减少牵牛两个重要花香调节转录因子ODO1和EOBII基因的表达，且该两基因的启动子区域均含有乙烯应答因子ERF的结合区域ERE box。我们推测乙烯可能通过其信号组分ERF调节ODO1和EOBII基因的表达来抑制花香合成。本申请项目拟筛选与ODO1和EOBII启动子结合的ERF转录因子，分析其时空表达、对乙烯的应答以及亚细胞定位；利用VIGS和转基因技术分别减少和增加ERF基因在植株中的表达，测定花香成分的合成量以及花香相关基因的表达量，鉴定ERF在花香合成调节中的功能。该研究对揭示乙烯调节花香合成的分子机理以及改良观赏植物花香品质具有重要意义。

花香是观赏植物的重要品质，矮牵牛等植物的花香合成受乙烯调节，但乙烯调节花香合成的分子机制还不清楚。本课题组拟揭示乙烯对花香的调节机理。本项目利用荧光定量PCR(qPCR)、转录组、蛋白组和泛素化组学分析表明外源乙烯处理导致花香转录因子ODO1、EOBI和EOBII的转录水平下调，PhDAHPS, PhDHQS, PhEPSPS, 3-dehydroquinate synthase, chorismate synthase (PhCS), PhPAL1, Ph4CL1, PhPAAS等7个花香相关基因的转录水平和蛋白水平均显著下调。乙烯处理32小时总蛋白含量开始下降，同时，乙烯处理也改变了乙烯合成相关酶（如ACO）和信号转导组分的蛋白水平的改变。Plantcare 软件分析显示ODO1基因启动子含有两个与乙烯响应相关的顺式作用元件，GCC Box和ERE Box，它们能够与ERF特异识别。EOBII启动子中也含有能与ERF特异识别的ERE Box顺式作用元件。构建了EOBII和ODO1基因启动子顺式作用元件ERE box 的酵母单杂交诱饵载体（pABAi-EOBII和pABAi-ODO1）。自激活分析发现pABAi-ODO1具有自激活现象而pABAi-EOBII没有自激活现象，pABAi-EOBII与矮牵牛cDNA文库进行酵母单杂交实验，结果发现，PhERF2、PhERF25、PhERF30可以与EOBII基因启动子互作。超表达PhERF2后，PhEOB1、PhEOBII和PhODO1以及6个花香合成酶基因的相对表达量均显著减少，超表达PhERF2还导致7种花香挥发物显著减少；PhERF2沉默后，EOB1和EOBII的表达呈现先升后降的趋势。PhERF25和PhERF30 沉默和超表达对花香基因表达表现出类似的影响。另qPCR分析表明PhERF6对花香合成相关基因的表达具有负向调节作用。本项目研究结果对提高花卉芳香品质的育种具有较重要意义。项目研究结果发表在Plant Physiology等期刊上，共7篇论文（其中SCI论文6篇）。