ERF对杏果实芳香味脱辅基类胡萝卜素形成的调控作用及机制研究

Apricot has unique aroma smell and is one of the main stone fruit tree. Some apocarotenoids with aroma smell are characteristic compounds of apricot, which contribute to its flower-odor. Our previous study found that carotenoid cleavage dioxygenases 1 (CCD1) is the key gene resposible for aroma apocarotenoids biosynthesis, while the mechanism of transcriptional regulation have remains to be elucidated. In this study, using apricot cultivars with different fruit colors, we plan to identify the ethylene response factor (ERF) candidate genes for regulation of aroma apocarotenoids production based on our four ERFs available. The gene screening will be carried out by analyzing the relationship between the carotenoids cleavage and the gene expression of PaERFs and PaCCD1, and integrating it with bioinformatic analysis. The putative functions of candidate genes will be verified by transgenic tomato and transient expression in apricot fruit. After cloning the promoter of PaCCD1, the interactions between PaERFs and PaCCD1 and the key binding domain will be confirmed by using yeast one-hybrid (Y1H), dual-luciferase reporter assay system (DLRAS) and electrophoretic mobility shift assay (EMSA). Finally, poromoter deletion and site-directed mutagenesis will be employed to reveal the regulation site and effect, with an aim to unravel how the PaERFs regulate biosynthesis of aroma apocarotenoids in apticot fruit. The results obtained will provide the basis for regulation of aroma quality and breeding.

杏是重要的核果类果树，其果实香味独特。一些具有芳香味的脱辅基类胡萝卜素是杏果实重要的“花香味”特征香气成分。我们前期发现，类胡萝卜素裂解双加氧酶（CCD1）是负责合成芳香味脱辅基类胡萝卜素的关键酶，但其转录调控机制尚不清楚。本项目拟在申请人近期获得的4个乙烯信号转导元件ERFs的基础上，以不同色泽杏品种的果实为材料，通过分析类胡萝卜素降解与PaERFs、PaCCD1基因表达的关系，并结合生物信息学方法，筛选参与调控芳香味脱辅基类胡萝卜素的候选转录因子ERFs；利用转基因番茄和果实瞬时表达进行功能验证；克隆PaCCD1的启动子，并借助酵母单杂交、双荧光素酶报告基因检测系统和凝胶迁移率分析法鉴定PaERFs与PaCCD1的互作关系及启动子核心区，使用启动子缺失、定点突变技术来确定具体调控位点与效应，系统阐明ERFs调控杏果实芳香味脱辅基类胡萝卜素形成的分子机制，为杏香气品质调控和育种提供依据。

芳香味脱辅基类胡萝卜素是决定杏果实“花香味”的特征香气成分，对其独特风味的形成具有重要作用。本研究基于现已发布的8个杏全基因组数据及前期转录组数据，利用生物信息学分析共筛选到5个与杏果实芳香味脱辅基类胡萝卜素代谢相关的转录因子ERF，包括PaERF98（PARG03662m01）、PaERF3（PARG10301m01）、PaERF28（PARG12147m01）、PaERF1（PARG24217m01）和PaERF2（PARG19277m01）。进一步基因表达分析发现，PaERF1与杏果实芳香味脱辅基类胡萝卜素代谢及PaCCD1的表达呈明显的正相关关系。在深色品种“佳娜丽”中瞬时超表达5个PaERFs候选基因，结果发现PaERF1超表达果实中叶黄质和β-胡萝卜素的含量显著降低而β-紫罗兰酮、二氢-β-紫罗兰酮和β-大马酮含量显著增加，在浅色品种“轮台小白杏”中沉默PaERF1则导致果实中叶黄质和β-胡萝卜素的含量显著积累而β-紫罗兰酮、二氢-β-紫罗兰酮和β-大马酮含量显著下降，表明PaERF1参与类胡萝卜素裂解形成杏果实芳香味脱辅基类胡萝卜素；进一步利用转基因番茄分析发现，过表达PaERF1可显著促进Tom番茄中二氢–β–紫罗兰酮、β–紫罗兰酮和β–大马酮含量的增加，证明PaERF1具有正向调控杏果实芳香味脱辅基类胡萝卜素积累的生物学功能。利用酵母单杂、荧光素酶检测分析发现，PaERF1能与PaCCD1启动子区结合，表明PaERF1能与PaCCD1互作。启动子删除和定点突变分析发现，PaCCD1启动子第3和第4 RAA-motif中的“CAACA”序列是PaERF1调控PaCCD1的核心作用位点。综上我们揭示了PaERF1通过PaCCD1正向调控杏果实芳香味脱辅基类胡萝卜素积累的分子机制。本项目执行期间，共发表相关论文（标注本项目资助）6篇，包括BMC Plant Biology、BMC Genomics、Frontiers in Plant Science、Horticultural Plant Journal和园艺学报等主流期刊。共培养研究生6名，毕业硕士4名。项目组成员参加国内外会议交流23人次。