百合苯丙烷类花香成分苯甲酸乙酯合成和节律释放的转录调控机制研究

The flower scent is one of important trait in ornamental plant. Circadian rhythm is an important regulatory mechanism for plant adaptation to predict changes in external environmental conditions. Lily is world widely cultivated fragrant flower. Ethyl benzoate is the main aroma components in lily flower and its release show rhythmically. Currently the transcriptional regulation of ethyl benzoate biosynthesis and rhythmical release are not clear. Screening and identification of cis-acting elements and trans-acting factor of transcriptional regulation of related gene controlling ethyl ester aroma biosynthesis and their rhythmical releases has important significance both in theory and in practice. We have very good background for this research: lily alcohol acyltransferase gene (LoAAT1) that closely related to flower scent was cloned and its function was identified by.transgenic tobacco and its prokaryotic expression in Escherichia coli, its flower specific expression pattern was also confirmed, we hope further to clone its promoter sequence, screen and identify its cis-acting element closely related with its expression by the deletion and mutation method; by yeast one-hybrid method to screen binding candidate transcription factors; by using barley stripe mosaic virus VIGS technique, RNAi methods and transgenic lily to determine the functions of candidate transcriptional factors. The purpose of this study is to realize the transcriptional regulation mechanism of ethyl ester biosynthesis and rhythmical release, which lays a theoretical foundation for the understanding of plant volatiles formation and release mechanism in molecular level, and provide available gene for molecular breeding of new varieties with fragrant traits.

花香是花卉重要的观赏品质之一，昼夜节律是植物适应可预知外界环境条件变化而产生的重要调节机制。百合是世界范围内广为栽培的香型切花，苯甲酸乙酯是其主要香气成分之一，其挥发具有明显的昼夜节律性，但其调节机制尚不明确。申请者在已经获得一个经原核表达鉴定、花部特异表达和烟草转基因鉴定证明其与苯甲酸乙酯合成密切相关的百合醇酰基转移酶基因(LoAAT1)的基础上，克隆其启动子序列，采用缺失和突变的方法筛选和鉴定与其节律表达密切相关的顺式作用元件；通过酵母单杂交和花香不同挥发节律时间点的转录组测序以及表达谱分析，筛选与节律表达密切相关候选转录因子；使用大麦条纹斑叶病毒VIGS沉默、RNAi花瓣干渋和百合正义超表达后测定苯甲酸乙酯等香气的节律挥发量等方法鉴定候选转录因子的功能。本研究旨在解析植物挥发性物质合成和节律释放调控的分子机制，为采用基因工程技术培育香花植物资源奠定基础。

百合是重要的香型切花，苯甲酸乙酯等酯类是其花香主要成分并呈节律释放，但控制花香成分合成和节律释放的关键酶基因在转录水平上是如何调控的，目前尚不清楚。我们前期研究表明，苯甲酸乙酯是‘西伯利亚’百合的主要花香成分之一，LoAAT1是调控百合苯甲酸乙酯生物合成的关键基因。本项目利用hiTAIL-PCR技术克隆了‘西伯利亚’百合LoAAT1的启动子序列，通过启动子片段缺失分析，在烟草中鉴定了LoAAT1启动子的花瓣特异性表达、伤害和茉莉酸甲酯诱导表达的特性，并鉴定到相关顺式作用元件所在区段。利用百合转录组测序，筛选了调控花香形成的相关转录因子，包括MYB1-3，以及调控因子FKF1和GI。荧光定量PCR分析了LoMYB1-3的时空表达特性及与LoAAT1表达和苯甲酸乙酯释放的相关性。证实了LoMYB1-3的核定位特性。酵母单杂和EMSA明确了LoMYB2/3可直接结合LoAAT1启动子的MBS元件，转录激活实验验证了LoMYB2可激活LoAAT1启动子，LoMYB3抑制LoAAT1的启动子活性。VIGS和百合花被正义表达验证了LoMYB2正调控LoAAT1的表达和苯甲酸乙酯的释放，而LoMYB3作用相反。此外，本项目还通过VIGS和百合花被正义表达分析了FKF1和GI在苯甲酸乙酯节律释放过程中的调控作用，通过酵母双杂和BiFC验证了FKF1可与GI和LoMYB3互作。.本项目研究了百合苯甲酸乙酯生物合成关键基因LoAAT1的启动子功能，筛选和确认与LoAAT1启动子顺式元件相结合的转录因子LoMYB1-3，进行了转录调节功能分析，明晰了苯甲酸乙酯等酯类合成和昼夜节律释放的作用机制。本研究为了解植物挥发性物质形成和释放的分子机制奠定一定的理论基础，可为采用基因工程的方法培育香花新种质提供理论指导。