OfbHLH82转录因子调控桂花花香核心挥发物芳樟醇合成的机制研究

单萜类芳香性挥发物芳樟醇是桂花花香的重要组成成分，其含量的高低对于桂花的花香品质具有决定性的影响，然而关于单萜类挥发物芳樟醇合成的转录调控机理仍有待阐明。我们前期通过对桂花花朵转录组和代谢组数据的关联分析以及亚细胞定位等实验，筛选出了一个表达水平与芳樟醇含量显著相关的核定位转录因子OfbHLH82。本研究拟先通过在桂花花瓣中瞬时过表达和VIGS沉默OfbHLH82，结合在花香研究模式植物矮牵牛中的超量表达，验证OfbHLH82在调控芳樟醇合成过程中的功能；然后利用DAP-seq和转录组测序技术获得OfbHLH82的下游靶基因，并采用酵母单杂交、EMSA以及双分子荧光素酶实验等技术对候选靶基因进行体外和体内验证；同时使用酵母双杂交系统筛选OfbHLH82的互作蛋白，并进一步利用该系统和BiFC等技术进行验证，解析OfbHLH82介导的芳樟醇合成的调控网络，为桂花花香的定向改良提供基因资源。

Linalool, as a member of the monoterpenoids aromatic volatiles, is a core composition of the floral scent in Osmanthus fragrans, and the content changes of it in the flowers can directly influence the commercial ornamental quality of O. fragrans. However, the molecular regulation mechanism of Osmanthus linalool synthesis is still unclear. In our previous study, according to the WGCNA correlation analysis of the transcriptome and GC-MS metabolome data of O. fragrans blooming flowers, as well as the subcellular localization experiment, a nuclear localized transcription factor gene OfbHLH82 was identified, of which the expression level was significantly correlated with the linalool content in different cultivars. Here, both O. fragrans and Petunia hybrida would be taken as the receptor to explore the function of OfbHLH82 during linalool synthetic process through transient overexpression, VIGS and stable transgenic techniques. Then, the DAP-seq and RNA-seq sequencing strategies would be used to obtain the down-stream regulated genes of OfbHLH82, and the candidate genes would be further selected in vivo/vitro by yeast one-hybrid system, dual-luciferase reporter assay and electrophoretic mobility shift assay. Meanwhile, the yeast two-hybrid library system and bimolecular fluorescence complementation analysis would be used to screen for the interaction proteins of OfbHLH82, by which the regulation network of OfbHLH82 mediated linalool synthesis would be well established in O. fragrans, and ideal transcription factor genes would be available for floral scent composition orientated molecular breeding.