桂花转录因子OfMYB14响应低温调控花开放的分子机理

Osmanthus fragrans, as one of the top ten famous flowers in China, plays important roles in landscaping and cultivation. We found that the flower opening of O. fragrans can be determined by changes in ambient temperature, especially, 19℃ significantly promotes their opening. R2R3-MYB transcription factors involved in processes of plant growth and development, it was found OfMYB14 were significantly differentially expressed at different opening stages under different temperature conditions. Genetic transformation of Arabidopsis thaliana showed that these lines were early in flower opening time and pod formation compared with wild types. Based on our obtained results, we planned to analysis OfMYB14 subcellular localization, transcriptional activation activity, expression patterns and further transfer to tobacco to definite OfMYB14 characteristics and biological functions. For analysis the gene networks, target genes of OfMYB14 and their promoter would be analyzed and cloned, EMSA and yeast one-hybrid were employed to detect their interactions. At the translation level, the proteins interacting with OfMYB14 were screened by yeast two-hybrid and validated in vivo and in vitro using BiFC and Pull-down, and their expression patterns were also detected. Finally, according to these results, we can remarkably clarify OfMYB14 how to respond to low temperature and regulate flower opening in O.fragrans. Furthermore, the proposal will shed light on the molecular mechanism of promoting flower opening in O.fragrans under low temperature, laying a theoretical foundation and practical guidance for the ambient temperature regulate flower opening and in O.fragrans. Our findings can enrich the molecular mechanism of plant flower opening and deepen the understanding of the biological functions of R2R3-MYB transcription factor, and lay a foundation for the regulation of flowering, fragrance and germplasm innovation of O. fragrans.

桂花是我国十大传统名花之一，在园林绿化中应用广泛。环境温度变化是影响桂花开放的重要因素，其调控机制尚不清楚。前期研究发现19℃低温显著促进桂花的开放，其中OfMYB14转录因子响应低温具有正调控开花的功能，然其分子机制有待探究。在此基础上，本项目拟通过（1）分析OfMYB14在桂花开放进程中的基因表达模式，遗传转化拟南芥和烟草明确其调控开花的生物学功能；（2）筛选OfMYB14调控的下游靶基因并分析其启动子序列，结合酵母单杂交和凝胶阻滞方法检测该蛋白与启动子的结合能力，进而明确OfMYB14调控的下游基因网络；（3）采用酵母双杂交技术筛选OfMYB14的互作蛋白，并通过双分子荧光互补和Pull-down方法验证，初步解析OfMYB14蛋白翻译后的调控机制。本研究将阐明OfMYB14响应环境低温参与调控桂花开放的作用机制，丰富花开放调控的分子机理，也为桂花花期、释香调控以及种质创新奠定基础。

桂花是我国十大传统名花之一，在园林绿化中应用广泛。前期大量的生理实验表明环境温度变化是影响桂花开放的重要因素，且已有研究表明R2R3-MYB转录因子广泛参与植物的生物学过程。本项目通过转录组测序发现R2R3类OfMYB14转录因子在桂花花开放过程中差异表达，在此基础上,本项目阐明了（1）OfMYB14在桂花开放进程中的基因表达模式，遗传转化拟南芥能够促进其开花进程；（2）建立了桂花愈伤瞬时转化体系；（3）采用酵母单杂交技术筛选OfMYB14转录因子调控的下游基因OfEXLA1，并通过Dual-LUC方法进行了验证，初步解析了OfMYB14蛋白翻译后的调控机制。本研究将阐明OfMYB14响应环境低温参与调控桂花开放的作用机制，丰富花开放调控的分子机理，也为桂花花期、释香调控以及种质创新奠定基础。