牡丹PsF-box1捕获PsDELLA打破花芽内休眠的分子机制

Flowering forcing culture during the Spring Festival is one of the most important contents of tree peony (Paeonia suffruticosa Andr.) industry in China, and the key technology is to break endo-dormancy of flower bud. Therefore, the analysis of endo-dormancy mechanism is the basis to resolve the practical problems of tree peony industry. More and more studies indicated that the activation of GA pathway is the key factor to break bud endo-dormancy in tree peony. It is well known that SCF complex binds GA-GID1(GA receptor)-DELLA after DELLA protein binds gibberellin and its receptor, and DELLA protein is degraded by ubiquitination pathway, then GA signal is transduced. The F-box protein is one of the key components of SCF complex, and its carboxyl terminal can specifically combine with target protein (DELLA protein). The family member of DELLA protein is the negative regulator in GA signal transduction, but the key domain of F-box protein binding DELLA protein is still unknown until now. We have obtained PsF-box1 gene by using 454 sequencing, and the results of microarray and real-time PCR analysis showed that it was induced by chilling treatment and exogenous gibberellin. At the same time, two PsDELLA genes were also obtained and the expression patterns were analyzed by real-time PCR, which indicated that both PsDELLA genes responded to chilling treatment and exogenous gibberellin. Based on these results, this project aims to verify the conservative function of PsF-box1 in GA signal transduction, and to identify which one of two PsDELLAs participates in GA signal transduction by in-situ hybridization analysis and functional complementation assay, to analyze the function of PsF-box1 during endo-dormancy release using VIGS technology to silence PsF-box1 in vitro by analysis the changes of sprouting state and the amounts of PsDELLA protein, and to screen the key domain of PsF-box1 protein binding with PsDELLA protein. The results will provide the theoretical basis of the molecular mechanism about PsF-box1 gene how to break bud endo-dormancy by participating GA pathway.

春节催花是牡丹产业的重要内容，其技术核心是解除花芽内休眠，因此解析内休眠机理是解决催花不良等生产问题的前提。越来越多证据表明，激活赤霉素途径是解除牡丹花芽内休眠的关键环节。已知DELLA与GA和GA受体结合后，通过结合SCF复合体而降解，从而传递赤霉素信号。F-box家族成员是SCF的关键组分之一，其C端负责结合DELLA，但目前仍不清楚其结合DELLA的关键结构域。前期课题组筛选出响应低温和赤霉素的PsF-box1及2个PsDELLA基因。本项目拟以此为切入点，结合原位杂交和功能互补实验验证PsF-box1参与赤霉素信号的功能保守性，鉴定出参与赤霉素信号的PsDELLA基因；利用VIGS技术沉默PsF-box1基因，解析其在花芽内休眠解除中的作用；鉴定PsF-box1蛋白结合PsDELLA蛋白的关键结构域。研究结果为阐明PsF-box1基因参与赤霉素途径打破内休眠的分子机理提供理论依据。

春节催花是牡丹产业的重要内容，其技术核心是解除花芽内休眠，因此解析内休眠机理是解决催花生产问题的前提。越来越多证据表明，激活赤霉素途径是解除牡丹花芽内休眠的关键。已知DELLA蛋白是GA途径的负调控因子，但牡丹芽休眠解除过程中起负调控作用的关键DELLA蛋白到目前为止尚未可知。本项目在前期筛选出响应低温的PsF-box1及2个PsDELLA基因为出发点，利用qRT-PCR分析发现PsF-box1受低温及GA3诱导呈上调表达趋势，但从转录水平无法鉴定参与GA途径的DELLA。利用cell-free半体内降解实验筛选到在低温及外源GA3处理后蛋白水平极显著下调的DELLA蛋白-PsRGL1，并检测到PsRGL1降解前强的泛素化信号。利用VIGS沉默牡丹芽中的PsRGL1基因，观测芽表型及芽休眠解除marker基因的表达变化表明PsRGL1负调控芽休眠解除。酵母双杂交、pull-down和BiFC实验表明PsRGL1与PsF-box1蛋白存在相互作用，且PsRGL1特异性结合在PsF-box1蛋白的C末端。酵母双杂交及pull-down结果表明PsF-box1可与PsSKP1结合，PsSKP1与拟南芥中SCF复合体的重要组分AtASK1高度同源，表明PsF-box1作为SCF复合体中的关键组分介导了PsRGL1的泛素化降解。遗传转化结果表明PsF-box1参与并促进芽休眠解除。以上结果为深入解析GA途径参与牡丹芽休眠解除的分子机制奠定了良好的理论基础，为牡丹春节催花品种选育提供候选基因。