牡丹芽内休眠解除是反季节生产和正常开花的前提，内休眠调控机理也是国际研究热点。越来越多的证据表明，GA合成的激活是低温诱导芽休眠解除的关键。miRNA是重要的基因表达调控子，尚未知其是否参与了赤霉素(GA)合成基因的调控。课题组前期已克隆了GA合成关键基因PsGA20ox，并通过生物信息学结合表达分析筛选到可能靶向PsGA20ox基因的PsmiR396b。本项目拟通过低温诱导的时空表达模式、Western及转基因分析等明确两者的靶标关系及调控方式；在明确PsmiR396b负调控PsGA20ox的基础上，利用VIGS沉默PsGA20ox分析牡丹花芽休眠状态、活性GA含量、休眠解除Marker基因等的变化及补施GA对休眠状态的影响，以期解析PsmiR396b/PsGA20ox调控内休眠解除的机理。项目拟探索一种GA合成调控的新机制，研究结果对深入理解低温、GA调控内休眠调控机理具有重要意义。

Endo-dormancy of deciduous plant is an adaptive mechanism to cold whether in winter or early spring, and also an obstacle to the anti-season culture of the perennial fruit tree and flowering plant. Regulation mechanism of endodormancy has been a research hotspot in recent years. To break dormancy of floral bud is necessary for forcing culture in spring festival, and the activation of gibberellins synthesis pathway is a keystone of dormancy release in tree peony. MicroRNA is of an important factor to regulate gene expression in many physiological and metabolic processes. However, miRNA targeting GA biosynthesis genes has not been reported to now. In the previous study, we had cloned PsGA20ox gene and analyzed its roles in dormancy regulation of tree peony, which might be a key point of dormancy release. Additionally, by the bioinformatics and real-time quantitative PCR analysis, a miRNA, PsmiR396b, probably targeting to PsGA20ox was obtained. Based on these above, the target relationship and regulative manner between miR396b and PsGA20ox will be identified through chilling inducible spatio-temporal expression patterns analysis, western blotting and transgenic study in this project. Overexpression of PsmiR396b is performed to confirm its negative regulation to PsGA20ox. Subsequently, PsGA20ox is silenced by VIGS technology in the floral bud of tree peony, and the dormancy status, the activated GA content of the buds and the expression pattern of the marker genes associated with dormancy release will be evaluated. Additionally, the effect of exogenous GA3 will be investigated to confirm the role of PsGA20ox in this process. Taken together, we aim to illuminate the regulation mechanism of PsmiR396b/PsGA20ox in chilling induced dormancy release. Our project will discover a novel regulation mechanism of GA biosynthesis in tree peony, and it is important to understand the regulation mechanism of chilling and GA in dormancy release.