唐菖蒲GhPP2C1在球茎休眠中的功能分析及其上游调控机制解析

Gladiolus is an important bulb flower both in China and abroad, and its off-season production can satisfy many market needs and presents vast economic benefits. However, corm dormancy is one of the main obstacles to Gladiolus off-season planting in China. Previous research has shown that ABA is an important phytohormone in corm dormancy, and our work has revealed key components of ABA signal transduction during the induction and release of corm dormancy. Using RNA-seq analysis, we identified a Gladiolus ABA co-receptor homologous gene, GhPP2C1, which was associate with dormancy release. In this proposal, the role of GhPP2C1 in dormancy release will be confirmed by gene function analysis and expression pattern. To further explore the transcriptional regulation of GhPP2C1, a yeast one-hybrid approach will be employed to screen a Gladiolus cDNA library. This will allow for the identification of upstream transcription factors that regulate GhPP2C1 expression. Subsequently, so as to select candidate transcription factors and confirm their effect on GhPP2C1 expression, several binding assays will be used including EMSA, transient transcriptional activation in Nicotiana benthamiana and qChIP. Ultimately, by combing the results obtained from overexpression and silencing candidate transcription factor genes, this work aims at dissecting the molecular mechanism of GhPP2C1-regulated Gladiolus corm dormancy. Thus, this proposal not only supports the identification of candidate genes to modulate corm dormancy via molecular breeding, but also lays academic foundation for our off-season Gladiolus corms.

唐菖蒲是国内外重要的球根花卉，其反季节切花生产的市场需求和经济效益巨大。然而，球茎休眠是我国唐菖蒲反季节种植的主要限制因素。课题组前期从调控唐菖蒲球茎休眠重要激素ABA入手，明确了脱落酸信号转导对球茎休眠诱导与解除起关键作用，通过转录组数据分析，筛选获得了ABA共受体同源基因GhPP2C1。本项目拟通过基因功能和表达验证，明晰GhPP2C1在球茎休眠解除中的功能。为深入研究GhPP2C1的转录调控机制，用酵母单杂筛库的方法，获得一批上游转录因子。拟通过结合验证的方法，筛选出重要的转录因子，并明确直接调控GhPP2C1的模式。此外，结合过表达与沉默分析，解析上游转录因子通过GhPP2C1调控球茎休眠解除的分子机制。本研究不仅能为采用分子育种手段改良唐菖蒲休眠特性提供基因储备，也为反季节种球的国产化奠定理论基础。

唐菖蒲是世界重要切花，其球茎休眠极大地限制了反季节切花生产。申请者通过转录组和外施激素处理试验证实脱落酸（ABA）和细胞分裂素在球茎休眠解除过程中有相反作用。基于转录组数据挖掘和基因沉默技术，证实了ABA共受体基因GhPP2C1可减少球茎内源ABA含量及减弱ABA信号传递，进而正调控休眠解除。通过酵母单杂筛库，获得一批GhPP2C1上游转录因子。其中，NAC转录因子GhNAC83直接负调控细胞分裂素合成基因GhIPT的表达和ABA信号共受体基因GhPP2C1的表达，减少体内细胞分裂素含量和ABA的信号响应，进而调控球茎休眠解除。鉴于目前尚无直接证据表明细胞分裂素相关基因参与调控球茎休眠解除，申请者采用基因沉默技术，沉默细胞分裂素相关基因GhIPT和GhCYP735A，并检测了球茎内源细胞分裂素各活性成分含量，结果证实了细胞分裂素合成基因在球茎休眠解除过程中发挥着重要的正调控作用。这些研究结果表明NAC转录因子家族成员可以同时直接调控ABA和细胞分裂素通路，进而调节球茎休眠解除的生理过程。这一发现不但丰富了ABA与细胞分裂素调控节点，也对于理解ABA和细胞分裂素的相反作用提供了新的证据。同时，为通过基因工程改良休眠特性提供了新的基因资源。