紫花地丁两型花发育过程中赤霉素及相关基因表达变化研究

Some plants develop a breeding system that produces both chasmogamous (CH) and cleistogamous (CL) flowers that is the results of adaptive evolutionary. However, the underlying molecular mechanism remains elusive. Viola philippica is a species with the typical CH-CL mixed breeding system. The photoperiod was the most crucial factor that effected the development of dimorphic flower. CH flowers were formed in response to short daylight, CL flowers in response to long daylight, with intermediate CL flowers produced during transitional periods. Previous studies have shown that the number and size of petals and stamens in CL flowers was lower and smaller than those of normally developed CH flowers, and the inhibition of the development of petals and stamens might be due to the downregulation of B-class MADS-box genes expression dosage by long daylight. In addition, some study indicated that active gibberellins could respond to the change of photoperiod, and be essential for the normal development of stamens and petals in floral buds. Moreover, the results of comparative transcriptomics of our preliminary study showed that there was significant difference of the expression of some gibberellins biosynthesis enzyme genes especially GA20ox, GA3ox and GA2ox between CH and CL flowers. So we suspect that the biosynthesis of active gibberellins might be also regulated by photoperiod and influence the development of the CH and CL flowers in V. philippica. In this project, under the different daylight, we will examine the endogenous active gibberellins and their precursor content, compare the gene expression pattern of GA20ox, GA3ox and GA2ox in different type flowers, and detect the phenotype change in exogenous system that was caused by gene silencing of GA20ox, GA3ox and GA2ox of V. philippica. Meantime, we also test exogenous gibberellins how to effect the dimorphic flower development and study the expression of B-class MADS-box genes in response to the gibberellins. Through the experiments above, our study will explore the molecular mechanism of gibberellins on the regulation of CH and CL flower development, and understand the significance of evolutionary biology about formation of dimorphic flower.

自然界一些植物具开放与闭锁花的混合繁育特征，是适应性进化的结果，但其形成的分子机制尚不清楚。紫花地丁是常见且具典型开放与闭锁花的两型自花受精植物，光周期是影响其两型花形成的关键生态因子。前期研究表明，开放花与闭锁花的差异主要体现在雄蕊与花瓣的大小数量上，与B类MADS-box基因的表达变异有关。而且，赤霉素可响应光周期变化并调控植物雄蕊与花瓣发育，结合我们比较转录组学研究结果，推测光周期可能还会通过调控赤霉素合成而影响两型花发育。本项目将在不同光周期下检测花芽内源活性赤霉素及前体含量，研究GA20ox、GA3ox与GA2ox等赤霉素合成相关基因在两型花发育过程中的表达模式及在外源体系中被沉默后花芽的表型变化，结合外施赤霉素等途径，揭示赤霉素对两型花发育的影响规律，并研究B类MADS-box基因对赤霉素的响应方式，探讨赤霉素对两型花发育调控的分子机制，理解两型花植物形成的进化生物学意义。

被子植物花适应性进化发育的机制是植物科学领域的前沿科学问题和研究热点。自然界有些植物在不同部位或不同环境下形成开放花（CH）与闭锁花（CL）两种形态完全不同的花，但环境和遗传因子相互作用下两型花进化发育的分子机制尚不清楚。前期研究发现，光周期是影响紫花地丁两型花形成的关键生态因子，在短日照下形成开放花，随光周期延长则诱导闭锁花的形成；前期研究已表明闭锁花中花瓣与雄蕊发育的抑制与B功能MADS-box基因表达的下调有关。该项目进一步揭示了赤霉素调控紫花地丁两型花发育的分子机制。研究发现，随着光周期的延长，活性赤霉素合成与活性赤霉素含量均有上升趋势，GA3-oxidase和GA20-oxidase在长日照下闭锁花中的表达量要高于短日照对应的开放花，而GA2-oxidase的表达趋势正好相反，闭锁花中的活性赤霉素含量均高于开放花；赤霉素合成关键酶基因GA20ox与GA3ox的时空表达推断赤霉素的合成位点主要在雄蕊与雌蕊中，且雌蕊中的表达量高于雄蕊，推测雌蕊中较高浓度的赤霉素会抑制雄蕊的发育及雄蕊中赤霉素的合成；外源赤霉素处理能抑制花发育与B功能MADS-box基因的表达，促进中短日照下闭锁花的形成。这些研究结果揭示了赤霉素在紫花地丁闭锁花的发育中起积极作用，为理解两型花进化发育提供了重要思路。