AP3/PI基因调控国兰花朵瓣型变异的分子机理研究

Chinese Cymbidium plant is a traditional famous flower in China. The variation in its floral morphology not only significantly improves the ornamental and economic value, but also directly promotes their industrial development. However, the molecular mechanism of their morphological variation remains unclear. Our research team dedicate on the selection of good traits as well as functional gene discovery of the orchid plant for more than ten years. We have obtained 117 cultivars with different floral morphology, and four duplicated AP3 genes and a PI-like gene were considered to be most important for their morphological variation. Through gene cloning and functional analysis, we found different subfamilies of AP3 genes regulate different flower organ development, to achieve specific control of the unique morphological variations of Chinese Cymbidium plant. Comparing to the most recent studies in Oncidium Gower Ramsey, we found different roles of these AP3 subfamily in Chinese Cymbidium, which illustrated the regulation of its floral organ morphogenesis more detailedly. However the specific functions of these AP3/PI genes and the regulatory mechanism underlying the variation in floral morphology need to be further analyzed. In this study, we will determine the functional mechanism of these AP3/PI genes associated with different floral structure development, which resulted in phenotypic variations of the orchid plant. On this basis, we will establish a molecular model to initially illustrate the regulation of floral morphology, which could offer important theoretical basis for molecular breeding and horticultural traits improvement of the orchid plants

国兰是我国十大传统名花之一，其独特和丰富的花朵瓣型变异不仅显著提高园艺观赏和经济价值，更为兰花花器官分化和发育研究提供了重要的材料。我们研究团队十多年致力于国兰瓣型变异品种选育和优良性状相关功能基因挖掘，已获得荷瓣、梅瓣、蝶瓣、少瓣、多瓣、树形花等变异品种材料117份；并通过转录组测序、基因克隆和功能初步分析，发现4个AP3和1个PI基因与花型变异紧密相关，不同AP3亚族基因之间存在精细分工从而控制不同瓣型的变异，但与文心兰等热带气生兰的最新研究结果有显著的不同，推测AP3亚族基因功能可能存在种属特异性。本项目将深入解析国兰AP3和PI基因的功能，并基于花器官发育四聚体模型，详细阐述它们对国兰花器官形态建成和瓣型变异的调控作用机制；以此为切入点，初步建立B类MADS-box基因调控的国兰花朵瓣型变异分子模型，作为兰科植物园艺性状改良及分子育种的重要理论基础。

国兰其独特和丰富的花朵瓣型变异不仅显著提高园艺观赏和经济价值，更为兰花花器官分化和发育研究提供了重要的材料。本项目深入解析国兰AP3和PI基因的功能，并基于花器官发育四聚体模型，详细阐述它们对国兰花器官形态建成和瓣型变异的调控作用机制；以此为切入点，初步建立B类MADS-box基因调控的国兰花朵瓣型变异分子模型，为兰科植物园艺性状改良及分子育种的奠定基础，主要研究结果如下：.1.通过克隆从建兰中分离出CePI、CeAP3-1、CeAP3-2、CeAP3-3和CeAP3-4共5个B类MADS-box基因。同源比对和进化树显示5个B类MADS-box基因与其他植物中对应B类基因都具有很高同源性。.2. CePI等B类基因在建兰中时空表达显示，CePI、CeAP3-1、CeAP3-2、CeAP3-3和CeAP3-4这5个B类MADS-box基因都集中在生殖器官花中高表达，而在营养器官根茎叶中低表达或几乎不表达。CeAP3-1在花芽发育前四个阶段表达量逐渐上升，在花开放时表达量陡增；CeAP3-2/3/4在花发育整个阶段表达量比较均匀，无明显差异。.3. CePI等B类基因在建兰不同瓣型中的实时定量显示，CeAP3-1主要在外轮花被片中高表达，可能调控萼片发育形成；CeAP3-2主要在唇瓣中集中表达，在唇瓣形成中其主要调控作用；CeAP3-3在内轮唇瓣和合蕊柱中相对较高表达，可能主要参与内轮唇瓣和合蕊柱的调控形成；CeAP3-4在各轮花器官中表达量差异不明显，可能辅助各轮花器官的形成。.4. CeAP3-2在拟南芥中高表达结果显示，高表达植株柱头呈现弯曲的状态，CeAP3-1和CeAP3-4在拟南芥中高表达无明显表型；而实时定量结果显示，高表达植株相比野生型，CeAP3-1/2/4表达量呈现出不同程度的升高。.5. 通过克隆从建兰中分离出CeSEP1、CeSEP2、CeSEP3和CeSEP4共4个E类MADS-box基因。同源比对和进化树显示4个E类MADS-box基因与蝴蝶兰中对应E类基因都具有很高同源性。在不同瓣型的建兰中分析了4个SEP基因的表达模式。.6. 采用酵母双杂交体系验证了AP3蛋白与SEP蛋白的互作关系，结果发现AP3-2能够与4个SEP蛋白分别发生互作。.7. 开发出一套高效省时的国兰原生质体制备技术，利用原生质体成功完成基因瞬时表达以及亚细胞定位实验。