蛋白激酶SPIS调控蒺藜苜蓿花器官决定的分子机理解析

Flower is one of the most complex and diverse morphology organs of plants. The determination of floral organ identity is always a fundamental question in biological science. Papilionoideae plants, which exhibit the unique morphological structure and developmental characteristics in their floral organs, attracted more and more attention from developmental biologists. To investigate the molecular basis of flower formation in papilionoids, we identified and characterized a special floral organ defect mutant sepals and petals increased severely (spis) in the model legume Medicago truncatula, which has the typical papilionoid flower, through forward screening in Tnt1 retrotransposon tagged lines of M. truncatula. The flowers of spis mutant show a significant increase in the number of sepals and petals, as well as loss of pistil and stamen, which are similar to the floral defect phenotype of the C functional gene deletion mutant of the classic ABC model. We cloned the SPIS gene by forward genetics approach. SPIS encodes an unknown function serine/threonine protein kinase with a conserved PKc (Catalytic domain of Protein Kinases) domain and further enzymatic activity assay shows that SPIS exhibits a phosphotransferase activity in vitro. In this project, we will further identify the direct targets and investigate the regulatory pathway of SPIS. Demonstrating the mechanism how SPIS orchestrates its function will have a significant contribution to understand the molecular mechanism of papilionoid flower development. In the meanwhile, this work may also benefit the genetic improvement of floral pattern in legume plants.

花是植物形态结构中最复杂多样的器官之一，花器官发育一直是生物学研究领域的重要科学问题。蝶形花亚科植物的花器官因其具有独特的形态构造和发育特性而倍受关注。为了研究蝶形花花器官形成的分子机制，我们通过定向筛选具有典型蝶形花特征的豆科模式植物蒺藜苜蓿的Tnt1插入突变体库，分离鉴定了一个特异性影响花器官决定的突变体spis，该突变体表现出花萼和花瓣数目显著增多，雌、雄蕊缺失等异常发育表型，与花发育ABC模型中C功能基因缺失突变体表型类似。我们通过正向遗传学的方法克隆了SPIS基因，该基因编码一个具有保守PKc结构域的功能未知的丝氨酸/苏氨酸蛋白激酶，体外酶活检测发现SPIS蛋白具有磷酸转移酶活性。本项目在此研究的基础上，拟通过鉴定SPIS的直接下游磷酸化底物以及其作用的调控通路，为解析蝶形花花器官决定的分子调控机制奠定理论基础，同时为豆科植物的花型改良提供理论依据。

花是植物形态结构中最复杂多样的器官之一，花器官发育一直是生物学研究领域的重要科学问题。蝶形花亚科植物的花器官因其具有独特的形态构造和发育特性而倍受关注。为了研究蝶形花花器官形成的分子机制，该研究通过定向筛选豆科植物蒺藜苜蓿Tnt1逆转座子插入突变体库，分离鉴定到一个雌、雄蕊缺失，花萼内部花瓣、花萼和花萼花瓣嵌合体增生，与蒺藜苜蓿花器官决定C功能基因AGAMOUASa (MtAGa)和MtAGb的双功能缺失突变体表型类似，该突变体还具有与蒺藜苜蓿terminal flower 1 (mttfl1)突变体类似的花序异常表型，即初级花序由无限生长转变为有限生长，因此将该突变体命名为agamous and terminal flower (agtfl)-1。此外，该突变体的叶片纵向伸长受到抑制，导致叶片呈现“心形叶”的表型。突变体的这些表型表明AGTFL在调控蒺藜苜蓿的花器官决定、花序和叶片形状发育等方面发挥着至关重要的作用。AGTFL基因编码一个核定位蛋白，在其N末端含有一个Myb/SANT-like DNA结合结构域，在其C末端有一个PKc激酶结构域。分子检测和遗传分析发现AGTFL通过影响MtAGa和MtAGb和花序发育关键调控基因MtTFL1的表达分别调控蒺藜苜蓿的花器官发育和花序形态建成。该研究为揭示蒺藜苜蓿及其近缘豆科植物花器官和花序发育调控机制提供新的研究思路, 同时为植物的花型和株型改良提供理论依据。相关研究成果在Journal of Integrative Plant Biology杂志上发表（第一标注），获授权国家发明专利1项。