矮牵牛FBP11基因互作蛋白的筛选及功能鉴定

As the core organ for generation of female gametophyte, ovule development has been fascinating and mysterious for scientist. Recently, there are some progresses in research for the ovule development especially in model plant of Arabidopsis, but it is far from enough to reveal the gene network behind the formation of this organ. Performing comparative functional studies in multiple species is necessary, which require a broader range of available model systems. Petunia is a good research model due to the recent sequencing of the petunia genome, and current studies have shown that there is an unknown pathway specifically existed in petunia for regulating the ovule development . The known FBP11 gene is identified as ovule identity gene and exclusively expressed in ovule during the whole process, even in the seed development after fertilization. Thus, FBP11 gene is considered as the point for studying the ovule development in petunia, especially for the determination of ovule identity. However, there was no other genes reported to be interacted with FBP11 gene except for E class genes, so there was very little known about the network environment of FBP11 gene. To resolve this problem, the unknown FBP11-interacting proteins will be obtained by the use of yeast two-hybrid technique, and confirmed by the combination of yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays in this study. Subsequently, the genes encoding the obtained proteins will be cloned and analyzed, such as the constructions of overexpression and RNAi vectors for functional analysis, in situ hybridization for expression pattern analysis, and the subcellular localization analysis. Finally, the regulatory relationship between the obtained gene and FBP11 gene will be discussed on the basis of quantitative Real-Time PCR analysis and the phenotype of double mutants. These results will facilitate the understanding of the precise mechanisms by which FBP11 gene perform its dual roles, and eventually enrich the related theory on ovule development in petunia.

结合拟南芥中胚珠发育的研究现状，展开模式植物矮牵牛中胚珠发育相关研究，将更有利于通过比较生物学分析来揭示其分子调控网络，而且现有研究表明矮牵牛中确实存在特异的未知途径对胚珠发育产生作用。FBP11是仅在胚珠内特异表达的基因，对胚珠发育以及受精后种子的发育均有重要作用，是研究矮牵牛胚珠发育尤其早期特性分化的关键。然而目前除了E类基因，没有其他FBP11互作基因的报道，缺乏对其所处分子调控网络的了解。本研究拟用酵母双杂交技术获得新的与FBP11蛋白产生互作的蛋白质，经酵母双杂交和BiFC双验证后，克隆该蛋白编码基因，构建其超表和干涉载体进行功能验证，利用组织原位杂交检测新基因的时空表达方式，并进行亚细胞定位分析，通过表达量分析和双突变体表型，探讨新基因与FBP11间的调控关系，以及二者互作产生的生物学意义。此研究将有利于了解FBP11基因参与的分子调控途径，丰富矮牵牛胚珠发育相关理论。

FBP11是矮牵牛中胚珠特异性表达基因，与FBP7、FBP6、PMADS3在胚珠发育中存在功能冗余，但目前缺乏对这些基因所处分子调控网络的了解。本课题利用4种不同大小花芽构建矮牵牛酵母双杂交cDNA文库，筛选并获得与FBP11蛋白产生互作的Ycf68蛋白，与FBP6蛋白互作的几丁质酶（chi）蛋白，利用pull-down技术验证了这一互作，并且BiFC验证表明FBP11-Ycf68、FBP6-chi互作均发生在细胞核和细胞膜；亚细胞定位显示Ycf68基因定位在细胞核和细胞膜，chi基因定位在内质网；Ycf68和chi基因的超量表达载体正在转化矮牵牛验证其功能。此外，本课题对包括蛋白文库构建所用花芽在内的5种不同大小花芽进行转录组和microRNA测序，获得不同花发育阶段差异表达的转录因子基因，WGCNA分析结果表明MADS、GATA、DOF等转录因子家族以及photosynthesis, light reaction、developmental process involved in reproduction等途径在花发育过程中担当重要角色；miRNA测序测得known miRNA45个，预测noval miRNA26个，其中MiR156、MiR319、MiR160、MiR393等家族成员在不同阶段表现出差异，mRNA-miRNA关联分析表明Trihelix转录因子基因可能是新miRNA靶基因，NAC转录因子基因可能是stu-miR399i-3p靶基因。本课题中FBP11与Ycf68、FBP6与chi互作结果能够为深入研究MADS-box基因在植物中的多样化功能提供参考，mRNA和miRNA测序结果则有助于进一步了解植物花发育过程中存在的分子调控网络。