油松LEAFY/NEEDLY靶基因筛选及其功能特异性研究

The LEAFY play a key role in the reproductive development regulatory networks in angiosperm plants, which integrate the reproductive development responses to the downward transmission of the endogenous and environmental signals. While the function of LEAFY homologous in the reproductive development of gymnosperms plants were still unclear, and few studies have identified the direct target genes of their. Based on our earlier investigations, we will investigate the function and mechanisms of the two LEAFY homologous, LEAFY and NEEDLY, in Pinus tabuliformis. First, we will identified the direct target genes of LEAFY homologous. Then we will explore the regulation mechanisms of the LEAFY to BC genes. Finally, we will further analyze the functional specificity of LEAFY and NEEDLY in the reproductive development of P. tabuliformis. In the course of the investigation, some new methods and techniques including gene expression profile analysis of transgenic cells, co-expression network analysis, regulatory element analysis will be used to determine the specific roles of LEAFY and NEEDLY in the reproductive development of gymnosperms plants, represented by P. tabuliformis. the results of this study will provide new insights into the roles and function of LEAFY homologous in the reproductive evolution of seed plants.

LEAFY是被子植物生殖发育调控网络中的核心调控因子，是整合内源与环境调控信号并向下传递引发生殖发育的节点。然而LEAFY同源基因在裸子植物生殖发育中的具体功能尚不十分清楚，特别是对其直接调控靶基因的筛选鉴定研究十分匮乏。在前期工作的基础上，本项目将以油松为材料，对其两类LEAFY同源基因（LEAFY ⁄ NEEDLY）的功能与作用机制进行解析：（1）筛选LEAFY同源基因的直接调控靶基因；（2）研究它们对油松BC类基因的调控及机制；（3）分析LEAFY与NEEDLY在油松生殖发育中的功能特异性。在本项目中，拟采用转基因表达谱分析、共表达网络分析、调控元件分析等新研究手段，旨在揭示以油松为代表的裸子植物LEAFY与NEEDLY在生殖发育调控中的具体功能，也为LEAFY同源基因在种子植物生殖进化中的地位与作用理论的发展提供新的数据支撑。

LEAFY是被子植物生殖发育调控网络中的核心调控因子，是整合内源与环境调控信号并向下传递引发生殖发育的节点。然而LEAFY同源基因在裸子植物生殖发育中的具体功能尚不十分清楚，特别是对其调控靶基因筛选及其功能特异性研究十分匮乏。本项目在建立油松愈伤组织诱导与遗传转化体系的基础上，在油松愈伤细胞内超表达了油松LEAFY同源基因PtLEAFY（PtLFY）与PtNEEDLY（PtNLY），通过RNA-seq分析筛选了其调控靶基因，发现PtLFY与PtNLY响应基因存在一定的相似性，表明其功能在油松中的确存在一定的冗余，但同时两者也有各自独特的响应基因，为两者功能的特异性提供了直接证据；油松PtLFY与PtNLY可以部分恢复拟南芥lfy-1突变体表型，其中PtNLY / lfy-1产生花器官结构较多，并可产生少量可育果荚，表明PtNLY在拟南芥中对花器官发育的调控功能更强。PtLFY与PtNLY均可结合AP1启动子元件，但与PtLFY相比，PtNLY可以形成更强的同源二聚体，同时PtNLY与油松UFO同源蛋白互作强度更强，表明其在调控B类基因表达中发挥主要作用。在不同油松组织样品中，PtLFY与PtNLY具有十分相似的表达模式，但几乎在所有样本中，PtNLY均具有更高的表达丰度。综合以上分析，表明PtLFY与PtNLY具有部分冗余的功能，同时也存在一定的功能特异性，其中PtNLY可能在针叶树发育过程中发挥更核心的调控功能。本研究为深入理解裸子植物LFY与NLY基因的功能分化提供了重要数据参考。