PtDAL1在油松营养生长时相转变中的功能与作用机制

The reproductive development is one of the most important biological events that affects the germplasm conservation, seed production and genetic improvement of higher plants. The vegetative phase change (transition from juvenile to adult period) is one of the most concerned issues in the research on reproductive development of perennial trees. Although the molecular regulation network of vegetative phase change with the miR156 as the core player in angiosperms has been well elucidated, we still know very little about what controls that process in perennial conifers, especially for the conifer specific regulatory mechanisms. Previous studies have indicating that DAL1 is a key candidate regulator of vegetative phase change in conifers. We will investigate the function and regulatory mechanisms of the PtDAL1 homologous in Pinus tabuliformis. First, we will identified the function of PtDAL1 and analyses its expression response to external and endogenous signals. Then we will identified the target genes, discover and verify the hierarchical gene regulatory networks of PtDAL1. Finally, we will explore the long distance transport characteristics of the molecular product of PtDAL1 gene and its downstream signals. In the course of the investigation, some new methods and techniques including protoplast transformation system of P. tabuliformis, RNA-seq, transcription factor directed hierarchical gene regulatory networks analysis, transgenic Arabidopsis micro-grafting, and grafting of different age P. tabuliformis will be used to determine the specific roles and regulatory mechanism of DAL1 in the vegetative phase change in conifers. The results of this investigation will provide new data support for the understanding of conifer specific reproductive development regulatory networks.

生殖发育是影响高等植物种质延续、良种生产与遗传改良过程的核心生物学问题，营养生长时相转变是多年生林木生殖发育研究最关注的问题之一。目前被子植物中以miR156为核心的营养生长时相转变分子调控网络已经被基本阐明，然而针叶树中的相关研究却十分滞后。前期研究表明，DAL1是针叶树营养生长时相转变关键候选调控因子，本项目拟以油松为材料，对PtDAL1的具体功能与作用机制进行研究：（1）鉴定PtDAL1的功能并观测其表达对外源或内源信号的响应;（2）鉴定PtDAL1在油松中的靶基因并构建其多层次调控网络;（3）阐明PtDAL1基因表达产物及其调控信号长距离运输特性。本项目旨在基于油松原生质体转化体系、RNA-seq分析、hGRNs网络构建、拟南芥微嫁接及不同龄级油松嫁接等技术的综合联用，以阐明针叶树DAL1在营养生长时相转变中的具体功能与作用机制，从而为针叶树特异的生殖发育调控网络提供新的数据支撑。

生殖发育是影响高等植物种质延续、良种生产与遗传改良过程的核心生物学问题，营养生长时相转变是多年生林木生殖发育研究最关注的问题之一。目前被子植物中以miR156为核心的营养生长时相转变分子调控网络已经被基本阐明，然而针叶树中的相关研究却十分滞后。本研究以我国重要乡土针叶树种油松为材料，筛选到针叶树保守的年龄标志因子DAL1，并对其具体功能与作用进制进行了系统研究。借助自主开发的Y2H-seq高通量互作蛋白筛选技术，共鉴定到135个油松PtDAL1直接相互作用蛋白，构建了以PtDAL1为核心的油松蛋白互作网络。在此基础上，揭示了针叶树首个年龄通路调控模块，解析了针叶树年龄标志因子DAL1通过与MADS11直接相互作用，将年龄信号在成年组织中精确转换为生殖发育信号的分子机制。筛选获得DAL1在油松全基因组范围下游靶位点，揭示了PtDAL1通过直接结合下游靶基因PtLFY、PtNLY启动子调控其表达，以启动生殖发育的分子调控机制。发现赤霉素信号通路是针叶树年龄通路的上游信号，通过直接调控DAL1的蛋白稳定性来介导年龄调控通路。证实DAL1ｍRNA及其编码蛋白并非直接可以长距离传递的年龄信号，其处于可移动信号的下游。本研究阐明了针叶树DAL1在营养生长时相转变中的具体功能与作用机制，深入解析了其上下游关键调控因子与蛋白互作网络，拓展了针叶树年龄途径与生殖发育研究的国际前沿，也为针叶树育种繁育新技术的研发与创新提供了思路与方向。