赤霉素诱导油松球花（孢子叶球）决定的分子机制研究

Flowering is a symbol of plant growth from vegetative growth to reproductive growth, which is the core of species continuity, system evolution and seed production. Gibberellin (GA) is one of the important pathway to induce higher plants flowering. In angiosperms the GA pathway to induce flowering has been defined, while in gymnosperms the reproductive organs induced by GA synthesis and regulation mechanism, signal transduction model and target gene determination etc are remains to be studied, which lead to a lack of effective theoretical support of GA in promoting conifer flowering and illuminating the reproductive organs evolution mechanism of seed plants. In this study we will take Pinus tabuliformis, the important native conifer tree species in northern China, as the object to explicit the active types and regulation mechanism of GA, clarify the system development and interaction model of the discovered 5 GID1 and 2 DELLA proteins on the GA signaling pathway and determine the targeted gene for strobilus initiation induced by the active GAs with the aid of already mastered new technology, such as yeast two-hybrid system, bimolecular fluorescence complementation (BiFC), callus genetic transformation of Pinus tabuliformis and RNA-seq high-throughput analysis etc. Based on this, we will reveal the molecular pathway between GA and the strobilus development, deepen and improve the molecular regulation network of strobilus initiation of Pinus tabuliformis. Results of this research will provide basic theory for reproductive control in seed orchard of Pinus tabuliformis, offer scientific basis for the GA induction pathway in gymnosperms represented by Pinus tabuliformis, and also supply experimental evidence for gymnosperms and angiosperms reproductive evolution.

成花决定是植物从营养生长转入生殖生长的标志，是物种延续、系统进化和良种生产的核心内容。赤霉素作为高等植物成花决定的重要途径之一，在被子植物中的诱导途径已经明晰，而裸子植物生殖器官诱导的赤霉素合成与调控机制、信号转导模式以及靶向基因确定等方面还尚待深入研究，导致赤霉素在促进针叶树球花决定以及种子植物生殖器官进化等方面还缺少理论支撑。本研究以我国重要的针叶树种油松为对象，借助已掌握的酵母双杂交、双分子荧光互补、油松愈伤遗传转化、RNA-seq高通量分析等技术，解析油松球花决定过程中活性赤霉素种类及其调控机制、阐明油松赤霉素信号通路5个GID1和2个DELLA蛋白的系统发育与互作模式、确定赤霉素诱导球花发端的靶向基因。以此为基础，构建赤霉素与油松球花决定之间的分子调控路径，深化和完善油松球花形成的分子调控网络，为当前油松种子园的生殖调控提供基本理论，也为裸子植物和被子植物的生殖进化提供实验证据。

生殖发育是针叶树遗传改良和良种生产的生物学基础，同时也是植物进化研究的核心问题。本研究以我国重要的针叶乡土树种油松为研究对象，开展了油松球花发育中赤霉素受体互作相关的候选基因筛选、赤霉素与球花发育之间的调控路径、PtLFY和PtNLY调控油松生殖发育差异的分子机制等研究，构建了赤霉素与油松球花决定之间的分子调控路径。主要研究结果：（1）油松基因组鉴定到9个PtNF-YAs、9个PtNF-YBs和10个PtNF-YCs基因。PtNF-YAs、PtNF-YBs和PtNF-YCs分别含有对应的保守结构域。PtNF-Ys在球花（雌雄）和根中表达较高，而在茎和花粉中表达较低。（2）PtCOL5和PtNF-YC1/4是参与油松球花发育重要候选调控因子。PtNF-YC1和PtNF-YC4与PtCOL5在球花发育中高度相关。（3）PtNF-YC1/4具有调控花发育功能。PtNF-YC1和PtNF-YC4异源转化拟南芥在短日照下推迟开花，说明PtNF-YC1/4影响拟南芥开花具有调控花发育功能。（4）PtNF-YC1/4和PtCOL5互作增强PtTFL2表达。PtNF-YC1/4和PtCOL5分别激活PtTFL2的表达，当PtNF-YC1/4和PtCOL5两者存在时能够增强PtTFL2的表达，说明在油松中PtNF-YC1/4能与PtCOL5互作形成复合体来激活PtTFL2。（5）PtNF-YC1/4-PtCOL5互作受PtDELLA抑制进而调控PtTFL2表达。酵母三杂交实验证实PtDELLA能阻碍PtNF-YC1/4和PtCOL5的结合，表明PtDELLA与PtNF-YC1/4竞争PtCOL5互作，在赤霉素信号转导中调控PtTFL2表达，影响油松球花发育。研究结果针叶树种子园良种生产和升级换代提供基本理论，也以油松为代表的裸子植物赤霉素诱导途径、生殖进化提供科学依据。