GbEMF2参与调控银杏开花时间的分子机制研究

The juvenile phase of Ginkgo biloba is quite long. Ginkgo plants typically do not reach sexual maturity until more than 20 years, severely limiting high quality ginkgo breeding. In our previous work, one of important flowering gene GbEMF2 was cloned from G. biloba, and two interacting proteins GbEIP1 and GbEIP2 were isolated from ginkgo by yeast two-hybrid method. The result of mutant complementation experiment showed that GbEMF2 could repress the formation of flower and delay the flowering time in Arabidopsis, implied that GbEMF2 might participate in regulating the flowering time in ginkgo. On the basis of previous work, the present study intends to screen the differential expressing gene among the mutant Arabidopsis (emf2) and transgenic Arabidopsis (35S:GbEMF2/emf2) over-expressed GbEMF2 in the mutant using RNA-seq, and identify the expression level by real-time PCR (qRT-PCR), to initially screen the target gene of GbEMF2. Furthermore, we will investigate the time and spatial expression pattern of GbEMF2, GbEIP1 and GbEIP2 in G. biloba, and analysis the function and regulatory mechanism of GbEIP1 and GbEIP2 involved in regulating the flowering-time by transgenic technology. The regulatory mechanism of GbEMF2 interacting GbEIP1 or GbEIP2 involved in the control of flowering time will be discussed in the present study. At last, we will construct the RNAi expression and overexpressing vectors of GbEMF2. The GbEMF2-RNAi vector and overexpressing vector will be transfromed into ginkgo callus to screen the differential expressed gene. The overexpressing vector of GbEMF2 will be transformed into Arabidopsis to analysis the function of GbEMF2 in regulating the flowering time. Also, on the basis of RNA-seq results of mutant and transgenic Arabidopsis, we will further screen the target gene of GbEMF2 combining the RNA-seq analysis results of transgenic and wild type ginkgo callus. The results of this study will provide the theory basic and technical support for shortening the juvenile phase by genetic engineering technology in G. biloba.

银杏童期特别长，从播种到开花结果需20年以上，这给银杏良种的选育带来了严重障碍。申请者前期从银杏中克隆了一个重要开花基因GbEMF2，并分离了该基因的2个互作蛋白GbEIP1、GbEIP2。突变体互补研究结果显示GbEMF2具有推迟拟南芥成花的功能，预示GbEMF2可能在银杏中也参与了开花时间的调控。据此，本项目拟开展以下研究：1）运用RNA-seq和实时定量PCR，在拟南芥突变体互补试验中筛选GbEMF2调控的下游靶基因；2）开展GbEMF2、GbEIP1、GbEIP2时空表达模式和转基因功能分析，探讨GbEMF2与GbEIP1、GbEIP2互作共同调控银杏开花时间的作用机制；3）构建GbEMF2的RNAi和超表达载体转化银杏愈伤组织和拟南芥，进一步筛选GbEMF2的靶基因，明确GbEMF2参与调控银杏开花时间的分子机制。本研究结果可为今后利用基因工程缩短银杏童期提供理论依据和技术支撑。

银杏（Ginkgo biloba）原产于我国，广泛应用于园林景观、医药、食品和保健等领域。银杏是一种幼年期（又称童期）特别长的中生代孑遗裸子植物，实生树一般需20-25年才能开花结果，这给银杏优良品种的选育带来了严重的障碍。因此在银杏上开展调控开花时间基因的研究，将会为利用基因工程技术大幅度缩短银杏童期奠定良好的基础。EMBRYONIC FLOWER（EMF）基因是近年来发现的抑制植物生殖发育的重要基因，EMF对成花基因的转录具有重要调控作用。本项目以银杏为试材，基于全长转录组数据克隆了1个银杏EMF基因家族成员GbEMF2，利用表达谱分析鉴定了GbEMF2参与银杏开花的功能；利用转基因技术进一步验证了GbEMF2抑制开花的作用，结合RNA-seq与酵母双杂交等技术筛选了GbEMF2下游靶基因以及互作蛋白，阐明了GbEMF2参与调控开花的分子机制。主要研究结果与数据如下：（1）开展了银杏GbEMF2的克隆、鉴定以及表达模式分析。从银杏中克隆鉴定了开花基因GbEMF2，并分析了GbEMF2的表达模式，为解析GbEMF2调控开花的功能提供了基础数据。（2）解析了银杏GbEMF2调控开花的功能以及分子机制。将GbEMF2基因转入拟南芥emf2突变体植株，发现emf2突变体植株的开花时间明显早于WT和GbEMF2的转基因植株。对转基因和突变体emf2以及野生型拟南芥进行RNA-seq分析，筛选了花器官发育、ABC转运蛋白、生长素途径、乙烯途径、脱落酸途径、细胞色素P450和转录因子等40个与开花相关的差异基因，进一步筛选了GbEMF2的下游靶基因，探明了GbEMF2调控开花的分子机制。（3）从银杏中分离验证了与GbEMF2发生互作的三个GbAS2、GbMSI1和GbTFL2蛋白，为阐明GbEMF2调控银杏开花的基因网络奠定了基础。（4）在完成GbEMF2调控功能解析的基础之上，本研究克隆了银杏开花途径中的3个重要基因GbCO、GbMADS9和GbFT，并通过转基因技术、表达谱分析以及转录组分析技术，揭示了银杏GbCO、GbFT和GbMADS9参与银杏开花调控的功能与分子机理。本项目克隆鉴定了包括GbEMF2等多个调控开花的重要基因，不仅丰富了银杏生殖器官发育的遗传学理论，而且为利用基因工程技术缩短银杏童期、开发银杏优良资源等提供了重要的靶标基因与技术支撑。