大豆中枢生育期基因耦合光周期的分子机理

The traditional genetic study and positional cloning demonstrated that maturity gene E1 is the central flowering repressor in soybean, and its expression is strongly suppressed under short-day condition and displaying a bimodal diurnal pattern under long-day condition. Therefore, understanding of the molecular mechanism underlying synchronization between maturity gene E1 and photoperiod is a key step towards uncovering of photoperiodic flowering pathway in soybean. Firstly, the key cis-elements in response to photoperiod in the promoter region of the E1 gene will be identified through serial deletion studies using expression vector containing conjugated report gene. These identified key elements or sequence will be used as a bait to identify associated transcription factors using various biotechnology e.g. yeast-one-hybrid system. The epigenetic landscape on methylations of DNA or modification of histone protein will be analyzed to reveal any association with the E1 expression. Expression study in the soybean transgenic soybean will be performed to make out any change in the circadian rhythm of central clock when the E1 gene is over-expressed. On the other hand, after screening of constructed soybean mutant library, we will identify mutant lines carrying dysfunctional clock genes(PPR,TOC, CCA1), and further investigate any changes in bimodal diurnal pattern of the E1 gene under long day condition in these mutant lines. Also, whether any key small RNA or lnc RNA is involved in E1 mediated phtoperodiod flowering pathway will be identified through sequencing and subsequent functional analysis. Taken together, the key molecular mechanism underlying synchronization between soybean maturity gene E1 and photoperiod will be understood. The fulfillment of this project will not only enrich regulatory pathways of photoperiodic flowering in plant, but also provide molecular markers or key genes for soybean molecular breeding.

经典遗传学研究及图位克隆结果表明：大豆生育期基因E1是大豆中枢开花抑制因子，在短日照条件下该基因不表达，在长日照条件下则呈现双峰型日周期节律。因而解析E1耦合光周期的分子机理是打通光周期调控大豆开花通路的关键。首先构建不同长度E1启动子区段连接报告基因的表达载体，确定启动子中响应光周期的关键区段，进一步利用酵母单杂等技术鉴定与其作用的转录因子。其次明确位于异染色质区域E1启动子DNA甲基化及组蛋白修饰等对E1表达的影响。进而研究E1过量表达对生物钟节律的影响；筛选并获得相关基因的突变体，明确生物钟基因(PPR、TOC、CCA1等)的功能缺失对E1及相关基因表达及节律特征的影响。通过测序及功能验证确定是否有特异小RNA及lnc RNA参与E1介导的光周期通路。明晰大豆中枢生育期基因E1与光周期耦合的关键分子机制，在理论上可丰富植物光周期调控途径的多样性，同时为大豆分子育种提供重要的分子元件。

E1基因作为开花抑制因子，可抑制大豆开花与成熟期，在调控大豆光周期通路中具有非常重要的作用。本项目以E1启动子序列为主要研究对象，利用酵母单杂交的方法鉴定出与E1基因上游启动子关键区段及与该区段特异性结合的与光反应相关候选基因。在大豆伽玛突变体库中鉴定出早花突变体，引致的突变基因为ELF3的同源基因及E1缺失型。通过该类突变体的基因表达特性的研究，明确了大豆生育期基因与生物钟基因间的耦合关系。结合本项目研究及本领域的最新研究进展，可以推断E1基因位于生物钟基因的下游，其表达受生物钟基因及光反应相关的基因的直接结合而调控，明确了大豆生育期基因耦合光周期的基本原理，为全面揭示大豆中重要的生育期E1通路奠定了坚实的基础。