大豆生育期基因E1的作用机理研究

The maturity locus E1 is the major gene underlying the flowering time and maturity in soybean, also involved in the photoperiodic response. Using positional cloning approach, the applicant and his colleagues have successfully identified molecular identity for the maturity E1 locus, which resides in the pericentromeric region and is difficult to identify. Previous studies revealed that this gene is a novel type of legume-specific transcription factor (TF). This research project will focus on the functional mechanism of the E1 gene with an emphasis on how E1 regulates GmFTs, the components of florigens, directly or indirectly. Given E1 gene has a nuclear localization signal (NLS) and DNA binding sites, we will first investigate whether the mutation in the E1 gene has any impact on subcellular distribution of the E1 protein, then to detect whether the E1 gene carries transcriptional activation or repression activity. Importantly, we use the chromatin immunoprecipitation-sequecing (ChIP-seq) to survey the genome-wide binding sites of the E1 protein. Also we use the XVE inducible expression system to investigate the direct target gene(s) of the E1 protein. The functional relationship between E1 and GmFTs (GmFT2a/GmFT5a) as well as other newly identified target sites will be validated by chromatin immunoprecipitation quantitative PCR (CHIP-qPCR), yeast one hybrid (Y1D), electrophoretic mobility shift assay (EMSA) and other technologies. I In addition, we also functionally characterize mutant lines of Medicago truncatula harboring mutations in homologs of E1 and its target sites to further verify functional relationship. Uncovering of how E1 regulates the downstream genes and GmFTs at molecular level will facilitate our understanding of regulatory mechanism of flowering time gene network in soybean, as well as the advance in molecular breeding of the new cultivars with designed flowering or maturity time.

大豆生育期基因E1是控制开花期与成熟期的主效基因，且与光周期反应密切相关。申请者已成功克隆出位于着丝点附近的E1基因，初步明确其为豆科植物特有的转录因子。本项目将集中解析尚未开展研究的E1基因的作用机理,在分子水平上明确E1与成花素GmFTs间的互作关系。基于E1含有核定位信号，首先明确E1的突变体对核定位的影响，进而分析其为转录激活或转录抑制作用。应用染色质免疫沉淀-高通量测序技术，在全基因组范围内解析E1基因的靶体位点；同时利用化学诱导型启动子XVE 系统来鉴定E1直接调控的下游基因。以GmFTs基因为重点,应用ChIP-qPCR、酵母单杂交、凝胶阻滞等技术来检验E1与靶体位点间互作关系。利用苜蓿突变体库存中E1及靶体位点的同源序列突变体进一步完成功能验证。在分子水平上明确E1调控下游基因及GmFTs的机理，对于揭示大豆开花基因的调控网络及开展生育期的分子育种具有重要理论与现实意义。

在克隆了大豆中主效生育期基因E1的基础上，通过本项目的研究明确了E1基因为豆科植物所特有的转录因子。基于E1含有核定位信号，首先明确E1基因的亚细胞定位水平，例如E1基因的突变对核定位的影响，明确了E1与e1-as及e1-b3a蛋白在核定位上差别并揭示了其与功能之间的关系。利用原生质体系统验证了E1与e1-as具有转录抑制功能。通过RNA-seq确定了E1基因调控的下游基因382个存在明显差异，经挑选验证证明了24个基因存在着差异，其中19个基因在E1超表达植株中为下调，5个基因为上调。其中E1稳定调控SPL基因家族基因Glyma.15G076200，不具有miR156识别的靶位点，与拟南芥中的SPL的同源基因明显不同。同时证明了8个AGL类基因受E1调控，并对其表达模式进行了分析，明确了其与E1之间的动态关系。针对E1蛋白制备了单克隆抗体，获得了特异性好的抗体，并利用之进行了Chip分析，并进行了基因组层面Chip-seq。综合Chip-seq测序结果与RNA-seq结果，共有52个基因在RNA-seq测序结果中显示明显差异同时在其启动子部分亦呈现峰值。同时，重点验证了E1基因在GmFT2a基因及AGL类基因Glyma.06g205800启动子区域存在富集，说明E1存在着对开花素及花原基分化重要基因的直接调控作用。以菜豆和蒺藜苜蓿为代表性植物，对E1基因家族的功能保守性和分化展开了研究。在大豆中超表达菜豆来源的PvE1L表现出与抑制开花的功能，而超表达蒺藜苜蓿来源的MtE1L则对大豆的开花无明显影响，但对大豆植株的株型产生了一定的影响。E1基因家族转入拟南芥或水稻中，未见明显的花期表型。对筛选获得的蒺藜苜蓿Tnt插入突变体mte1l进一步分析，MtE1L突变引致植株晚花，表明MtE1L在蒺藜苜蓿具有促进开花的功能。本研究揭示来源大豆近缘种菜豆的PvE1L基因与来源于蒺藜苜蓿的MtE1L在调控开花的功能上出现了明显的分化，且功能分化与E1基因家族的进化及基因组扩增相关联。