GmGBP1基因参与大豆光周期、温度和赤霉素协同调控开花时间的分子机制研究

Soybean is a thermophilic and photoperiod-sensitive short-day plant, which narrows the adaptability of soybean vairiety and affects the effective exertion of yield level. GmGBP1 transcription factor is a very important promoting factor in soybean photoperiod and gibberellin (GA) controlling flowering pathways and the expression level is also induced by high temperature. The objectives of the present work aim to utilize the GmGBP1-overexpressing and GmGBP1-interferring transgenic plants and the control wild-type soybean as experiment materials to identify the alterations of photoperiod, temperature, photo-thermal comprehensive response sensitivity as well as GA response sensitivity treated by different photoperiod and temperature, or GA to clarify whether it is involved in the photo-thermal interaction and GA pathways regulating flowering time. On the other hand, data were integrated and analyzed to discover novel key genes by combining the ChIP-seq and RNA-seq data of GmGBP1 regulating the downstream target genes on the whole genomic and transcriptional levels, as well as the CoIP-MS data of protein interaction on the proteomic level. The research will be of significant meaning of theory and production by constructing regulatory network of GmGBP1 involved in the photoperiod, temperature and GA controlling flowering time and it will also provide new experimental data for improving soybean wide adaptability by the approach of genetic engineering.

大豆是喜温和光周期敏感的短日照作物，这一特性降低了大豆品种的适应性，影响产量水平的有效发挥。GmGBP1转录因子是大豆光周期和赤霉素开花途径中重要的促进因子，并且高温诱导该基因表达。本研究拟以GmGBP1过量表达、干涉植株及其对照野生型大豆为试验材料，一方面根据不同光温或赤霉素处理鉴定光周期、温度和光温综合反应敏感性，及对赤霉素反应敏感性的变化，阐明该基因是否作为整合因子参与光温互作和赤霉素途径调控大豆开花时间。另一方面，结合全基因组和转录组水平上ChIP-seq和RNA-seq获得的GmGBP1调控下游靶基因数据，以及蛋白质组水平CoIP-MS获得的蛋白互作数据，进行数据分析和整合，从而挖掘出新的关键基因，并最终构建GmGBP1参与大豆光周期、温度及GA调控开花时间信号传导网络系统，为通过基因工程手段来改良大豆广适性提供新的实验数据，具有重要的理论和生产实践意义。

大豆属于光周期敏感的短日照作物，这一特性降低了品种的适应性，影响产量水平的有效发挥。GmGBP1是大豆光周期调节开花的重要促进因子。在大豆中，SKIP有两个同源基因分别命名为GmGBP1与GmGBP2，二者氨基酸同源率达到96.73%。利用高效的大豆遗传转化技术，获得GmGBP1-ox过表达大豆植株,进行光周期反应基因功能验证，在长短日照下T3代转基因植株与野生型植株相比，株高明显高于野生型植株，开花早；GmGBP-i干涉植株相反。对大豆进行外施赤霉素处理，发现转基因大豆植株对赤霉素的反应敏感度显著高于野生型植株。蛋白半定量结果表明短日照和赤霉素均能诱导GmGBP1蛋白表达，上述结果说明GmGBP1转录因子为大豆光周期开花途径中的开花促进因子，并参与赤霉素促进大豆株高反应。利用ChIP-seq技术构建ChIP DNA文库及相关数据分析，鉴定出GmGBP1蛋白DNA-结合基序PIF4/PIF5(bHLH)-binding motif即PBE-box (CATGTG)，GmGBP1通过与GmSAUR启动子PBE-box基序结合并抑制GmSAUR表达，RNA-seq测序表明GmGBP1能够抑制E2基因表达，并促进GmFT2a基因表达从而促进开花。利用BiFC和CoIP技术鉴定出GmGBP1互作蛋白GmGAMYB，获得GmGAMYB-ox过表达大豆植株，也表现出早花、早熟特征。确定了GmGBP1通过与GmGAMYB互作通过促进GmFULc的表达从而促进大豆开花和成熟。本研究阐明了GmGBP1基因作为正调节子参与光周期调控开花效应和GA效应的分子机制。