光周期调节基因GmFT4在大豆中的功能研究

Since the major maturity gene E1 is a legume-specific transcription factor in soybean, the main pathway that regulate soybean photoperiod response and floweirng is different from that in Arabidopis or rice. Expression profiling demonstrated that E1 can upregualte GmFT4. In term of expression pattern under SD/LD conditions, GmFT4 showed an antagonistic relation to GmFT2a/5a that have been characterized as soybean "Florigen", an activator of flowering. Although GmFT4 is phylogenetically belonging to FT family based on the key domain in the protein, it might function antagonistically to FT, as a repressor of flowering like TFL in Arabidopsis. The function of GmFT4 in regulating flowering time in soybean has not yet been reported, the function of GmFT4 is the main target of this study.The expression pattern in different tissues and organs at different developmental stage will be studied. The funtion of GmFT4 will be fully elucidate via overexpression and RNA interference(RNAi) in soybean. When the GmFT4 is transformed into various E1 genotypes, the expression profilings of GmFT2a/5a, E1, and GmFT4 in the transgenic soybean will be analyzed to reveal functional connections. The key residues will be swapped by site-directed mutagenesis to determine the key residues leading to the functional divergence between FT and GmFT4. This study on the function GmFT4 protein will facilitate our understanding of the precise regulation mechansim of the floweing in soybean.

控制大豆生育期的核心基因E1是豆科植物特有的转录因子，据此我们推测大豆可能存在着独特的开花分子调控机制。我们首次图位克隆了E1基因，E1超量表达转基因大豆的表达谱分析显示E1可以明显上调GmFT4的表达，然而GmFT4基因在长日/短日条件下的表达特征与已明确的大豆成花素基因GmFT2a/5a相反，呈负相关关系。据此我们推测，GmFT4虽然属于FT家族蛋白，但功能可能与GmFT2a/5a相反。目前尚未有GmFT4功能和其在开花调控中作用的报道。本研究将利用Real-time PCR分析该基因的时空表达特征；利用过表达、RNAi技术系统全面地解析GmFT4在大豆中的功能；通过对不同E1基因型大豆品种的遗传转化及转基因植株中GmFT2a/5a的表达量分析，明确该基因在E1-FT通路中扮演的角色；通过定点突变技术确定GmFT4功能转变的关键位点。本研究对于揭示大豆特有的开花调控机制具有重要意义。

大豆的开花分子调节机制有自己独特性。GmFT4受大豆生育期核心基因E1调控，E1是豆科植物特有的转录因子，GmFT4与以往的FT不同，是个开花抑制子，可见E1-GmFT4是大豆生育期调控特有通路，深入研究GmFT4对于揭示大豆光周期调节机制具有重要的意义。本项目利用Real-time PCR详细分析了该基因的时空表达特征，发现GmFT4主要在叶片中表达，且表达量与大豆的开花期明显正相关。GmFT4在一日的表达呈现有规律的节律性，且这种节律性表达严格受光调控，光照可以促进其表达，黑暗则抑制其表达。采用RNAi将GmFT4抑制后，大豆开花提前，将GmFT4在大豆中过表达后，大豆开花显著延迟，同时结荚习性由亚有限变为无限。大豆中的FT之间也存在调控关系，在具有开花促进作用的GmFT2a/5a过表达植株中，GmFT4显著下调，说明GmFT4受GmFT2a/5a调控，但GmFT4的过表达植株中，GmFT2a/5a的表达不发生变化，说明GmFT4不调控GmFT2a/5a的表达。通过蛋白序列比对，预测GmFT4第161位氨基酸可能是其功能发生逆转的关键。然而将该位点突变后，基因功能确未发生改变。根据我们的研究并结合最新的文献资料，我们认为大豆中的FT类基因不像拟南芥中的FT/TFL可以用一两个氨基酸来划分其功能。GmFT4可显著抑制分生组织开花决定基因GmAP1a、GmAP1b、GmAP1c、GmAP1d、及8个MDAS-Box基因的表达。