野生大豆GsWDF1基因交叉调控抗旱和早花分子机制研究

Stress tolerance(reducing stomatal and cuticular conductance) and stress avoidance(earlier stage transition from the vegetative growth to reproductive growthg) are main mechanisms that plant have evolved to adapt to drought stress. In our previous study, GsWDF1, a WRKY transcription factor gene, was isolated from Glycine soja, which is highly tolerant to drought and is an ideal candidate plant for isolating drought-tolerance-related genes. By analyzing transgenic Arabidopsis plants that heterogonous overexpression GsWDF1, GsWDF1 was throught to be an integrator of stress tolerance and stress avoidance for Glycine soja to resist drought.In order to further characterize the molecular mechanism of cross-regulating drought and flowering in soybean by GsWDF1, this program was conducted.GsWDF1 gene over-expression and RNAi soybean lines will be used to elucidate the roles of GsWDF1 in stomatal development, cuticle formation, drought tolerance and flowering time, whether GsWDF1 mediating soybean ABA signal pathway、cuticle development、the autonomous flowering pathway will be studied by qRT-PCR ,ChIP-seq will be used with the objective of isolating the target gene that GsWDF1 directly regulates. Ultimately we will characterize the molecular mechanism of cross-regulating drought and flowering in soybean by GsWDF1. The results will help us to understand the molecular mechanism of Glycine soja to resist drought, and will provide new theoretical guidance to study plant resistance to stress.

胁迫耐受(如降低气孔、表皮导度)和胁迫逃避(如提前进入生殖期)，是植物应对干旱等逆境的基本策略。野生大豆抗逆性强，前期研究中我们从野生大豆中克隆了一个WRKY类转录因子基因GsWDF1。在拟南芥中研究表明，它介入多个信号通路过程，提高抗旱性和早花。申请者推测，GsWDF1是野生大豆通过胁迫耐受和生殖逃避来抵御干旱的交叉因子。为深入研究GsWDF1交叉调控抗旱和早花分子机制，本项目以GsWDF1基因过量表达和RNAi大豆为材料，研究GsWDF1对大豆气孔发育、角质层形成和开花时间的影响，评价抗旱和早花功能；采用qPCR技术，研究GsWDF1是否介入大豆ABA信号通路、角质层形成和自主开花途径调控；采用ChIP-seq技术，研究GsWDF1直接调控的下游基因。解析出大豆GsWDF1基因交叉调控抗旱和早花分子机制。研究结果将有助于深入认识野生大豆抗旱分子机制，为植物应对逆境机理研究提供新理论。

GsWDF1基因属于WRKY家族，来源于野生大豆，将GsWDF1在拟南芥中过表达，发现GsWDF1具有调控植物抗旱和早花的功能，深入研究GsWDF1在豆科植物中交叉调控抗旱与早花的分子机制对揭示豆科植物的干旱逃逸机理具有重要价值。本研究以不同生育期大豆品种为材料，通过q-PCR研究发现GsWDF1基因在不同大豆品种间表达量稳定，在叶片中表达量最高，在开花前期表达量达到顶峰，因此GsWDF1基因表达与大豆生殖生长起始相关。GsWDF1基因表达不受光周期影响，但可以被多种非生物胁迫诱导，因此GsWDF1基因与非生物胁迫引起的生殖逃逸现象有关。将GsWDF1基因转化早熟和中晚熟大豆品种，发现GsWDF1基因能提高大豆的抗旱性，并使早熟和中晚熟的大豆品种都能提前开花。抗旱机理研究发现，GsWDF1基因使植物的气孔密度降低、开度减小。通过RNA-seq和qPCR技术发现，GsWDF1基因调控多个ABA信号通路基因表达，从而调控了气孔的密度和开度；GsWDF1基因调控β-酮脂酰-辅酶A合成酶（β-ketoacyl-CoA synthase, KCS）和蜡质合成基因表达，从而影响角质层合成；GsWDF1基因调控了两个MADS-Box基因表达，从而促使植物提前开花。我们综合RNA-seq结果和启动子区分析结果，经过qPCR验证筛选GsWDF1直接调控下游候选基因。以融合myc标签的GsWDF1基因过表达大豆为材料，通过Chip-qPCR和凝胶阻滞实验发现，GsWDF1通过结合到Glyma.12g198200启动子上的w-box元件直接调控Glyma.12g198200（酪氨酸蛋白激酶）的表达，从而初步阐明了GsWDF1调控大豆抗旱和早花的分子机理。