FaMAX2介导干旱抑制苇状羊茅分蘖发育的分子机制

Tillering is a key component that affects forage grass production. Drought stress prominently inhibits tiller development but the molecular mechanism was largely unknown. F-box protein MAX2 has been previously shown to be the key determinant factor controlling plant growth responses to environmental signals. However, whether drought-inhibition of tiller formation and growth is mediated by this gene is unknown. Preliminary analysis of FaMAX2 responses to drought stress in crowns that control tiller formation and tiller outgrowth by RNA-seq analysis from drought-stressed Festuca arundinacea suggested that this gene could play key roles in regulating tiller formation and outgrowth under drought stress. This project will focus on molecular pathways regulated by FaMAX2 that may controlling tiller formation as effected by drought stress by cloning promoter sequence of FaMAX2 and screening upstream factors that regulates its transcription by yeast one hybrid, characterizing the function of newly screened factors by transient assay in protoplast, EMSA and transformation assay in Festuca arundinacea. In addition, we will create RNAi: FaMAX2 transgenic lines, identifying downstream signaling pathways to answer how FaMAX2 regulates tiller growth under drought stress by RNA-seq and bioinformatic analysis. Such information will provide insights into understanding the molecular mechanism controlling tiller development in grasses, and provide theoretical and practical significance for improving forage grass production by breeding new cultivars with more tillers under drought stress.

苇状羊茅（Festuca arundinacea）是重要的禾本科牧草，干旱胁迫显著抑制其分蘖发育。已有研究显示F-box蛋白MAX2是介导环境信号与植物分蘖发育的重要调控因子，该蛋白的研究大多集中在互作机制上，其上下游调控途径尚不清楚。本课题组前期通过转录组测序获得的苇状羊茅FaMAX2基因受干旱上调表达。在此基础上，本项目拟以FaMAX2基因为切入点，展开两方面研究：（1）获得RNAi：FaMAX2抑制表达的转基因植株，结合转录组测序和生物信息学手段解析FaMAX2介导干旱抑制分蘖的下游途径；（2）通过启动子缺失突变获得FaMAX2基因启动子中的干旱诱导元件，采用酵母单杂交文库筛选获得FaMAX2的上游调控因子并明确其功能，从而揭示FaMAX2响应干旱的上游通路。研究结果可望阐明MAX2介导干旱抑制苇状羊茅分蘖发育的分子机理，为培育多蘖型牧草新种质、提高干旱胁迫下牧草产量提供理论依据。

分蘖是构成禾本科牧草产量的重要农艺性状。干旱胁迫显著抑制植株分蘖的产生与生长，进而影响牧草产量。研究发现，MAX2基因不仅调控分枝/分蘖发育，还参与干旱胁迫。但项目前期实验发现干旱胁迫下FaMAX2的表达显著上调，表明其可能介导干旱胁迫抑制分枝/分蘖发育。在此基础上，我们克隆了苇状羊茅MAX2基因，对其表达模式进行了分析，表明FaMAX2主要在分蘖节表达（产生分蘖芽的部位），且其定位于细胞核。随后我们克隆了FaMAX2上游1500bp启动子区序列，并且进行片段截断的构建，同时构建干旱胁迫下苇状羊茅分蘖节cDNA酵母单杂交文库，经文库筛选获得3个候选上游因子。然而，后期一对一验证并未证实其特异互作。随后我们采用Biacore T200 系统筛选FaMAX2候选互作蛋白48个，并进行酵母一对一互作验证。同时，我们进行了FaMAX2在苇状羊茅和水稻中的抑制及过量表达转基因实验，并且获得了相应的转基因株系。抑制表达FaMAX2降低植物株高，增多其分蘖；而过量表达FaMAX2则使转基因水稻表现出分蘖减少的表型。进一步分析发现，分蘖芽激活基因在转基因株系中下调，分蘖芽休眠相关基因在转基因株系中显著上调。负控制分蘖发育的两个因子，TB1和IPA1，在转基因株系中显著上调。本项目的实施，获得了活体中与FaMAX2互作的候选蛋白，并且揭示了FaMAX2的下游调控因子，初步阐明MAX2介导干旱抑制苇状羊茅分蘖发育的分子机理，为培育多蘖型牧草新种质、提高干旱胁迫下牧草产量提供理论依据。