小麦中MSR（methionine sulfoxide reduetase）基因在植物耐逆中的作用机制研究

Drought and high salinity are important factors limiting growth and yield of plant. Therefore, it is one of the key strategies in the future to characterize more abiotic resistance related genes for crop improvement. It was not reported about the molecular mechanism of MSR gene although its family has been cloned from Arabidopsis and rice. MSR genes can be induced by various abiotic stresses, and MSR can clear the excess ROS in plant cell, and also can catalyze the reduction of R and S MetSO (free and peptide) oxidized by ROS in vitro, suggesting they could play important roles in stress response. We characterized two abiotic stress-induced MSR genes, TaMSRB3.1 and TaMSRA4.1, from a wheat somatic hybridid cv.SR3 with high salt resistance, and found that overexpression of them in Arabidopsis and wheat could enhance their abiotic stress tolerance. In this project, we will clone MSR gene family and detect the response profiles of TaMSRB3.1 and TaMSRA4.1 to various abiotic stresses, characterize their substrates, identify the important genes regulated by TaMsrB3.1 and TaMsrA4.1, discover the relationship between them and ROS/ABA pathways, and isolate transcripnal factors regulating the expression of TaMSRB3.1 and TaMSRA4.1. It will primarily elucidate the underlying mechanism of MSR genes in abiotic stress tolerance in plants.

干旱和高盐等逆境是限制植物产量的重要因素。因此，鉴定抗逆相关基因用于作物改良是未来抗逆育种的关键策略之一。MSR基因已在拟南芥和水稻等植物中克隆，但其作用机制未见报道。MSR能被多种非生物胁迫诱导表达，而且MSR可以清除机体内过量的ROS，也可以体外催化因ROS氧化的R和S型MetSO的还原，暗示其在植物胁迫应答中发挥作用。申请人从体细胞渐渗系耐盐新品种SR3中克隆了非生物胁迫响应基因TaMSRB3.1和TaMSRA4.1，其在拟南芥和小麦中过表达能提高耐盐、抗旱、抗氧化能力。本申请拟系统并首次克隆小麦MSR基因家族，揭示TaMSRB3.1和TaMSRA4.1的在非生物胁迫下的响应特征，鉴定其体内底物，研究该基因调控的下游基因及其与ABA和ROS途径的关联，发现调控TaMsrB3.1和TaMsrA4.1表达的重要转录因子，初步阐明其在应答非生物胁迫的作用机制。

本研究以山融三号甲硫氨酸亚砜还原酶基因TaMSRA4.1和-B3.1为研究对象，利用小麦过表达体和拟南芥相关遗传材料，对TaMSRA4.1的基因功能和作用机制进行了全面详细的研究。本研究系统养久了小麦MSR基因家族的序列，并在基因结构，蛋白特征、分子进化、功能结构域及酶学性质等方面进行分析；发现TaMSRA4.1和B3.1蛋白主要定位于叶绿体中；它们的表达并无组织特异性，但在叶和茎中表达较高，且能够被盐、旱胁迫所诱导。以扬麦20、TaMSRA4.1和-B3.1小麦过表达系及拟南芥野生型Col-0、TaMSRA4.1/B3.1拟南芥过表达系及msra4拟南芥突变体等为材料，分别进行不同程度的盐旱处理观察野生型和过表达体的表型实验，结果发现TaMSRA4.1能够显著的提高小麦及拟南芥对盐旱胁迫的抗性;B3.1能提高植物的抗旱性。通过对小麦及拟南芥各个株系进行ROS染色实验发现，它们能够显著的降低小麦及拟南芥在盐旱处理下的ROS积累，减轻氧化损伤，增强氧化清除酶类如SOD及CAT等的酶活，降低MDA含量。通过检测ROS通路的marker gene，结果发现它们通过抑制ROS信号传导通路相关基因及ROS产生相关基因，促进ROS清除相关基因的表达来维持ROS平衡。通过气孔运动实验发现TaMSRA4.1能够提高小麦及拟南芥气孔对于ABA的敏感性，对ABA通路相关Marker gene的检测及TaMSRA4.1/aba2表型实验结果表明，TaMSRA4.1能够分别调控ABA的内源合成及下游逆境胁迫响应基因的表达。. 通过生物信息学预测分析血红素加氧酶TaHO1可能是TaMSRA4.1/B3.1潜在互作蛋白。通过酵母双杂、BiFC、Co-IP等实验证明它们与TaHO1能够互作结合，并且结合区域为TaHO1蛋白的Heme Domain（Heme区）。通过体外底物酶活反应实验证明，TaHO1是TaMSRA4.1/B3.1的底物。药理学表型实验及遗传实验证实，它们是通过与TaHO1的互作，一方面作用于ROS通路，另一方面作用于ROS通路发挥抗逆作用，二者对盐旱响应的差异需要进一步研究。