拟南芥STRF1蛋白参与植物盐胁迫信号转导途径的机理研究

Salt stress is one of the major detrimental abiotic stresses that affect plant growth and development, consequently decreased the yield of crops. The ubiquitin/proteasome system has been demonstrated to play critical roles in salt stress response of plant. In order to study functions of some novel E3 ligases involved in salt stress signaling, we focus mainly on the mutant line of E3 (strf1), which is insensitive to salt stress through sceening the mutant pool. Our preliminary results indicated STRF1 was involved in salt stress response pathway; STRF1 was localized on the plasma membrane and endosomes；STRF1 affected endocytosis in plant. To deeply reveal STRF1's function in plant salt stress response, yeast two-hybrid was applied to sreen protiens interacting with STRF1. We had proved that IntP1 was the protien interacting with STRF1. We will confirm the interaction between STRF1 and IntP1 in vitro and in vivo through Pull-down, Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. The degradation of IntP1 through the 26S proteasome in the presence of STRF1 and the stability of IntP1 protein in strf1 mutant will be investigated. Furthermore, the phenotype of IntP1 overexpressing plants stressed by salt will be analyzed. The study might provide some clues to investigate the role of STRF1/IntP1 complex in salt signaling of plant. Since STRF1 affected endocytosis in plant and endocytosis, which is important in salt stress signaling of plant, we will investigate whether STRF1 mediated salt stress signaling transduction through membrance trafficking. This study will role out the new molecular mechanism of plant salt stress response. And as a guidance, it can be applied to develop the new strategy to improve salt tolerance of crops.

盐胁迫是影响植物生长发育并造成农作物大规模减产的主要原因之一，泛素介导的26S 蛋白酶体降解途径在植物参与盐胁迫信号转导中起着重要的作用。为探索未知泛素连接酶E3参与盐胁迫的机制，本研究对筛选到的E3耐盐突变体(strf1)进行深入研究。项目前期工作表明该蛋白（STRF1）参与了植物对盐胁迫的响应；其定位于质膜和内涵体上，调控了植物细胞的内吞过程。本项目已筛选到STRF1的相互作用蛋白IntP1，拟运用Pull-down、Co-IP等体外和体内实验分析STRF1和IntP1的相互关系，并通过对STRF1/IntP1复合体功能的研究来揭示STRF1参与植物盐胁迫响应的分子机理；由于STRF1参与了植物细胞的内吞过程，且内吞过程在植物应对盐胁迫中起着重要作用，本项目将探索STRF1蛋白是否是通过影响植物的膜运输来参与盐胁迫的信号转导途径；该相关研究将为农作物的改良提供理论依据。

盐胁迫是影响植物生长发育并造成农作物大规模减产的主要原因之一，泛素介导的26S 蛋白酶体降解途径在植物参与盐胁迫信号转导中起着重要的作用。为探索未知泛素连接酶E3 参与盐胁迫的机制，本研究对筛选到的E3 耐盐突变体(strf1)进行深入研究。通过遗传学和分子生物学的证据表明strf1突变体是由于STRF1单基因突变造成的。与WT相比，strf1突变体在种子萌发及萌发后生长对NaCl均有耐性，同时对KCl和甘露醇的处理也表现为耐性，而STRF1的互补转基因株系对NaCl和甘露醇的处理均是敏感的，这些结果表明STRF1蛋白确实参与了植物盐胁迫和(或)渗透胁迫的响应过程。体外生化试验表明STRF1蛋白是一个有活性的E3连接酶。亚细胞定位的结果表明该蛋白不仅定位在质膜上还定位在高尔基体(Golgi)和PVC这些内体(Endosomes)上。FM 4-64的染色结果发现，strf1突变体对FM 4-64的积累无论是在正常条件下还是盐处理后均高于野生型，这一结果表明STRF1蛋白影响了植物的内吞(Endocytosis)过程。同时，我们还检测了strf1突变体和WT中一些与膜运输(Membrane Trafficking)相关基因在盐处理前后的表达情况，结果表明STRF1基因调控了相关基因的表达量，尤其是AtRab7的表达量在strf1突变体中是被上调表达的。结果与已知的AtRab7高表达植物在含有NaCl和甘露醇平板上均表现为抗性的表型相吻合，并且该研究验证了NADPH氧化酶产生的ROS参与了这些胁迫过程。因此，我们也对strf1突变体中的ROS含量和编码NADPH氧化酶的RbohD基因进行检测。发现盐处理后，strf1突变体中H2O2的积累低于野生型，并且qPCR的结果表明，strf1突变体中编码NADPH氧化酶的RbohD基因的表达量与野生型相比有下调的趋势，这些结果与AtRab7高表达植物的结果是一致的。由此我们认为，STRF1蛋白可能是通过影响植物的膜运输过程来调控植物ROS的产生进而参与盐胁迫的信号转导过程。