小麦促分裂原蛋白激酶TaMPK3/MPK4级联通路介导植株抵御非生物逆境分子机制

The mitogen-activated protein kinase (MAPK) cascades are consisted of components of MAPKKK, MAPKK, and MAPK and involved in mediating the transduction of distinct signaling cues after sequential phosphorylation reactions. They play important roles in regulating plant growth, development and plant tolerance to diverse environmental stresses. In this project, two wheat MAPK member referred to TaMPK3 and TaMPK4 that we previously identified to act as essential regulators in plant tolerance to a set of stresses, such as drought, high salinity, nitrogen and phosphorus deprivations, will be subjected to further investigation. The main research contents are as follows: we will identify the upstream components interacted with TaMPK3 and TaMPK4 on which to establish the cascade modules comprised by these wheat MAPK members. The phosphorylation mechanism of these MAPK cascade members will be further dissected. Based on generating the fusion proteins of the MAPK cascade gene with GFP, a green fluorescence reporter gene together with further fluorescence signal detection for these fusions and bimolecular fluorescence complementation (BiFC) analysis, we will define the acting positions of the MAPK cascade members. Using gene chip approach, we will investigate the plant transcroptome characterization under stresses of drought, high salinity, nitrogen and phosphorus deprivations regulated by TaMPK3 and TaMPK4, on which to establish the gene networks specifically controlled by these MAPK genes. Furthermore, we will experimentally validate the putative interactions of an array of proteins encoded by key regulatory genes in gene networks with TaMPK3 and TaMPK4. The functions of these regulatory genes in mediating plant tolerance to diverse stresses will be further determined. Together, we will dissect the distinct MAPK cascade module covering TaMPK3 and TaMPK4 and elucidate the molecular mechanisms of these MAPK cascade pathways in regulating plant tolerance to drought, high salinity, nitrogen and phosphorus deprivations.

促分裂原活化蛋白激酶级联途径（MPKKK-MPKK-MPK）在介导植株非生物逆境信号转导中发挥重要作用。本课题以前期鉴定的在介导植株抵御干旱、高盐、低氮和低磷逆境中发挥重要功能的小麦MAP激酶TaMPK3和TaMPK4基因为基础，鉴定与供试基因编码蛋白互作的上游组分，构建TaMPK3和TaMPK4级联通路，解析通路中各组分磷酸化机制；通过对TaMPK3和TaMPK4及上游组分基因与报告基因GFP融合蛋白荧光检测结合双分子荧光互补（BiFC）分析，揭示TaMPK3和TaMPK4级联通路介导胁迫信号转导的亚细胞部位；利用基因芯片阐明TaMPK3和TaMPK4调控逆境下的转录谱特征，对网络中关键调节基因编码蛋白与TaMPK3和TaMPK4的互作进行验证，进一步对上述基因介导植株抵御逆境的功能进行鉴定。通过研究，解析TaMPK3和TaMPK4构建的MPK特定级联通路及其介导植株抵御逆境分子机制。

本课题针对迄今小麦促分裂原活化蛋白激酶（MAPK）基因介导植株抵御逆境研究尚少现状，以2个小麦MAPK基因TaMPK3和TaMPK4为基础，鉴定了供试基因分子特征及介导植株抵御逆境分子机制。研究结果如下。 .1. TaMPK3与TaMAPKK4互作，后者与TaMAPKKK1互作，构建TaMAPKKK1-TaMAPKK4-TaMPK3级联通路；TaMPK4与TaMAPKK2互作，TaMAPKK2与TaMAPKKK;A互作，构建TaMAPKKK;A-TaMAPKK2-TaMPK4通路。.2. TaMPK3和TaMPK4具有磷酸化能力。供试基因转录后，受到上游通路组分激活，介导植株对特定逆境适应过程。 .3. 通过双分子荧光互补（BIFC）检测供试基因与互作组分定位，TaMPK4与TaMAPKK2在核内互作，TaMAPKK2与TaMAPKKK;A在胞质互作。上述蛋白在细胞特定部位参与蛋白磷酸化和信号转导。 .4. TaMPK3增强植株抵御低氮和盐分胁迫逆境能力，TaMPK4介导植株抵御盐分胁迫。上述基因调控根体建成和氮素吸收、脱落酸（ABA）信号通路及活性氧稳态，参与植株对特定逆境适应过程。.5. 与对照相比，TaMPK3和TaMPK4使植株体内众多基因转录发生改变。TaMPK3低氮下特异调控上调基因303个，下调基因366个；盐分下特异调控上调基因805个，下调基因124个。盐胁迫下，TaMPK4特异调控上调基因213个，下调基因82个。上述基因富集生物调节、物质运输、逆境响应、代谢、信号转导、转录调节、蛋白结合和抗氧化过程。.6. TaMAPK3与转录因子TaNF-YC1、TaWRKY46以及鸟嘌呤结合亚基TaNBP1互作，TaMAPK4与转录因子TaERF9互作。供试小麦MAPK基因与上述下游因子互作，参与植株抵御逆境过程。