MAPK级联途径在调控毛果杨干旱、高盐胁迫信号转导中的作用及其机制的研究

The mitogen-activated protein kinase (MAPK) cascades are the central parts of the regulation network of plant signal transduction in response to external stress stimuli. To date, a complete MAPK signal pathway has not yet been established in poplar. In our previous study, one MAPKK gene PtMKK3 and two MAPK genes PtMPK6-2 and PtMPK7 were found to be induced by drought and high salinity in Populus trichocarpa. Furthermore, the PtMKK3 was detected to interact with both PtMPK6-2 and PtMPK7 by yeast two-hybrid (Y2H) assay. With the help of the established research platforms such as poplar genetic transformation system, protoplast transient expression system, and etc., the following research work will be carried out in this project, which includes: comprehensively analyzing the functions of PtMKK3、PtMPK6-2 and PtMPK7, screening the differentially expressed genes between the transgenic and wild type plants, identifying their downstream target genes, and making clear whether PtMPK6-2/PtMPK7 could be activated by PtMKK3 in the drought and high salinity stress signal transduction pathways or not. And then, the above results will be further integrated and analyzed, and the important nodes will be further verified. Based on the data obtained, we will build the basic frame of the mechanisms under the PtMKK3-PtMPK6-2/PtMPK7 kinase cascades regulating the drought and high salinity stress signal transduction of poplar. Our study will offer insights into the molecular mechanism of MAPK cascades regulating the plant stress responses, and lay foundations for the improvement of stress resistance in poplar via genetic engineering.

在植物逆境信号转导网络中，MAPK级联途径处于中心环节。迄今，尚未在杨树中确定一条完整的MAPK信号通路。前期研究显示，毛果杨PtMKK3、PtMPK6-2和PtMPK7均受干旱和高盐诱导，并通过酵母双杂交检测到PtMKK3与PtMPK6-2、PtMPK7均有相互作用。在此基础上，本项目借助于已建立的毛果杨原生质体瞬时表达系统和遗传转化体系等研究平台，全面解析PtMKK3、PtMPK6-2和PtMPK7功能，并分析转基因和野生型植株之间基因表达谱的差异，寻找其下游目标基因；探明在毛果杨干旱、高盐等逆境响应中PtMPK6-2/PtMPK7能否被PtMKK3激活。将所得数据整合分析，并对重要节点进一步验证，建立PtMKK3-PtMPK6-2/PtMPK7级联途径调控毛果杨干旱、高盐信号转导作用机制的基本框架，以期为深入理解MAPK级联途径调控植物逆境响应机制提供新资料。

在植物逆境信号转导网络中，MAPK级联途径处于中心环节。迄今，尚未在杨树中确定一条完整的MAPK信号通路。为深入理解MAPK级联途径调控杨树逆境响应机制，同时也为林木抗逆改良提供重要基因资源，本项目主要开展以下研究工作：.（1）.系统分析毛果杨中的11个MAPKK基因PtMKKs对非生物胁迫及激素的响应模式，结果显示，11个PtMKKs 基因表达水平在不同非生物胁迫和激素处理后表现出不同的变化趋势，其中值得关注的是PtMKK3和PtMKK4均可被PEG、NaCl和H2O2处理诱导表达，并且PtMKK4对三种胁迫相关激素处理均响应。.（2）.选取PtMKK4进行功能鉴定。结果显示，PtMKK4基因的过表达显著增强了转基因杨树的耐旱性，表明PtMKK4在介导杨树干旱胁迫信号转导途径中发挥重要作用。.（3）.筛选、鉴定与PtMKK4互作的下游靶标PtMPKs。利用酵母双杂交和双分子荧光互补鉴定出PtMKK4与PtMPK6存在相互作用关系。并且，PtMPK6可同样被PEG和NaCl处理诱导表达，与PtMKK4响应模式相似，暗示在高盐和干旱胁迫信号转导途径中PtMPK6可能是PtMKK4的下游组分。.（4）.RNA Seq分析PtMKK4过表达转基因植株与野生型对照之间基因表达的差异，进一步比较上调基因对应的同源基因在抗逆胡杨和非抗逆毛果杨中对高盐和干旱响应模式的差异，选取在胡杨中上调幅度高于毛果杨中的一个候选基因肌醇-1-磷酸合酶基因PeMIPS1（Populus euphratica MIPS1）进行功能分析。借助于肌醇合成缺陷突变体功能互补实验和亚细胞定位分析，证实PeMIPS1编码的蛋白为胞质型肌醇-1-磷酸合酶;定量PCR分析结果显示，PeMIPS1不仅被NaCl和PEG处理诱导表达，也能被CuSO4处理高水平诱导；过表达PeMIPS1转基因杨树对高盐和Cu胁迫的耐受性明显高于野生型。进一步研究结果表明，过表达PeMIPS1可增强转基因植株中AsA/GSH循环的速率提高其抗氧化能力，从而降低了胁迫引起的氧化伤害。此外，MI还可通过增加非酶抗氧化剂AsA的积累来增强清除ROS的能力。.（5）.将以上鉴定出的PtMKK4和PeMIPS1应用于速生林‘欧美杨107’抗逆遗传改良。优化‘欧美杨107’遗传转化体系，现已获得过表达PtMKK4和PeMIPS1的转基因“欧美杨1