干旱和低温胁迫下玉米ZmMPK17基因功能分析及ZmMKK3/CaMs-ZmMPK17级联途径的鉴定

Zea mays L., Zhengdan 958, is selected as the plant material in this subject. The kinase activities of ZmMPK17 and ZmMKK3 were investigated by in-gel kinase assays in response to drought, low temperature, and stress-related signaling molecules in maize seedlings (Zea mays L.cv Zhengdan 958). To test whether ZmMPK17 interact with ZmMKK3 and CaMs, we analyzed their interactions using yeast two-hybrid assays, bimolecular fluorescence complementation (BiFC) assays, in vitro and in vivo kinase assays, Co-IP and so on. In response to drought and low temperature stress, ZmMPK17 was activated by ZmMKK3 and CaMs, respectively. Using pZP211 expression vector, we generated transgenic maize with overexpression of sense- and antisense-ZmMPK17, ZmMPK17/ZmMKK3 and ZmMPK17/CaM. The physiological characteristics of transgenic maize and molecular mechanism of ZmMPK17 gene and the ZmMKK3/CaM-ZmMPK17 cascades were investigated in response to drought, low temperature and pathogens infection. This project provides important theoretical basis and the research methods for elucidating mechanisms of signal transduction and improving the comprehensive resistance of maize.

本项目选择玉米品种郑单958为材料，通过激酶试验分析干旱、低温及逆境信号分子对ZmMPK17和ZmMKK3 激酶活性的影响。采用酵母双杂交、双分子荧光互补、体内和体外激酶试验及免疫共沉淀等方法，证明ZmMPK17与ZmMKK3、CaMs之间的互作；在响应干旱和低温逆境时，ZmMPK17可以分别被ZmMKK3和CaMs激活。利用pZP211表达载体，转化玉米获得转ZmMPK17 正、反义及ZmMKK3/ZmMPK17、CaM/ZmMPK17二元转基因植株。在干旱、低温及病原菌侵染条件下研究转基因玉米的抗逆生理特性，探究ZmMPK17基因和ZmMKK3/CaM-ZmMPK17级联途径提高玉米抗逆性的分子机制。该项目为最终探明禾谷类作物玉米的抗逆信号转导机理和改良玉米作物的综合抗逆性，提供重要的理论依据和研究方法。

本项目研究了ZmMPK17，ZmMPK5，ZmSIMK1和ZmMKK1基因的功能及抗逆生理特性。不同胁迫及信号分子处理后均能诱导ZmMPK17，ZmMPK5，ZmSIMK1和ZmMKK1在玉米中的表达变化。将ZmMPK17，ZmSIMK1和ZmMKK1转化模式植物烟草和拟南芥，分析这些基因的功能。在渗透和低温胁迫下，ZmMPK17通过调控抗氧化防御系统及渗透物质的积累，增强了对渗透和低温胁迫的抗性。病原菌CMV和PVY侵染烟草后，ZmMPK17通过激活SA介导的抗逆信号转导途径，增强了对病原菌的抗性。在烟草中过表达ZmSIMK1后增强了烟草对干旱和丁香假单包杆菌（Pst DC3000）的抗性。高盐和干旱条件下，过表达ZmMKK1拟南芥通过ROS和依赖ABA途径，增强了对高盐和干旱胁迫的抗性。利用酵母双杂交实验发现，ZmMPK17并没有与CaMs和ZmMKKs发生互作，并且从cDNA文库中也没有筛选到与ZmMPK17互作的蛋白。ZmMPK17与其它蛋白的相互作用，还需要通过其它方法做进一步的验证。 通过酵母双杂交实验发现ZmMKK1能够与ZmMEKK1发生互作。构建PZP211-ZmMKK1正反义表达载体，进行同源转化玉米。qRT-PCR分析表明，ZmMKK1在转基因玉米中稳定表达。