棉花激酶GhMKK6基因的功能分析及其级联信号途径的鉴定

Mitogen-activated protein kinase (MAPK) signaling cascades play important roles in plant stress responses. Early we isolated a MKK gene from cotton, GhMKK6, which was strongly induced by pathogen infection and salt stress. In this project, we plan to elucidate the biological functions of GhMKK6 in plant disease resistance and salt tolerance through the analysis of the physiological and biochemical indexes of the transgenic plants under pathogen infection and salt stress. Furthermore, we use the yeast two-hybrid technology, bimolecular fluorescence complementation and in-gel kinase assay to identify the interaction-proteins of GhMKK6, such as upstream receptors MKKK, downstream MAPK, and the downstream targets of MAPK, and establish the MAPK pathways that GhMKK6 involved in regulation of plant disease resistance and salt tolerance, respectively. In addition, we use yeast one-hybrid technology to separate the upstream transcription factor of GhMKK6 and analyze its expression patterns. Finally, transcription factor genes and all of the components of GhMKK6 signaling cascades are transformed into Nicotiana benthamiana to illuminate their biological functions. This study can not only clarify the mechanism of GhMKK6 involved in the regulation of plant disease resistance and salt tolerance at molecular level, but also provide a theoretical basis for the genetic improvement of cotton under adversity stress.

促分裂原活化蛋白激酶（MAPK）级联信号途径在植物胁迫反应中起重要作用。前期我们从棉花中分离出一个MKK基因（GhMKK6），表达分析及启动子活性测定表明GhMKK6的表达明显受病原物及盐诱导。本项目在此基础上，试图根据对病原物侵染及盐胁迫下转基因植株各项生理生化指标的分析，明确GhMKK6在植物抗病性及耐盐性等方面的生物学功能；并通过酵母双杂交技术、双分子荧光互补实验、凝胶激酶活性测定，分离GhMKK6的上下游级联组分及MAPK的下游靶蛋白，分别建立GhMKK6参与调控植物抗病性及耐盐性的级联信号途径。同时利用酵母单杂交技术分离GhMKK6的转录调控因子，解析GhMKK6的表达调控特点；最后将GhMKK6级联信号途径的各组分及其转录调控因子基因分别转化本生烟，探讨各组分的生物学功能。从分子水平上阐明GhMKK6参与调控植物抗病性及耐盐性的作用机制，为棉花的抗逆遗传性状改良提供理论依据。

MAPK级联信号通路是植物中重要的信号转导通路，广泛参与调控植物对逆境胁迫的防卫反应。然而，目前关于MAPK级联信号通路在重要经济作物棉花上的研究较少。本项目以陆地棉为实验材料，利用棉花转基因技术，获得了超表达GhMKK6的转基因棉花，并通过对转基因棉花的功能分析，揭示了GhMKK6在调控棉花抗盐性及抗病性方面的生物学功能；通过转录组测序、酵母双杂交筛库等技术，筛选出GhMKK6上游的MAPKKK基因及下游的MAPK基因；通过在模式植物中对这些基因的生物学功能分析，鉴定出了调控棉花抗盐性及抗病性的MAPK级联信号通路；利用双荧光素酶报告系统，明确了调控GhMKK6表达的核心调控区域；通过酵母单杂交技术，筛选出多个调控GhMKK6表达的重要转录因子，并通过对转录因子的功能分析，揭示了GhMKK6的表达调控机制；通过对ghr-miR5272a的生物学功能分析，阐明了ghr-miR5272a与GhMKK6介导的MAPK级联信号通路组成的反馈调节系统，进一步解析了GhMKK6调控棉花免疫反应的分子机制。通过本项目的研究，系统的阐明了GhMKK6介导的MAPK级联信号通路调控棉花抗盐性及抗病性的分子机制，为棉花遗传性状改良提供了理论依据。