OsMKK1和OsMKK6基因在水稻多重抗逆信号传导通路中的调控功能研究

In the recent years, natural adverse environmental conditions, such as low temperature, drought and salt stresses, frequently affect the growth, development and yield of rice in China. Many lines of evidences have shown that MAPK cascade and associated regulatory genes influence the tolerances to the mutiple abiotic and biotic stresses in higher plants.Our previous study has confirmed that, OsMKK6-OsMPK3 is involved in the cold stress tolerance signaling in rice, it probably also participate in the salt stress tolerance. On the other hand,as an another novel type A MAPKK gene, OsMKK1 can enhance both the cold and drought stress tolerances in the overexpressing transgenic rice plant lines.However,the downstream target proteins and transcription factors still remain to be identified now. In this proposal, OsMKK1 andd OsMKK6 are planned to develop the monovalent and divalent overexpressors as well as RNAi-repressors in rice to identify their differentialtolerances to the low temperature, drought and salt stresses.And then,by the means of BiFC (Bimolecular Interaction fluoresecne Complementation) and Co-IP (Co-immuneprecipitation), both OsMKK1 and OsMKK6 will be detected their genuine in vivo interactions with OsMPK3, OsMPK4 and OsMPK6,respectively. After that, By using the microarray gene differential expression analysis, yeast two-hybrid screening, and in vivo MAPK kinase activity assays, their respective specific target proteins and transcription factor genes will be screened out. The accomplishment of this proposal will dissect the regulatory functions of OsMKK1 and OsMKK6 genes in the MAPKK-MAPK-target protein pathways involved in the mutiple abiotic stress tolerances in rice for the first time, and provide novel avenue to the mutiple stress genetic improvements with regulon egineering in rice.

近年来，低温、干旱和盐害等自然灾害频繁影响水稻的生长发育和产量。研究表明，MAPK级联途径及其调控基因影响植物对生物和非生物逆境的抗性。我们前期研究发现，OsMKK6激活OsMPK3介导的水稻耐冷害信号传导过程。同时，它参与了水稻的耐盐性反应。另一个A类MAPKK基因OsMKK1超表达增强了水稻的耐旱性和耐冷性。但是，它们的下游靶蛋白和转录因子还未鉴定。为此，本项目拟创建OsMKK1和OsMKK6基因的单价、双价超表达与RNAi抑制表达转基因植株，研究其多重耐逆性差异；利用BiFC和Co-IP方法，在水稻活体中证实它们与OsMPK3、OsMPK4和OsMPK6之间的特异性真实互作; 利用芯片表达分析、酵母双杂交体系和MAPK磷酸化检测方法，筛选出MAPK的特异靶蛋白或转录因子，揭示它们在水稻多重抗逆信号传导MAPKK-MAPK-靶蛋白途径中的调控功能，为水稻多抗性的遗传改良提供新途径。

植物MAPKK是决定MAPK信号转导级联途径的关键蛋白。在水稻中，A类MAPKK基因OsMKK1和OsMKK6对多重非生物逆境的抗性和特异靶蛋白或调控因子还不明确。首先，利用Y2H文库筛选、成对Y2H和BiFC技术，鉴定了2个与OsMKK1互作的蛋白ATP sulfurylase 2和GDSL esterase/lipase，4个与OsMKK6互作的蛋白synaptotagmin-5、glyceraldehyde-3-phosphate dehydrogenase 2、CBL-interacting protein kinase 1和protein AE7，这为深化OsMKK1和OSMKK6在调控水稻多重耐性的复杂调控网络提供了研究支点。其次，利用成对杂交、BiFC和体外激酶检测方法，首次鉴定了OsDMI3与OsMKK1、OsMKK6的特异性互作，首次在水稻中将CaM信号转导途径与OsMKK1和OsMKK1和OsMKK6途径联系起来，揭示了CaM与MAPKK存在Crosstalk的分子机制；再次，利用Y2H、BiFC、体外激酶活性检测技术，鉴定了OsMKK1- OsMPK4/6途径，以及OsMKK6-OsMPK3/4途径，证实了该途径在植物中的高度保守性，为在其他植物中研究该途径提供了借鉴。转基因株系萌发期耐盐性和耐冷性实验结果表明，超表达OsMKK1和OsMKK6基因可以明显增强水稻种子萌发期的耐冷性，超表达 OsCaM1-1基因可以增强水稻萌发期的耐盐性和侧生根的伸长。这些结果表明，这些蛋白参与了OsMKK1和OsMKK6基因在水稻的硫代谢，脂类代谢，细胞膜稳定性调节，糖代谢，钙信号，细胞分裂等过程。这些共同和特异调节的靶蛋白基因参与了水稻的多重抗性，本项目为研究OsMKK1和OsMKK6蛋白的信号转导途径和复杂调控网络提供了基因资源和技术支撑。