小麦CDPK与TabZIP60转录因子在调控植物盐胁迫中的相互作用研究

Abstract: Salt stress directly affects the growth, development and yield of wheat. Our knowledge on the regulation mechanism of salt stress is still poor in wheat. The sucrose non-fermentaing related protein kinase (SnRK) and calcium dependent protein kinases (CDPK) have been known to regulate downstream AREB/ABF transcription factors. Previously, Our studies showed that TabZIP60 belonged to AREB/ABF subfamily of bZIP transcription factor and played role in the response to salt stress. So TabZIP60 is likely to be the downstream substrate of CDPK. But the interaction between wheat CDPK and TabZIP60 transcription factor in the regulation of plant salt stress has not been reported. In this work, we plan to investigate the effect of CDPK on the expression of TabZIP60 and its downstream ABA related genes and physiological index through the CDPK inhibitor LaCl3, and screen TaCDPK interacting with TabZIP60 transcription factor and making its phosphorylation in vitro, and study the phosphorylation of TabZIP60 transcription factor by TaCDPK, the effects of TaCDPK on the expression of ABA related genes and physiological index in transgenic wheat under salt stress. Through the above studies, We expect to uncover the interaction between wheat CDPK and TabZIP60 transcription factor in the regulation of plant salt stress, and provide valuable gene resource for wheat breeding and offer important theoretical significance for further research of wheat salt stress signal transduction network system.

摘要：盐胁迫直接影响小麦的生长发育和产量，目前对其调控机制研究很少。已知SnRK和CDPK调控AREB/ABF转录因子，我们前期研究发现TabZIP60是响应盐胁迫的AREB/ABF转录因子，因此TabZIP60很可能是CDPK的作用底物。但目前关于小麦CDPK与TabZIP60在调控植物盐胁迫中的相互作用研究未见报道。本项目拟通过三氯化镧抑制剂研究CDPK对TabZIP60及其下游ABA相关基因表达和生理指标的影响；筛选与TabZIP60转录因子体外互作并使其发生磷酸化反应的TaCDPK；研究盐胁迫下该TaCDPK对转基因小麦体内TabZIP60蛋白的磷酸化反应及其下游ABA相关基因表达和生理指标的影响。通过以上研究以期阐明小麦CDPK与TabZIP60转录因子在调控植物盐胁迫中的相互作用，为小麦新品种的培育提供宝贵基因资源，同时对研究小麦盐胁迫信号转导的网络体系具有重要的理论意义。

TabZIP60是参与盐胁迫反应的小麦ABF/AREB转录因子，研究表明拟南芥CDPK激酶与ABF/AREB转录因子相互作用并使其磷酸化以响应逆境胁迫反应。但小麦CDPK与TabZIP60在调控植物盐胁迫中的相互作用研究未见报道。本项目首先通过RT-PCR方法获得13个与盐胁迫相关、分布第Ⅰ-Ⅳ亚家族的小麦TaCDPK基因。RT-qPCR分析TaCDPK5、TaCDPK9-1和TaCDPK30基因表达被ABA、NaCl和PEG诱导增强。用NaCl和NaCl+LaCl3分别处理小麦陇春27，结果表明NaCl+LaCl3处理下TabZIP60、TaNCED基因表达量和ABA含量比NaCl处理下明显降低。其次利用酵母双杂交和BiFC方法，筛选到TaCDPK5、TaCDPK9-1和TaCDPK30激酶分别与TabZIP60相互作用。TaCDPK30激酶不能与102位丝氨酸突变的TabZIP60相互作用。通过Pro-Q方法分析了TaCDPK5和TaCDPK9-1激酶分别磷酸化TabZIP60蛋白，而且TaCDPK9-1以钙依赖方式磷酸化TabZIP60蛋白。最后用NaCl和NaCl+LaCl3分别处理野生型和转TabZIP60基因小麦，结果表明NaCl处理下，TaNCED1基因表达量和ABA含量在转基因小麦中明显高于野生型对照，在NaCl+LaCl3处理下，TaNCED1基因表达在野生型中低于转基因植株。综合以上体外和体内研究结果，表明TaCDPK与TabZIP60转录因子相互作用并使其磷酸化，有活性的TabZIP60转录激活下游TaNCED基因表达进而提高ABA含量增强小麦抗盐反应。该研究结果不仅阐明了小麦TabZIP60转录因子通过ABA信号参与盐胁迫反应的调控机制，而且也为后期小麦抗逆育种提供理论依据和优良的转基因小麦。