水稻中SAPK8/9/10与OsRbohB/E蛋白互作在ABA诱导H_2O_2产生中的作用  

Functional analysis of the interaction between SAPK and OsRboh in abscisic acid-induced H_2O_2 production in rice

作　　者：宋文敏 孙嫚嫚 蒋明义[1] SONG Wenmin;SUN Manman;JIANG Mingyi;(College of Life Sciences,Nanjing Agricultural University)

机构地区：[1]南京农业大学生命科学学院,江苏南京210095

出　　处：《南京农业大学学报》2018年第2期321-329,共9页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(2012CB114300)

摘　　要：[目的]本文旨在验证水稻中SAPK8/9/10与OsRbohB/E蛋白的互作及其在ABA诱导的H_2O_2产生中的作用。[方法]先采用酵母双杂交系统进行初步的SAPK8/9/10与OsRbohB/E蛋白的互作分析,然后采用双分子荧光互补(Bi FC)、GST-pull down和体外磷酸化技术验证其互作。为了进一步探讨SAPKs与OsRbohs在ABA信号转导中的作用,采用水稻原生质体瞬时体系分析ABA处理时SAPKs与OsRbohs对H_2O_2产生的影响。[结果]SAPK8/9/10与OsRbohB和OsRbohE在体内、外存在相互作用,且OsRbohs是SAPKs磷酸化的底物。与对照组相比,水稻原生质体瞬时过表达SAPK9/10(SAPKs-OE)和OsRbohB/E(OsRbohs-OE)组中H_2O_2的产生明显增加,且ABA处理后增加趋势增强;瞬时沉默SAPKs(ds-SAPKs)和OsRbohs(ds-OsRbohs)组中H_2O_2的产生明显下调,且ABA诱导的H_2O_2增加在这些原生质体中也均被抑制;SAPKs-OE+ds-OsRbohs、ds-SAPKs+OsRbohs-OE组中H_2O_2的产生表现出不同程度的减少。[结论]SAPKs与OsRbohs共同参与调节ABA诱导的H_2O_2的产生。[Objectives]The aim of this study is to identify the interaction between rice sucrose nonfermenting 1-related protein kinase 2 SAPKs and rice NADPH oxidases OsRbohs,and its role in ABA-induced H2O2 production in rice.[Methods]To clarify the interactions between SAPK8/9/10 and OsRbohB or OsRbohE,a yeast two-hybrid system was firstly used,and then glutathione S-transferase（GST） pull-down assay,bimolecular fluorescence complementation（Bi FC） analysis and in vitro phosphorylation test were performed to confirm the interactions.To investigate the roles of SAPKs and OsRbohs in ABA-induced H2O2 production,a transient gene expression analysis and a transient RNA interference（RNAi） test in rice protoplasts were also used.[Results]Our results showed that OsRbohB/E interacted with SAKP8/9/10,and they were phosphorylated by SAPK8/9/10,indicating that OsRbohB/E were phosphorylation substrates of SAPK8/9/10.Further,under the control conditions,the transient over-expression of SAPK9/10 or OsRbohB/E increased the production of H2O2 in the protoplasts,and the RNAi silencing of SAPK9/10 or OsRbohB/E decreased the production of H2O2.ABA treatment induced a significant increase in the production of H2O2 in the control protoplasts,and the increases were further enhanced in the protoplasts transiently expressing SAPK9/10 or OsRbohB/E,but were inhibited in the protoplasts transiently silenced SAPK9/10 or OsRbohB/E.Moreover,the transient expression analysis in combination with the transient RNAi test in protoplasts also showed that both SAPKs and OsRbohs were required for ABA-induced H2O2 production.[Conclusions]These results indicate that both SAPKs and OsRbohs coordinately regulate the production of H2O2 in ABA signaling.

关 键 词：脱落酸 应激激活蛋白激酶 OsRboh 蛋白互作 过氧化氢 

分 类 号：Q945[生物学—植物学]