茉莉酸信号转录抑制关键组分JAZ与NINJA互作体系建立及复合体结晶条件筛选  

Establishment of the system for in vivo and in vitro interaction between JAZ and NINJA in jasmonate signaling,and crystallization for JAZ and NINJA complex

作　　者：曹晓蔓 张亚光 张峰 CAO Xiaoman;ZHANG Yaguang;ZHANG Feng(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学植物保护学院,江苏南京210095

出　　处：《南京农业大学学报》2022年第1期86-93,共8页Journal of Nanjing Agricultural University

基　　金：教育部霍英东青年教师基金项目(161022);江苏省六大人才高峰高层次人才项目(NY-035);中组部万人计划“青年拔尖”人才项目。

摘　　要：[目的]本文旨在建立JAZ与NINJA蛋白离体和活体互作以及2个蛋白复合体晶体筛选的体系,并获得2个蛋白复合体晶体。[方法]采用酵母双杂交和双分子荧光互补法用于活体条件下JAZ与NINJA蛋白互作区域的验证;在离体条件下,通过氢氘交换质谱(HDX)和Alpha Screen验证JAZ与NINJA蛋白的互作区域并进一步筛选2个蛋白互作的最小区域;使用原核(大肠杆菌)表达系统、亲和层析和凝胶过滤层析纯化等手段获得NINJA截短蛋白。将NINJA截短蛋白与JAZ多肽按照物质的量比为1∶1.5混合孵育一段时间,通过坐滴结晶方法进行复合体结晶,最终获得复合体晶体。[结果]成功创建了JAZ与NINJA离体和活体互作体系;获得JAZ多肽与NINJA(342~406)的复合体晶体的条件分别是0.2 mol·L^(-1) CaCl_(2)·2H_(2)O、0.1 mol·L^(-1) BIS-Tris(pH6.5)、45%2-甲基-2,4-戊二醇与0.2 mol·L^(-1)醋酸铵、0.1 mol·L^(-1) Tris(pH8.5)、45%2-甲基-2,4-戊二醇。[结论]JAZ蛋白ZIM结构域的TIFY基序和羧基端与NINJA(342~406)是JAZ与NINJA蛋白互作所必需的;JAZ蛋白ZIM结构域是JAZ同源和异源互作所必需的;成功获得了JAZ与NINJA复合体晶体。[Objectives]This article aimed to establish the interaction system for JAZ and NINJA in vitro and in vivo,the crystal screening system for JAZ and NINJA complex,and obtain the complex crystals for two proteins.[Methods]Yeast two-hybrid and bimolecular fluorescent complimentary methods were used to verify the regions that contribute to interaction between JAZ and NINJA in vivo.The discovered region was verified by hydrogen-deuterium exchange mass spectrometry(HDX)and Alpha Screen assay,we also used HDX and Alpha Screen assay to find the minimum region that contributed for the sufficient interaction between JAZ and NINJA in vitro;Prokaryotic(Escherichia coli)expression system,affinity chromatography and gel filtration chromatography purification were used to obtain NINJA truncated protein.We mixed NINJA truncated proteins and JAZ peptides with a mole ratio of 1∶1.5 and incubated for a period of time.Finally,we obtained the complex crystals through the sitting drop method for crystallization.[Results]We established the interaction system between JAZ and NINJA in vitro and in vivo successfully;we obtained the complex crystal for JAZ peptide and NINJA(342-406)under the conditions of 0.2 mol·L^(-1) CaCl_(2)·2H_(2)O,0.1 mol·L^(-1) BIS-Tris(pH6.5),45%2-methyl-2,4-pentanediol;0.2 mol·L^(-1) ammonium acetate,0.1 mol·L^(-1) Tris(pH8.5),45%2-methyl-2,4-pentanediol,respectively.[Conclusions]TIFY motif and carboxyl terminal of ZIM domain in JAZ and NINJA(342-406)were necessary for the interaction between JAZ and NINJA;ZIM domain in JAZ was necessary for JAZ homologous and heterologous interaction;we obtained NINJA and JAZ complex crystals successfully.

关 键 词：茉莉酸 JAZ NINJA ZIM结构域 互作 晶体学 

分 类 号：Q71[生物学—分子生物学]