小麦ERF类转录因子W17的结合特异性及亚细胞定位分析  

Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

作　　者：赵云祥[1] 徐兆师[2] 陈明[2] 李连城[2] 陈耀锋[1] 邱志刚[2] 熊祥进[2] 马有志[2] 

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100 [2]中国农业科学院作物科学研究所/国家农作物基因资源与基因改良重大科学工程/农业部作物遗传育种重点开放实验室,北京100081

出　　处：《中国农业科学》2008年第6期1575-1582,共8页Scientia Agricultura Sinica

基　　金：国家高技术研究发展计划(“863”计划)项目(2007AA10Z130);国家自然科学基金项目(30700504)

摘　　要：【目的】构建ERF(ethylene-responsive element binding factor)转录因子基因W17的亚细胞定位载体和原核表达载体,验证W17是否具有核定位功能,阐明W17与GCC、DRE探针的体外结合特性,利用GUS瞬时表达系统分析W17蛋白的体内结合特性和转录激活功能,初步预测W17在植物胁迫信号传导途径中的作用。【方法】构建W17/163hGFP亚细胞定位载体,基因枪转化洋葱表皮细胞,暗培养24h后共聚焦显微镜下观察。构建W17/pGEX-4T-1原核表达载体,转入大肠杆菌BL21(DE3),IPTG(0.5mmol·L-1,3h)诱导,GST纯化柱纯化,纯化的融合蛋白与[γ-32P]ATP标记的GCC、DRE探针混合进行凝胶阻滞试验。构建GUS瞬时表达系统,通过农杆菌介导转化烟草,X-Gluc染色、酒精脱色后体视显微镜下观察。【结果】W17基因具有核定位功能,纯化的融合蛋白GST/W17能与正常GCC、DRE探针体外特异结合,与突变GCC、DRE探针不结合,在植物体内与GCC特异结合并能激活下游GUS基因表达。【结论】W17通过自身的NLS进入核内行使功能,参与了GCC-box调控的生物胁迫信号传导途径,还可能参与了非生物胁迫(盐胁迫)传导途径。【Objective】This study aims to detect the subcellular localization of ERF（ethylene-responsive element binding factor）transcription factor W17 protein,the interaction between W17 and cis-acting regulatory elements GCC box and DRE in vitro,the binding and transactivating ability in vivo,and the role of W17 in higher plant stress-signal pathway. 【Method】Recombinant plasmid W17/163 hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol·L^-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns,and then subjected to gel retardation assay with a ^32P-labeled GCC or DRE sequence. Different reporters and effector plasmids were introduced into tobacco leaves through agroinfiltration,then transformed leaves were stained by X-Gluc,faded with 75% alcohol and monitored under a Stereozooming microscope. 【Result】The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC box and DRE element,but had no interaction with either the mutant DRE or GCC box; W17 protein can bind to GCC box and transactive downstream GUS gene in vivo. 【Conclusion】 W17 can localize into the nuclei,and it may be involved not only in biotic stresses controlled by GCC box,but also in abiotic stresses （eg. salt） induced signaling pathway.

关 键 词：ERF/AP2结构域 ERF DRE元件 GCC-BOX 亚细胞定位 小麦 

分 类 号：S512.1[农业科学—作物学]