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

作　　者：ZHAO Yun-xiang LIU Pei XU Zhao-shi CHEN Ming LI Lian-cheng CHEN Yao-feng XIONG Xiang-jin MA You-zhi 

机构地区：[1]Institute of Crop Science, Chinese Academy of Agricultural Sciences/The National Key Facility for Crop Gene Resources and Genetic Improvement （NFCR1）/Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Beijing 100081, P.R.China [2]Agronomy College, Northwest A ＆ F University, Yangling 712100, P.R.China

出　　处：《Agricultural Sciences in China》2008年第6期647-655,共9页中国农业科学（英文版）

基　　金：financially supported by the National 863 Program of China(2007AA10Z130);the National Natural Science Foundation of China(30700504).

摘　　要：The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 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. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/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. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. 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 at 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. 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 （e.g., salt-） induced signaling pathway.The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 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. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/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. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. 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 at 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. 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 （e.g., salt-） induced signaling pathway.

关 键 词：ERF/AP2 domain ERF DRE element GCC-BOX subcellular localization 

分 类 号：S512.1[农业科学—作物学]