关键词: ERF/AP2结构域, ERF, DRE元件, GCC-box, 亚细胞定位, 小麦

Abstract: 【 Objective】The 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, and the role of W17 in higher plant stress-signal pathway. 【Methods】Recombinant plasmid W17/163hGFP 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.【Results】The GFP fused with W17 protein were localized in the neclei; SDS-PAGE demonstrated that the fused protein 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. 【Conclusion】W17 can localize into the nuclei and may be involved not only in pathogen- (biotic stress), but also in salt- (abiotic stress) induced signaling pathway.

Key words: ERF/AP2 domain, ERF, DRE element, GCC-box, subcellular localization