title: Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution.
abstract: In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCCbinding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3- hy15.
authors.affiliation: Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
authors.forename: Xiao-Fen
authors.initials: XF
authors.lastname: Jin
authors.forename: Bo
authors.initials: B
authors.lastname: Zhu
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Hai-hua
authors.initials: HH
authors.lastname: Jiang
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Zhuang
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D029688:Brassica napus / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050436:Regulatory Elements, Transcriptional; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D050656:Transcription Factor AP-2