Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution.
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.
Jin
Xiao-Fen
XF
Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
Zhu
Bo
B
Peng
Ri-He
RH
Jiang
Hai-hua
HH
Chen
Jian-Min
JM
Zhuang
Jing
J
Zhang
Jian
J
Yao
Quan-Hong
QH
Xiong
Ai-Sheng
AS
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
D050656:Transcription Factor AP-2