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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

  • Jin, Xiao-Fen (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences) ;
  • Zhu, Bo (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences) ;
  • Peng, Ri-He (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences) ;
  • Jiang, Hai-Hua (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences) ;
  • Chen, Jian-Min (College of Life Science and Technology, Yangzhou University) ;
  • Zhuang, Jing (Life Science, Alberta Research Council) ;
  • Zhang, Jian (Life Science, Alberta Research Council) ;
  • Yao, Quan-Hong (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences) ;
  • Xiong, Ai-Sheng (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences)
  • Received : 2010.06.30
  • Accepted : 2010.07.24
  • Published : 2010.08.31
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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 GCC-binding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3-hy15.

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References

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