In Vitro Molecular Evolution For DNA Binding Domain Of Transcription Factor NtERF2

Ethylene response element binding factor NtERF2 belongs to thesubfamily of the highly conserved EREBP/AP2 transcription factorssuper family found in plants specifically. NtERF2 participates inregulation of many genes that are involved in many importantphysiological and developmental processes of tobacco. NtERF2 bindsspecifically the target gene 5'-upstream GCC box motif through itsDNA binding domain (ERF domain), and mediates the regulation ofexpression of pathogen-related (PR) genes. Thus, studies on theconservative nature and DNA binding specificity of ERF domain havebeen the interesting topic. In this work, wild erebp2 cDNA wasmutated using error-porn polymerase chain reaction (EP-PCR)technique to generate construct erebp2 mutant library. Throughimproved southwestern blotting screening method, we havesuccessfully acquired mutant NtERF2s which didn't bind and bindweakly to GCC box. Two of the mutants NtERF2s were found to havetwo amino acid residues changed respectively. Both amino acidresidues affected seriously the binding affinity of NtERF2 to GCC box.We discussed the possible mechanism through which those amino acidresidues affect specific bind to GCC box.