A Novel Quick Method for Detecting Target DNA Binding Sites of Protein

DREB (dehydration responsive element binding protein) transcription factors play important roles in stress response and regulation of plant growth and development. In general, the traditional method of DNase I foot-printing is used to identify DNA binding of transcription factor. The DNA probes were labeled with isotope and then polyacrylamide gel electrophoresis was performed to separately digested labeled-DNA fragments, which had low resolution ratio and complexity in operation, and was not suitable for detecting interactions in large scales. To explore the transcriptional regulation mechanism of GmDREB3, the DNase I foot-printing method was improved. Combing with electrophoretic mobility shift assay (EMSA), DNA was labeled by fluorescence instead of isotope and the capillary electrophoresis instead of polyacrylamide gel electrophoresis was used to detect the digested DNA fragments and further identify DNA binding region of GmMYB1 in promoter of GmDREB3 gene. DNA binding site of GmMYB1 in GmDREB3 promoter was confirmed via the method of restriction enzyme digesting DNA. To further confirm binding element in GmDREB3 promoter, we used a putative DNA binding element of GmMYB1 to complete EMSA, indicating that GmMYB1 can bind target DNA element in vitro. In short, compared with classic DNase I foot-printing, the modified method is more rapid, accurate and reliable, and will be a high throughput method to identify interactions between proteins and target DNA sites.