Figure 5.

Electrophoretic mobility shift assay (EMSA) of BrWRKY65 binding to the W-box element and promoters of BrNYC1, BrSGR1, and BrDIN1 containing W-box element. The probe sequences corresponding to each of the target gene promoters are shown, with red letters representing the W-box and the mutant W-box. The purified recombinant GST-BrWRKY65 protein was incubated with probes, and the protein–DNA complexes were separated on native polyacrylamide gels. GST protein alone was used as the negative control. − represents absence, while + represents presence. Unlabeled or mutant probes at different concentrations (from 100 to 1000 times) were added to the reaction mixture for competition and testing binding specificity. Arrows indicate the position of shifted bands.