Recombinant protein production and EMSA

To express and purify the recombinant protein, the full-length OpWRKY2 ORF fragment was cloned into the BamH I and Hind III sites of the pCold-TF vector. The constructs were verified by DNA sequencing and transformed into Escherichia coli strain Rosetta (DE3) cells to produce His-tagged fusion proteins. The pCold-TF empty vector without OpWRKY2 was used as the negative control. Transformed Rosetta cell cultures used for the expression of HIS recombinant protein were induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM at an optical density of approximately 0.6 at 600 nm. After induction for 14 h at 16 °C, Rosetta cells were harvested by centrifugation and purified using Ni-NTA (nitrilotriacetic acid) agarose (Invitrogen, USA) as previously described¹⁶. To investigate the ability of the OpWRKY2 protein to bind to the W-box in the OpTDC promoter, the 3090 bp upstream region of the OpTDC gene was analyzed. For Electrophoretic mobility shift assay (EMSA) experiments, DNA probes were designed based on the native OpTDC promoter sequence (−2565 to −2552 relative to the ATG) containing a single W-box. Complementary oligonucleotides labeled with biotin at the 5’ end of each strand were synthesized and annealed to produce double-stranded probes for EMSA. EMSAs were performed as previously described¹⁶.