Recombinant TcbZIP60 Protein Production and DNA-Binding Assay

The ORF without the TcbZIP60 terminator codon was used to generate the pET6×HN-C vector protein, which was fused into the N-terminal frame of 6×His, and the vector pET6×HN-C-TcbZIP60 was then transformed into E. coli strain Rosetta (DE3). For the induced recombinant protein, 0.5 mM IPTG was used, and the cultures were incubated at 18°C for 16 h. Then, Ni²⁺–nitrilotriacetic acid was used to purify the recombinant proteins. For the electrophoretic mobility shift assay (EMSA), the promoter fragments of TcCHS and TcAOC containing E-box or G-box cis-regulatory elements labeled with fluorescein amidite (FAM) as probes, the same but unlabeled DNA fragments, and cis-element mutant DNA fragments were used as competitors in the assay. After performing the EMSA assays, FAM-labeled DNA was detected from the chemiluminescent signal.