Electrophoretic mobility shift assay
The PtrWRKY75 cDNA was cloned into the PET28a vector, then the
construct was inserted by heat shock into Escherichia coli strain
BL21. Production of the His-PtrWRKY75 fusion protein was induced by 0.2
Mm isopropyl β-D-1-thiogalactopyranoside.
Oligonucleotide probes (PAL1 W-box: TTGACC) were synthesized by
Sangon (Beijing, China) and labeled with biotin using an EMSA Probe
Biotin Labeling Kit (GS008, Beyotime Biotechnology, Jiangsu, China). For
the mutated probe, the single mutation site was located in the core
sequence of the W-box (changed from TGAC to TAAC). The EMSA was
performed using a Chemiluminescent EMSA kit (GS009, Beyotime
Biotechnology, Beijing, China) in accordance with the manufacturer’s
instructions. Briefly, biotin-labeled probes were incubated with the
fusion proteins in binding buffer for 30 min at room temperature (20–25
°C). The reaction products were transferred to 4% polyacrylamide gel,
then electrophoresed in 0.5× Tris-borate-EDTA (TBE) buffer for 60 min.
All oligonucleotide sequences are listed in Table S2.