Electrophoresis mobility shift assay (EMSA)
The CDS of CsHSFA1b and CsHSFA2 were cloned into pCold
vector to construct the fusion protein with His-tag fusion vector and
then introduced into BL21 (DE3) Escherichia coli . His-beads
(70501, Beaver, China) were used to purify the fusion protein
His-CsHSFA1b/CsHSFA1bA2 in accordance with the manufacturer’s
instructions. DNA fragments from the CsJAZ6 promoter that
contained HSE motifs were amplified using biotin-labeled primers (Table
S1). His-tagged CsHSFA1b and CsHSFA2 proteins were incubated in binding
buffer with biotin-labeled probes for 20 min at room temperature. The
LightShift Chemiluminescent EMSA kit (Thermo Scientific, Waltham, MA,
USA) was used to detect the igration of biotin-labeled probes.