For protein expression and purification, 1 to 942 bp of EIN3 CDS containing the DNA-binding domain and full-length CDS of ORE1/ANAC092 were cloned into pMAL-c5g (New England Biolabs). The empty vector pMAL-c5g was used for MBP expression alone as a negative control in EMSA. Plasmids were transformed into Escherichia coli strain Rosetta (DE3) pLysS (Merck). The expression of proteins was induced by 1mM isopropyl thio-β-D-galactoside (IPTG) at 20°C for 10 hr in 200 mL LB medium. Cells were then collected and sonicated. Protein was purified with Amylose resin (New England Biolabs) following the manufacturer’s instructions.
EMSA was carried out using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). Briefly, 400 ng purified protein was incubated with 12.5 fmol 5’-biotin-labeled probe DNA and 1 μg poly (dI-dC) in binding buffer for 15 min. Binding reactions were resolved on a polyacrylamide gel and electrophoretically transferred to nylon membrane. The transferred DNA was cross-linked to membrane by use of CL-1000 Ultraviolet Crosslinker (UVP, Upland, CA, USA). Biotin-labeled DNA was detected by chemiluminescence and exposed with a ChemiScope 3500 Mini Imaging System (Clinx Science Instruments, China).
EMSA was carried out using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). Briefly, 400 ng purified protein was incubated with 12.5 fmol 5’-biotin-labeled probe DNA and 1 μg poly (dI-dC) in binding buffer for 15 min. Binding reactions were resolved on a polyacrylamide gel and electrophoretically transferred to nylon membrane. The transferred DNA was cross-linked to membrane by use of CL-1000 Ultraviolet Crosslinker (UVP, Upland, CA, USA). Biotin-labeled DNA was detected by chemiluminescence and exposed with a ChemiScope 3500 Mini Imaging System (Clinx Science Instruments, China).