Ni nta 6ff

Full length CDS of OsCCA1 was cloned into the pET-28a vector and transformed into BL21 strain of Escherichia coli to express His-CCA1 fusion protein. The expression and purification of recombinant protein were performed using Ni-NTA 6FF (Sangon Biotech). The promoter sequence of the OsNRT2.2 was cloned and purified from Wild-type rice DNA. The EMSA assays were performed as described previously [39 ]. P6 (100 bp DNA fragment containing MYB binding element), P7 (100 bp DNA fragment containing MYB binding element) and P8 (64 bp DNA fragment containing MYB binding element) were incubated with His-OsCCA1 protein in binding buffer (10 mM Tris–HCl [pH 7.5], 50 mM KCl, 1 mM DTT) at 4℃ for 40 min, respectively. 4% TBE-polyacrylamide gels were used for electrophoretic assay. Electrophoresis was performed in TBE buffer at 120 V for 60 min at 4 ℃. Gels were stained with ethidium bromide (0.5 mg/mL) and photographed (Fig. S5, Fig. S6). The related primers are listed in Table S2.

Specific primers were designed to clone the cDNA that encodes XaffOBP9 (Supplementary Table S1). The PCR products were inserted into the pET-28a (+) vector using NotI and NcoI restriction endonucleases. The plasmid containing the correct insert fragment was subsequently transformed into Escherichia coli BL21 (DE3) cells. The recombinant protein was induced at 28°C for 6 h by 1 mM isopropyl β-d-l-thiogalactopyranoside (IPTG) when the OD600 value reached 0.6. The suspension was sonicated and then separated into supernatant and sediment by centrifugation (11,000 rpm, 20 min, 4 C). The protein was then purified using Ni-NTA 6FF (Sangon Biotech, Shanghai, China) in a graded imidazole series of 0 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 400 mM, 600 mM for washing and desalted using Dialysis Membrane (Sangon Biotech, Shanghai, China). The molecular weight and purity of the XaffOBP9 proteins were checked using 15% SDS-PAGE.