Escherichia coli bl21 competent cells

According to the genomic DNA sequence of A. hydrophila OmpA gene (GenBank accession number: AF146597), the primers were designed to construct the recombinant plasmid including the coding region of OmpA gene (Supplemental Table 1). The PCR amplified product was digested and cloned into pET-32a vector, and then transformed into Escherichia coli BL21 competent cells (TransGen Biotech, Beijing, China). The recombinant OmpA protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mM at 37°C for 10 h, which was purified using a Ni-Agarose His-tagged Protein Purification Kit (CoWin Biosciences, Jiangsu, China). Then, the protein concentration was detected by BCA Protein Assay Kit (CoWin Biosciences).

Coding DNA sequence of SPL35 was inserted into the pMAL‐c2x vector to generate the construct MBP‐SPL35. CDSs of OsUBC5a, Delta‐COP1 and Delta‐COP2 were cloned into the pGEX4T‐1 vector to generate GST‐OsUBC5a, GST‐Delta‐COP1 and GST‐Delta‐COP2 respectively. The constructs, together with the empty vector, were transformed into Escherichia coli BL21 competent cells (TransGen Biotech, Beijing, China) to express protein. Recombinant proteins from E. coli lysates were purified using the corresponding resins [amylose resin for MBP purification (New England Biolabs, Beijing, China), and GST‐binding resin for GST purification (Merck, Beijing, China)]. GST or GST fusion proteins coupled to beads were incubated with MBP or MBP‐SPL35. In vitro GST pull‐down analysis was performed as described previously (Wang et al., 2009). For protein detection, anti‐GST antibody (1:1000), anti‐MBP antibody (1:500) and goat anti‐mouse HRP‐conjugated antibody (1:5000; MBL International, Beijing, China) were used. Three independent repeats were performed. Primers used are listed in Table S1.