Cloning and Expression of IlWRKY22 in I. laevigata

Total RNA was extracted from the tepals of I. laevigata using a kit (Kangweishiji, Taizhou, China), and the integrity was checked by electrophoresis. Frist-strand cDNA was synthesized using the Primer Script TM RT kit (TakaRa, Beijing, China). IlWRKY22 (accession number: ON399552) was cloned using KOD-plus-neo (ToYoBo, Shanghai, China) with primers (Ruibo Kexing, Harbin, China) listed in Table S1. The cloned sequences were inserted into the Cloning vector pEASY^®-Blunt Zero Cloning Kit (TransGen, Beijing, China) and transformed into Escherichia coli DH5α (WEIDI, Shanghai, China). The pCAMBIA1300: IlWRKY22-GFP carrier was constructed for heterologous expression through homologous recombination. Homologous recombination primers (Ruibo Kexing, Harbin, China) listed in Table S1. The pCAMBIA1300 vector were digested with SalI and BamHI, which were then used to create pCAMBIA1300-IlWRKY22-GFP using ClonExpressII (Vazyme, Nanjing, China). The recombinant vector was transformed into E. coli DH5α (WEIDI, Shanghai, China) for sequencing. Positive plasmids were transferred into Agrobacterium tumefaciens GV3101 (WEIDI, Shanghai, China) for subsequent infection experiments. Bioinformatics analysis of IlWRKY22 was performed as described [26 (link)].