Molecular Characterization of WRKY Transcription Factors

Two groups of specific primers (Supplementary Table 1B) were designed with Primer Premier 5.0 software (Premier, Canada), and the complete coding sequences of the IgWRKY50 and IgWRKY32 genes were amplified twice. The PCR products were connected to the pEASYT3 cloning vector (TransGen Biotech, Beijing, China) and then sequenced by the company (Sangon Biotech, Shanghai, China). The open reading frames of IgWRKY50 and IgWRKY32 were found by NCBI ORF Finder.¹ The protein functional domains of IgWRKY50 and IgWRKY32 were predicted by SMART software (Letunic and Bork, 2018 (link); Letunic et al., 2021 (link)).² The phylogenetic tree of amino acid sequences of other species with homology to IgWRKY50 and IgWRKY32 genes was constructed by MEGA 6 software using the neighbor-joining (NJ) method with 1,000 bootstrap replicates (Tamura et al., 2013 (link)). The accession numbers of the WRKYs are listed in Supplementary Table 2. The tertiary structure prediction of IgWRKY50 and IgWRKY32 proteins was completed by SWISS-MODEL software (Biasini et al., 2014 (link)), and the three-dimensional structure model of proteins was constructed by the I-TASSER website³ and Chimera 1.11.2 software⁴ (Pettersen et al., 2004 (link)).