3.1 Isolation of NtMYB12 genes from N. tabacumgenome
Six MYB12 homologous genes were cloned and sequenced fromN. tabacum genome, and their sequence information was shown in Supplementary Table S1. Phylogenetic tree was constructed with 24 known R2R3-MYB TFs, which has been clustered into PA-related subgroup (subgroup 5/SG5), anthocyanin-related subgroup (SG6), and flavonol-related subgroup (SG7) (N. Wang et al., 2017). All the 6 NtMYB proteins belonged to SG7, and were divided into 3 pairs (Figure 1a). The two members within each pair shared highest sequence identities (Supplementary Table S2). One pair genes close to AtMYB111 were named NtMYB111a/NtMYB111b , while the other two pairs were named NtMYB12a/NtMYB12b andNtMYB11a/NtMYB11b , respectively. Sequence alignment showed that the R2-R3 domains were highly conserved among the 6 NtMYB proteins at the N -terminus, but their C terminal sequences were diverse (Figure 1b). NtMYB11b, NtMYB111a, and NtMYB111b contained the typical SG7 motif ([K/R][R/x][R/K]xGRT[S/x][R/G]xx[M/x]K), while NtMYB12a, NtMYB12b, and NtMYB11a contained both SG7 motif and SG7-2 motif ([W/x][L/x]LS) (Figure 1b), indicating their putative function in regulating flavonol biosynthesis. Gene structure analyses showed that NtMYB12a gene shared similar exon/intron organization with NtMYB12b gene, while those within other two pairs were diverse (Figure 1c). Therefore, NtMYB12a and NtMYB12b were a pair of homologous genes with high similarity, and their respective functions needs to be clarified.