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.