2.2 Gene cloning and bioinformatics
The coding sequences and amino acid sequences of Arabidopsis AtMYB12 (At2g47460), AtMYB11 (At3g62610), and AtMYB111 (At5g49330) were used as query sequences to make blast in the NCBI database. Six homologous proteins were identified (XP_016480310.1, XP_016482665.1, XP_016508470.1, XP_016487451.1, XP_016444804.1, and XP_009790561.1) in the N. tabacum database, and the full length gene sequences and coding sequences were amplified and sequenced with gene-specific primers (Supplementary Table S1). Exon/intron structure of each gene was determined by aligning the CDS and genomic DNA sequences with Clustal X (version 1.83). Multiple sequence alignment of the NtMYB proteins was performed using the DNAMAN (version 6.0) software with default gap penalties (Jeanmougin, Thompson, Gouy, Higgins, & Gibson, 1998). The phylogenetic tree was built up using the MEGA 5.0 software (Tamura et al., 2011) and the following protein accessions in GenBank: AtMYB90 (AAG42002), AtMYB75 (AAG42001), AtMYB113 (NP_176811), PhAN2 (AAF66727,Petunia hybrid), MdMYB10 (XP_028963317.1, Malus domestica), VvMYBA1 (BAD18977.1, Vitis vinifera), VvMYBA2 (BAD18978.1), MdMYB11 (AAZ20431.1), MdMYB12 (XP_008337875.1), GhMYB38 (AAK19618.1, Gossypium hirsutum), VvMYBPA2 (ACK56131.1), AtMYB123 (Q9FJA2), MdMYB9 (NP_001280749.1), PmMBF1 (AAA82943.1, Picea mariana), MdMYB22 (AAZ20438.1), SlMYB12 (ACB46530, Solanum lycopersicum), ZmP (P27898, Zea maize), GhMYB1 (CAD87007.1,Gerbera hybrid cultivar), LjMYB12 (BAF74782.1, Lotus japonicas), and VvMYBF1 (FJ948477).