Identification and functional characterization of SbMYB306 as a R2R3-MYB regulator of anthocyanin biosynthesis in Scutellaria baicalensis Georgi

Scutellaria baicalensis Georgi is a perennial herb belonging to the family Labiatae. Its roots are rich in flavonoids and possess heat-clearing, dampness-drying, fire-purging, and detoxifying properties. Under natural conditions, purple petals predominate in this species, whereas purple-red and white variants are rare. Previous transcriptomic and metabolomic analyses of flowers with different colors have mapped the anthocyanin biosynthetic pathway in S. baicalensis and identified differentially expressed candidate genes. However, the transcriptional regulation of anthocyanin structural genes remains unclear, and the SbR2R3-MYB gene family has not yet been systematically characterized. To address this knowledge gap, a genome-wide analysis of the SbR2R3-MYB family was conducted, with a particular focus on candidate genes involved in anthocyanin biosynthesis. A total of 55 members were identified and characterized based on their physicochemical properties, phylogenetic relationships, gene structures, sequence conservation, chromosomal distributions, and collinearity. Bioinformatic analyses identified SbMYB306 and SbbHLH96 as key regulatory genes and SbANS and SbDFR as anthocyanin-related structural genes for further investigation. Multiple assays, including yeast one-hybrid (Y1H), yeast two-hybrid (Y2H), dual-luciferase reporter, bimolecular fluorescence complementation (BiFC), and β-glucuronidase (GUS) activity assays, demonstrated that SbMYB306 and the SbMYB306–SbbHLH96 complex regulate key enzyme-encoding genes involved in flower color formation. Heterologous overexpression of SbMYB306 in tobacco enhanced petal pigmentation and increased anthocyanin accumulation. These findings establish SbMYB306 as a positive regulator of anthocyanin biosynthesis, support the evolutionary conservation of the anthocyanin biosynthetic pathway, and provide a theoretical basis for further investigation of flower color formation in S. baicalensis .