Identification and functional analysis of GbMYB15 as a negative regulator in flavonoid biosynthesis of Ginkgo biloba

Flavonoids, which possess diverse pharmacological properties, are abundant in Ginkgo biloba leaves and contribute to the prevention of cardiovascular and cerebrovascular diseases. R2R3-MYB transcription factors are crucial regulators of flavonoid metabolism, yet their functions, particularly those involving negative regulation in G. biloba, remain poorly understood. In this study, GbMYB15, a gene implicated in flavonoid biosynthesis, was cloned based on preliminary transcriptome data. Its functional role was investigated through bioinformatics analysis, transient overexpression in G. biloba, and stable heterologous expression in Arabidopsis thaliana. The results showed that the GbMYB15 protein contains a C1 activation motif. Under exogenous ABA and MeJA treatments, the expression level of GbMYB15 was negatively correlated with the total flavonoid content. Overexpression of GbMYB15 in A. thaliana and G. biloba significantly reduced the total flavonoid content, confirming that GbMYB15 is a negative regulator of total flavonoid synthesis. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and luciferase complementation imaging assays confirmed that GbMYB15 interacts with the energy metabolism-related protein Gb34759. Transient overexpression experiments in G. biloba leaves further showed that the interaction between GbMYB15 and Gb34759 enhances the negative regulatory capacity of GbMYB15. Dual-luciferase reporter assays indicated that GbDFR2 is a candidate downstream structural gene potentially regulated by GbMYB15 in flavonoid biosynthesis. Based on these results, this study hypothesizes a regulatory model: GbMYB15 may affect total flavonoid content by modulating the flux of the GbDFR2-associated pathway, and that the interaction between GbMYB15 and Gb34759 may enhance this regulatory effect. This work provides a theoretical foundation for future genetic engineering efforts aimed at enhancing flavonoid content in G. biloba.