A novel miR396-GbMYB41 module regulates flavonoid biosynthesis in Ginkgo biloba

Flavonoids, major bioactive constituents of Ginkgo biloba extract (EGB), possess cure cardiovascular, antioxidant, antitumor, and anti-inflammatory properties, and they play vital roles in plant growth and stress responses. While MYB transcription factors are known regulators of flavonoid biosynthesis, the miRNA–MYB network in G. biloba remains unclear. Here, multi-omics and weighted gene co-expression network analysis identified GbMYB41 as a key regulator positively correlated with flavonoid content under tissue-specific and methyl jasmonate/abscisic acid treatments. GbMYB41 overexpression in Arabidopsis thaliana increased flavonoid levels by approximately 300 % and significantly upregulated the expression levels of structural genes ( PAL , C4H , 4CL , F3H , FLS , OMT ). Further, we discovered that the novel non-coding RNA miR396 negatively regulates flavonoid biosynthesis by repressing GbMYB41 expression. Consequently, we identified the miR396-GbMYB41 module as a key regulatory factor for flavonoid biosynthesis in G. biloba . In summary, this study reveals the interaction between miR396 and its target gene GbMYB41 in G. biloba , demonstrating GbMYB41 ’ s role as a positive regulator of flavonoid biosynthesis and elucidating the mechanism whereby miR396 targets GbMYB41 to modulate this process. Overall, our findings reveal a previously uncharacterized flavonoid biosynthesis and regulatory mechanism provide a theoretical framework for improving flavonoid content in G. biloba through genetic engineering. • GbMYB41 was selected as a candidate gene based on co-expression and correlation analysis. • GbMYB41 up-regulated flavonoid biosynthesis in G. biloba . • miR396 bound to and inhibited the expression of GbMYB41 . • Revealing the miR396-GbMYB41 network associated with flavonoid metabolism in G. biloba .