Phylogenetic Analysis of R2R3-MYB Family Genes in Tetrastigma hemsleyanum Diels et Gilg and Roles of ThMYB4 and ThMYB7 in Flavonoid Biosynthesis

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is an extensively used Chinese folk herb with multiple bioactivities. Among these bioactivities, flavonoids are recognized as the representative active ingredients. We previously found an elevated accumulation of flavonoids in T. hemsleyanum under water stress; however, the mechanism remains unclear. R2R3-MYB transcription factors play vital roles in the plant response to environmental stress and the regulation of secondary metabolites. Herein, a systematic transcriptome identification of R2R3-MYB family genes under water stress in T. hemsleyanum was performed to explore their potential function in the biosynthesis of flavonoids. A total of 26 R2R3-MYB genes were identified, most of which were clustered into functional branches of abiotic stress. ThMYB4 and ThMYB7 were then screened out to be associated with the biosynthesis of flavonoids through a protein-protein interaction prediction. An expression correlation analysis based on RNA-seq further confirmed that ThMYB4 and ThMYB7 were positively related to the flavonoid biosynthetic pathway genes of T. hemsleyanum. In ThMYB4- and ThMYB7-overexpression hairy roots, it was found that the expression of ThCHS and ThCHI was significantly increased, suggesting that ThMYB4 and ThMYB7 may act as regulators in flavonoid biosynthesis. This will shed new light on the promotion of flavonoid production and the medicinal value of T. hemsleyanum by manipulating transcription factors.