Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related Genes in Blueberry ( L.).

Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related -acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of -elements and TFBSs varied greatly between the two groups. Quantitative real-time PCR results showed that expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these showed certain spatiotemporal specificity. Additionally, might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.