Cloning and characterization of the cDNA and promoter of UDP-glucose:flavonoid 3-O-glucosyltransferase gene from a purple-fleshed sweet potato

Abstract

UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) catalyses the transfer of the glucosyl moiety from UDP-glucose to the 3-hydroxyl group of anthocyanidins to produce the first stable intermediate in the anthocyanin biosynthesis pathway. A full-length UF3GT cDNA (designated IbUF3GT) was isolated and characterized from a purple-fleshed sweet potato (Ipomoea batatas cv. Yamakawamurasaki). The cDNA was 1541bp in length and contained a 1359bp open reading frame encoding 452 amino acids with a calculated molecular mass of 48.8kDa and an isoelectric point of 5.64. Bioinformatics analyses revealed that IbUF3GT had high homology with UFGTs from other plant species. To verify the function of IbUF3GT, the gene was introduced into the UF3GT T-DNA insert mutant of Arabidopsis thaliana. Transformed Arabidopsis plants were obtained with purple cotyledon, hypocotyl and stems. A comparison of the anthocyanin contents of transgenic lines with the Arabidopsis mutants indicated that IbUF3GT encoded a functional protein for anthocyanin biosynthesis. The gene expression and anthocyanin accumulation analyses revealed that the IbUF3GT gene expression pattern matched the accumulation pattern of anthocyanins in different tissues and cultivars and in different developmental stages of the sweet potato root, suggesting that IbUF3GT was a key enzyme involved in anthocyanin accumulation in the purple-fleshed sweet potato. Furthermore, the IbUF3GT promoter sequence (designated pIbUF3GT; 758bp in length) was cloned from the purple-fleshed sweet potato cv. Yamakawamurasaki. Structural analysis of pIbUF3GT suggested that the IbUF3GT transcript level might be regulated by the transcription factors MYB and MYC, responsive factors to light, salicylic acid (SA) and gibberellic acid (GA). Tobacco leaves transiently expressing full-length and four 5′-deletions of pIbUF3GT fused with GUS indicated that pIbUF3GT had promoter activity and that the key elements related to promoter activity were located in the region from −758bp to −633bp in pIbUF3GT.