Mining and functional characterization of a flavonoid glucosyltransferase (OtUGT) involved in 7-O-glucosylation of apigenin in Ocimum tenuiflorum

Abstract

The genome data of Ocimum tenuiflorum (CIM-Ayu) were utilized for mining novel flavonoid biosynthesis-related gene(s). The search using the unannotated gene sequences yielded an uncharacterized flavonoid glucosyltransferase gene (OtUGT), which was selected for characterization. Its bioinformatics-based analysis predicted it to be an OtUGT. qRT-PCR analysis of this OtUGT indicated its highest expression in O. tenuiflorum in comparison to other Ocimum species. In O. tenuiflorum, its expression was highest in the leaf tissue compared to the trichome, stem, flower, and root. The gene expression was found to be sensitive to MeJA and light. Heterologous expression of the OtUGT protein was induced in BL21(DE3) strain of Escherichia coli. The purified recombinant protein was used for assay with different flavonoid and sugar donor substrates, whereby it showed activity only with apigenin and UDP-α-d-glucose. The product formed was apigenin 7-O-glucoside (apigetrin), which was confirmed through TLC, HPLC, LC-QTOF mass spectrometry, and NMR analyses. Transient overexpression and VIGS of OtUGT in O. tenuiflorum were appropriately reflected in terms of alterations in apigetrin levels in the plant. Apigetrin content increased by up to 2.5-fold in transgenic Nicotiana tabacum lines generated by transformation with Agrobacterium tumefaciens carrying the pBI121-OtUGT construct. Hairy roots generated from leaf explants of O. tenuiflorum through transformation with A. rhizogenes carrying the pBI121-OtUGT construct were not found to be advantageous in terms of apigetrin content. Since apigetrin possesses high medicinal value, elucidation of its biosynthesis in O. tenuiflorum will enable its economical production through optimization in the plant or heterologous systems in the future.