Abstract：Grape hyacinth (Muscari armeniacum) is an important ornamental bulbous plant with increasing economic, ornamental and scientific importance because of their outstanding blue color. Anthocyanins are principal flower pigments in M. armeniacum, and indispensible in plant biology and human/animal diets. Whereas anthocyanin synthesis is well characterized in numerous plants, the mechanism underlying the blue appearance of M. armeniacum is still far from understanding for the little knowledge of gene information of this plant. Here, two dihydroflavonol 4-reductase (DFR) genes, which encode a later enzyme for anthocyanin formation, were isolated from Muscari armeniacum petals using RACE techniques and designated as MaDFR2a and MaDFR2b(GenBank accession No. KJ619963 and KJ619964), respectively. Sequence analysis of cDNAs revealed that the full length sequence was 1 395 bp, containing a length of 76 bp 5' untranslated region and 215 bp 3' untranslated region，both of them contained a 1 101 bp open reading frame (ORF), which encoded a protein of 366 amino acids. The similarity of the two DFR was 98%. Homology analysis showed that the deduced DFR proteins were highly homologous to other DFR proteins from different plant species. The cluster analysis results showed that the two DFR genes clustered together with monocotyledons firstly. Bioinformatics showed that two DFR proteins were both hydrophilic proteins, containing two transmembrane domains and neither of them had signal peptide. Subcellular localization analysis showed that two DFR genes located in the whole cells. α-Helix and random coil were primary secondary structural components of the two DFR genes. The tertiary structures of two DFR proteins were similar. Both of them had a fissure which could do some catalytic reactions. High performance liquid chromatography (HPLC) analysis showed that high contents of color anthocyanins were detected in pigmented flower organs, while no anthocyanins were detected in root, stem, leaf and unpigmented flower buds. The result of Real-time fluorescence quantitative PCR analysis demonstrated that the DFRs expression was presented in flowers but little or very weak expression was also detected in the other tissues (leaf, stem and root). When flower pigmentation started, a steep rise of two DFR genes expression occurred and peaked at fully coloured flowers. Subsequently a slight decrease in the MaDFR2a and MaDFR2b expression was presented at later stage. In contrast, a relatively high-level of MaDFR2b expression was found in both unpigmented or fully-pigmented buds. This result suggested that DFR can play a role in the color formation of blue grape hyacinth.
