Cloning And Expression Analysis Of DFR Genes From Grape Hyacinths

Grape hyacinth (Muscari) is an important perennial herbaceous bulbous plant whichintroduced from Europe. Except for a few white cultivars, flower colour of these plants variesfrom blue to purple. So it is an ideal material to study the mechanism of blue color formationin monocotyledons. The current researches on this plant are mainly about propagation, genetictransformation, flower regulation and anatomic structure.The information on the molecularbasis of anthocyanin metabolic was seldom reported. Previous studies showed that the colordifference in grape hyacinth is depended on the presence of anthocyanin in the palisadetissues of petals. Anthocyanins are the principal flower pigments in Muscari, and areindispensible in plant biology and human/animal diets. Anthocyanin synthesis pathway is wellcharacterized in numerous plants，and dihydroflavonol4-reductase (DFR) is a crucial latergene for anthocyanin formation. In this report, a blue flower cDNA library of M. armeniacumand a white flower library of Muscari botryoides cv. White Magic were used for transcriptomesequencing. Through a combination of chemical analysis with bioinformatics, threedihydroflavonol4-reductase (DFR) genes were cloned from Muscari armeniacum petals. Wealso did some sequence alignment, Phylogenetic analysis and subcellular localization of DFR;We analysed the expression levels of DFR in the flowers, leaves, stems and roots usingreal-time PCR. The main results are as followings.1. we cloned three dihydroflavonol4-reductase (DFR) genes from Muscari armeniacumpetals, and designated as MaDFR1, MaDFR2a and MaDFR2b respectively.Sequence analysis of cDNAs have revealed that MaDFR1contained a984bp of open readingframe (ORF), which encoded a protein of327amino acids; The full length sequence ofMaDFR2a and MaDFR2b was1,395bp, containing a length of76bp of5’untranslatedregion and215bp of3’ untranslated region, both of them contained a1,101bp of ORF, whichencoded a protein of366amino acids. The similarity of MaDFR2a and MaDFR2b was98%.In contrast, the similarity of MaDFR2a and MaDFR2b with MaDFR1was just41%.Homology analysis showed that the deduced DFR proteins of MaDFR2a and MaDFR2b werehighly homologous to other DFR proteins from different plant species. 2. Bioinformatics analysis showed that three DFR proteins were hydrophilic proteins,containing two transmembrane domains and neither of them had signal peptide. Serine,threonine, tyrosine are phosphorylation sites of the three DFR proteins. α-Helix and randomcoil were primary secondary structural components of the three DFR genes. The tertiarystructures of three DFR proteins were similarly. All of them had typical hand motif structuresand fissures which could do some catalytic reactions. NADP (H) binding sites located in theN-terminus of the surface fissure; Bioinformatic analysis showed that the three DFR proteinshad the typical functional domains of DFR protein and belonging to the NADB_Rossmannsuperfamily.3. We constructed pBI221-MaDFR1-GFP, pBI221-MaDFR2a-GFP,pBI221-MaDFR2b-GFP vectors for subcellular localization, and transformed them into onionepidermal cells using microprojectile bombardment. Under a laser scanning confocalmicroscope, we found that all the three DFR genes located in the whole cell.4. The expression patterns of DFR in Muscari armeniacumandMuscari botryoides cv.White Magic were investigated. We analysis the spatial and temporal expression patterns ofDFR using real-time PCR. The result showed that the DFRs expression was presented inflowers but reduced or absent in the other tissues (leaf, stem and root). The expression level ofMaDFR1was higher in Muscari botryoides cv. White Magic, and MaDFR1activity reachedits peak at the fully-pigmented buds, and decreased at later flower development stages. Infully-opened flowers of Muscari armeniacum, the expression level of MaDFR1was higherthan that in white varieties. The expression trend of MaDFR2a and MaDFR2b was consistent,but differ in expression level.5. High performance liquid chromatography (HPLC) analysis showed that high contentsof color anthocyanins were detected in pigmented flower organs, while no anthocyanins weredetected in root, stem, leaf and unpigmented flower buds.