Cloning And Expression Analysis Of FLS Genes From Grape Hyacinths

Grape hyacinth(Muscari) is an important perennial herbaceous bulbous plant which introduced from Europe. Except for a few white cultivars, flower colour of these plants varies from blue to purple. The current researches about thisplant are mainly focused on propagation, genetic transformation, flower regμLation and anatomic structure.The information on the molecular basis of flavonoids metabolic pathway has not been reported almostly. Flavonols synthesis pathway is an important branch of flavonoids way, and flavonol synthetase(flavonol synthase, FLS) is a key enzyme in plant’s flavonols synthesis.In this study, a blue flower c DNA library of Muscari botryoides armeniacum and a white flower library of Muscari botryoides cv. album were used for transcriptome sequencing. Through a combination of chemical analysis with bioinformatics, two flavonol synthase(FLS) genes were cloned both from the petal of Muscari botryoides armeniacum and Muscari botryoides cv. album. We also did some sequence alignment, Phylogenetic analysis, subcellular localization and Prokaryotic expression of FLS; We analysed the expression levels of FLS in the flowers, leaves, stems and roots using real-time PCR as well as the change of genes’ sexpressin level and contents of the flavonol under environmental stresses. The main results are as followings.1. we cloned two flavonol synthase(FLS) genes from Muscari botryoides armeniacum petals and Muscari botryoides cv. Album petal, designated as Ma FLS-a, Ma FLS-b respectively. The full length ORF of Ma FLS-a and Ma FLS-b was 999 bp, which encoded a protein of 332 amino acids. The protein similarity of Ma FLS-a and Ma FLS-bwas 100%, and only in the 468th(C-T) and 642th(G- A)nucleotide their sequenceswere different,2. Bioinformatics analysis showed that the FLS protein was hydrophodic proteins, and did not had signal peptide or transmembrane domain. Serine, threonine, tyrosine are phosphorylation sites of the the FLS protein. α-Helix and random coil were primary secondary structural components of the the FLS gene. The FLS proteincontained several typical conserved elements found in the 2-oxoglutarate-Fe(II)-dioxygenase superfamily(2-ODD), and inferred residues for coordination of ferrous iron(His204, Asp206 and His265) and 2-oxoglutarate binding(Arg274 and Ser276) were also found.3. We constructed a p BI221-Ma FLS-GFPvector for subcellular localization, and importedit into the epidermal cells of onion using microprojectile bombardment. Under a laser scanning confocal microscope, we found that the FLS protein located in the whole cell,especially in the the cytoplasm.4.We built a p ET- Ma FLS prokaryotic expression vector, and transformed the vectorinto BL21(DE3), inducted by IPTG.The results show that the FLS proteincould be expressed in vitro.5.Withthe use of Real-time fluorescent quantitative PCRtechnology, we analysedthe expression level of FLS gene with the treatment of salicylic acid and sodium chloride.Results show that under the condition of high salt and hormone, showed varying degrees of differences in transcription. Salicylic acid(SA) could significantly increasethe expression level of Ma FLS-a in leaves and stems, which is8.36 times and 5.87 times larger than the control group respectively. Howeverthe Ma FLS-aexpression level inroots and leaveswere reduced than the control. With the treatment of Sodium chloride(Na Cl) the FLSs expression wereincreased in roots but reduced in the other tissues(leaves, stems and flowers). The above results suggest that Ma FLS- a gene was tissue specificity and could be induced, and salicylic acid(SA) could stimulate the expression of FLS according to the corresponding signal, thus protect the plant from dangerous.6. The expression patterns of FLS in Muscari botryoides armeniacum and Muscari botryoides cv. album were investigated. We analysis the spatial and temporal expression patterns of FLS using real-time PCR. The result showed that the FLSs expression were presented in flowers but reduced or absent in the other tissues(leaf, stem and root). The expression level of FLS was higher in Muscari botryoides cv. Album than Muscari botryoides armeniacum.7. 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 accumulation of flavonols was in agreementwith the expression level of FLS gene. And the content was more higher in Muscari botryoides cv. album than in Muscari botryoides armeniacum.