Characterization Of DFR And FLS Genes Regulating Of The Anthocyanin Accumulation In Blue Grape Hyacinth

Grape hyacinth(Muscari spp.)is an important ornamental bulb flower and widely used in garden quilts and home pots.Because of its unique blue tones and patterns,it is an ideal materials for studying the mechanism of blue flower coloration.The formation of flower color in grape hyacinth is mainly dependent on flavonoids,especially the blue anthocyanin derivatives.In our previous study,the dihydroflavonol-4-reductase(DFR)and flavonol synthase(FLS)may regulate the metabolic flux of anthocyanin synthesis pathway contributing to the formation of blue color in grape hyacinth.In this study,the Muscari.aucheri'Dark Eyes'was used as the typical blue material to study the mechanism of flower color formation mediated by MaDFR and MaFLS genes.The main results were as follows:1.Two DFR genes from‘Dark Eyes'were cloned and named as MaDFR(MK937098)and MaDFR1(KJ619964).The full-length of DFR open reading boxs are 1101 bp encoding366 amino acids.The molecular weights of the proteins were approximately 40.93 KDa and41.00 KDa,respectively.Comparative analysis revealed that the amino acid homology was97.27%between MaDFR and MaDFR1.Furthermore,The deduced amino acid of the cloned MaDFR1 had 100%identities with previously reported MaDFRb from M.aucheri‘White Beauty'.Alignment analysis among DFR protein sequences from other species,MaDFR and MaDFR1 both had a conserved the NADPH-binding motif and a substrate-binding domain.Cluster analysis indicated that they belonged to the monocot DFR family.Both were localized in the cytoplasm,and their transcriptional expressions showed tissue-specific,especially in flowers,positively correlating with the accumulation of delphinidin.2.In vitro prokaryotic expression and enzyme activity assay showed that both of MaDFR and MaDFR1 catalyzed three dihydroflavonols,dihydrokaempferol(DHK),dihydroquercetin(DHQ)and dihydromyricetin(DHM),to yield the corresponding leucopelargonidin.Moreover,they preferentially used the DHM as the substrate.In addition,amino acid point mutation experiments confirmed that the asparagine(N)at position 135 and the glutamic acid(E)at position 146 determined the catalytic activity of the MaDFR and MaDFR1 proteins.3.MaDFR and MaDFR1 were overexpressed in tobaccos by Agrobacterium-mediated tobacco leaf discs.Compared with the control,the corollas of OE-MaDFR and OE-MaDFR1transgenic lines turned deep red.HPLC analysis showed that the content of total anthocyanins in the OE-MaDFR and OE-MaDFR1 transgenic lines was significantly higher than the control.In addition,UPLC-Triple-TOF/MS analysis confirmed that the Cy3R was the main kind of anthocyanins both in the transgenic and control lines,and no new substances were formed.Real-time quantitative qPCR analysis showed that the expression of genes involved in flavonoid biosynthetic pathway were up-regulated in OE-MaDFR lines but not the flavonoid 3'5'hydroxylase(F3'5'H)gene.While in the OE-MaDFR1 lines,CHS and CHI,upstream structural genes,were down-regulated,which may be influenced by the feedback mechanism of the flavonoid pathway in tobacco.The F3'5'H gene was not expressed in the OE-MaDFR,OE-MaDFR1 and control lines.This may be one of the reasons why delphinidin(Dp)could not be detected in the transgenic lines.4.The upstream promotor sequence of DFR gene was cloned according to the genome walking method.Cis-acting elements analysis indicated that the 1518 bp promoter region contained CAAT-box,TATA-box,and color related elements including light responsive element,MYB-motif and MYC-motif type transcription factor binding elements.The full-length and different deletions of MaDFR promoter were fused to tne N-terminal of GUS reporter gene.The recombinant vectors were transformed into Agrobacterium tumefaciens GV3101 strain and stably transformed into tobacco.Histochemical analysis and GUS activity analysis were performed.The results showed that the MaDFR promoter was only expressed in the corolla,and the GUS activity was significantly attenuated after the deletion of-231 bp--407 bp.Dual luciferase assay analysis indicated that the transcription factor MaMYBA binds to the-231 bp to-407 bp region of the MaDFR promoter.5.The full-length cDNA sequence of MaFLS was obtained by Rapid amplification of cDNA ends.The full length of MaFLS mRNA was 1418 bp and the coding region was 993bp,encoding 330 amino acids,with the molecular weight of 36.45 kDa.Genbank accession number was MH636605.Amino acid sequence alignment and cluster analysis showed that MaFLS had the conserved ferrous iron-binding residues(His 216,Asp 218 and His 272)and the 2-oxoglutarate binding residues(Arg 282 and Ser 284),which indicated that MaFLS belonged to the soluble Fe2~+/2-ODD protein family.In addition,MaFLS had five key amino acids(Tyr 127,Phe 129,Lys 197,Phe 288,Ser 290)that were identified as potential active DHQ binding site residues.MaFLS contained FLS-specific motifs"PxxxIRxxxEQP"and"SxxTxLVP".The transcriptional expression of MaFLS was tissue-specific and preferentially expressed in the S1(newly formed flower buds with a little color)and S2(development of petals with some color)stages of flower development,but its expression was not correlated with the accumulation of total flavonol in grape hyacinth.6.Compared with the pink flowers in wild-type tobacco pink,the OE-MaFLS transgenic lines exhibited different degrees of color change,including pale pink to almost white,light pink and pink.The content of anthocyanins in the corolla of OE-MaFLS lines was significantly lower than that of in the control,while the accumulation of total flavonols was significantly increased in OE-MaFLS lines.qPCR analysis showed that the expression patterns of NtCHS,NtF3H,NtDFR,NtANS and NtAN2 were significantly decreased in the transgenic lines,While the expression of NtFLS gene was significantly up-regulated.In summary,MaDFR altered metabolic flux flowing to the accumulation of blue delphinidin by preferentially catalyzing dihydromyricetin;Competed with MaDFR,MaFLS catalyzed common substrates(dihydroflavonol)to form the copigmentation-flavonol,MaDFR and MaFLS worked together to regulate the blue flower development in grape hyacinth.