| Ophiorrhiza japonica（O.japonica）,a perennial herb,belongs to the genus Ophiorrhiza,and its whole plant can be used as a medicinal material.O.japonica has significant effects in many aspects,such as promoting blood circulation,disperding blood stasis,removing phlegm,regulating menstruation and stopping bleeding.Its corolla is white,shaped like a funnel with cylindrical stems,mainly distributed in southern China.There are many important secondary metabolites in plants,flavonoids are one of them.Anthocyanins,the end product of flavonoid synthesis pathway,play an irreplaceable role in the formation of flower color.Dihydroflavonol 4-reductase（DFR）,an important regulatory enzyme in anthocyanin biosynthesis pathway,is responsible for the catalytic reduction of dihydroflavonol to colorless anthocyanidins.Subsequently,the colorless anthocyanidins were converted in to anthocyanins through the catalysis of anthocyanin synthase and flavonoid glycosyltransferase.Analysis of anthocyanin components showed that there were four main anthocyanins in O.japonica: cyanidin 3-glucoside,peonidin derivatives,cyanidin 3-rutin and petunia derivatives,but pelargonidin derivatives were not detected.Quantitative analysis showed that the content of total anthocyanin decreased gradually during flowering process,and the content of cyanidin 3-rutin was the highest among the above four anthocyanins.For different tissues,the contents of anthocyanin were the highest in sepals,the lowest in leaves,but not detected in roots.Previous studies have shown that the presence or absence of pelargonidin derivatives in plants is due to the different substrate selectivity of DFR.Therefore,based on the identification of anthocyanins in O.japonica,DFR gene was studied in this paper.Firstly,three DFR genes（OjDFR1/OjDFR2/OjDFR3）were successfully cloned from O.japonica.Subsequently,bioinformatics analysis,expression level analysis,enzyme activity detection in vitro and genetic transformation of Arabidopsis thaliana and tobacco were performed to verify the functions of these three genes.The main results are as follows:1.Qualitative and quantitative analysis of anthocyanin of O.japonicaHPLC analysis of anthocyanins in the petals of O.japonica showed that there were four kinds of anthocyanins detected in its petals: cyanidin3-glucoside,peonidin derivatives,cyanidin 3-rutin and petunia derivatives.During flower development,the accumulation of anthocyanin decreased gradually,and the content of cyanidin 3-rutin was always at the highest level.2.Bioinformatics analysis of OjDFRsBioinformatics analysis showed that the open reading frame length of OjDFR1,OjDFR2 and OjDFR3 were 1011 bp,1071 bp and 1074 bp,encoding 336,356 and 357 amino acids,respectively.The predicted molecular weights were 36.96 KD,39.16 KD,39.27 KD,and the isoelectric points were 6.08,5.09,5.08,respectively.OjDFR1 and OjDFR3 were stable proteins,while OjDFR2 was unstable.In addition,these three proteins all have highly conserved NADP binding motifs and substrate binding domains at their N-terminal,and their 134 th amino acid residues are Asn,Thr and Thr,respectively.The three OjDFRs proteins were all composed of 20 amino acids without signal peptides.Phylogenetic analysis showed that OjDFR1 was most closely related to Nicotiana tabacum DFR,OjDFR2 and OjDFR3 were most closely related to Matthiola incana and Ipomoea batatas Poir DFR respectively.3.Expression analysis of OjDFRsThe results of expression analysis showed that the three genes showed significant spatial and temporal specificity.During flower development,the expression of OjDFR1 reached the peak at the first stage and then decreased gradually.OjDFR2 and OjDFR3 were also expressed highest at stage 1,but their transcription levels were not significantly correlated with anthocyanin accumulation patterns.Subsequently,the expressions of three genes were examined in different tissues.The results revealed that these three genes were expressed in all tissues,but OjDFR1 was mainly expressed in flower,stem,leaf,scape and calyx,while OjDFR2 was mainly expressed in stem,scape and calyx,and OjDFR3 was mainly expressed in root,stem,leaf,scape and calyx.Furthermore,the expression of OjDFR1 in scape and calyx that accumulates higher anthocyanin was significantly higher than that of OjDFR2 and OjDFR3.These data suggest that OjDFR1 may play a key role in anthocyanin biosynthesis in O.japonica.4.Construction of prokaryotic expression vectors of OjDFRs and detection of enzyme activity in vitroOjDFRs were respectively inserted into pET-32a（+）vector and the recombinant plasmid was transferred into BL21（DE3）for inducing the expression of soluble recombinant protein.Recombinant protein with good purity and concentration was obtained by preparation,dialysis and concentration.Then,the activity of OjDFRs was determined by HPLC using dihydroquercetin（DHQ）,dihydrokahanol（DHK）and dihydromyricetin（DHM）as substrates.The results displayed that only OjDFR1 had the catalytic activity of DFR,and could effectively catalyze the formation of Leucocyanidin and Leucodelphinidin,but had no catalytic activity towards DHK,while OjDFR2 and OjDFR3 have no catalytic activity towards above three substrates.5.Construction of eukaryotic expression vector of OjDFRs and their genetic transformationOjDFRs was inserted into pBI121 vector and transferred into GV3101 competent cells.The recombinant strains were then transferred into Arabidopsis thaliana tt3-1 mutant.Results showed that only OjDFR1 could successfully restore the anthocyanin synthesis of Arabidopsis thaliana tt3-1 mutant,while OjDFR2 and OjDFR3 could not.In order to verify whether OjDFR1 can affect the formation of flower color in plants,this gene was overexpressed in tobacco.The results showed that the petals of transgenic tobacco were darker than that of the wild type,and anthocyanin accumulation was also significantly increased in transgenic tobacco,suggesting that OjDFR1 could regulate the synthesis of anthocyanin in tobacco and affect the presentation of flower color. |
PDF Full Text Request