Cloning,Tissue Expression Analysis And Enzymatic Function Identification Of FtFLS4 And FtFLS5 From Fagopyrum Tataticum

As a kind of food and medicine plant,tartary buckwheat(Fagopyrum tataricum)is rich in rutin compound.Synthase synthase(Flavonol Synthase,FLS)is a key enzyme in the biosynthesis of flavonols,can catalyze the formation of quercetin,kaempferol and myricetin;the quercetin and kaempferol would be further glycosylated into rutin.In this study,flowering plants of tartary buckwheat as research materials,using RT-PCR molecular biology technology,successfully obtained FtFLS homologous gene,and predict its function by using biological information technology.The expression of FtFLS in Escherichia coli was studied by using prokaryotic expression technology;using fluorescence quantitative PCR technology,analyzed the expression of FtFLS Gene in the stem,leaves and flower of tartary buckwheat in transcription level;The content of flavonols in stems,leaves and flowers of flowering buckwheat was determined by HPLC;using ultraviolet spectrophotometry technique to compared the enzymatic activity of FtFLS on three kinds of substrates,analyzed the optimal substrate in the synthesis of rutin of tartary buckwheat.The main results of this study are as follows:1.Using RT-PCR technology,we successfully obtained the ORF sequence of flavone synthase from tartary buckwheat,named FtFLS4 and FtFLS5.The cDNA of FtFLS4 gene are 1026bP in length,encoding 341 amino acids.The theoretical molecular weight of 39.2 kDa,predicted isoelectric point(pI)was 5.79,the molecular formula is C1777H2739N463O515S11;the cDNA of FtFLS5 gene are 1068bP in length,encoding 355 amino acids.The theoretical molecular weight of 40.085 kDa,isoelectric forecast point(pI)was 7.18,the molecular formula C1816H2821N487O514S12;The results of molecular identification showed that the obtained sequence is a member of the 2-ODD protein family,which is the homologous gene of FLS from Tartary buckwheat.2.We constructed prokaryotic expression plasmids of FtFLS4-pET-30(b),FtFLS5-pET-30(b)and expressed them in E.coil,detected two about 45kDa fusion proteins respectively by electrophoresis,which consistent with predicted molecular weight;The enzyme activity of purified protein using affinity chromatography,was determinded by TLC and UV spectrophotometric,the results showed that FtFLS4 and FtFLS5 proteins can transform flavanonols(dihydroquercetin,dihydrokaempferol and dihydromyricetin)into flavonol;the total enzyme activity of FtFLS4 is 0.630×10-3IU,1.2×10-3IU and 0.476×10-3IU;FtFLS5 is 0.602×10-3IU,0.981×10-3IU and 0.303×10 3IU,respectively.3.The content of flavonols in stems,leaves and flowers of flowering buckwheat was determined by AICl3 and HPLC,the results show that the highest content of total flavonoids in the flower bud of tartary buckwheat is 5.47%(DW),in leaves is 2.67%(DW),and in stems is 0.17%(DW);Myricetin exists only in leaf(0.32mg/g)and flowers(0.23 mg/g);Quercetin exists only in leaves(0.08 mg/g)and flowers(5.85 mg/g);the higher content of kaempferol in leaves(0.40 mg/g),the low content of kaempferol in flowers(0.07 mg/g)and stem(0.05 mg/g).Fluorescent quantitative PCR was used to detect the expression of five FtFLS homologous genes in the corresponding tissues.The results showed that five FtFLS homologous genes were expressed in the stems,leaf and flower of tartary buckwheat,and there exhibited tissue specificity.In the stem,the expression of FtFLSl and FtFLS3 is the highest,FtFLS2 and FtFLS4 is lower,almost no expression of FtFLS5;in the leaves,the expression level is:FtFLS3>FtFLSl>FtFLS2>FtFLS4>FtFLS5;in the flower,the expression level is FtFLS3>FtFLS1>FtFLS2>FtFLS4>FtFLS5.SPSS Statistics was used to analyze the correlation between flavonol content and the relative expression of FtFLS,the results show that the content of rutin with FtFLSl and FtFLS5 are highly correlative and positively correlated,with FtFLS2,FtFLS3,FtFLS4 are lower correlative;the content of myricetin with FtFLS2 and FtFLS4 are highly correlative and positively correlated,with FtFLSl,FtFLS3 and FtFLS5 are lower correlative;the content of quercetin with FtFLS1 and FtFLS5 are highly correlative and positively correlated,others are negatively correlated;the content of kaempferol with FtFLS2,FtFLS3 and FtFLS4 are highly correlative and positively correlated,others are negatively correlated,others are negatively correlated.