Cloning And Functional Identification Of Flavonol Synthase Homologous Gene Promoters From Tartary Buckwheat

Tatary buckwheat?Fagopyrum tataricum?L.?Gaertn?,classfied into the family of polygonaceae and genus of Fagopyrum,is an excellent medicinal and nutrient-rich crop containing abundance of rutin?quercetin-3-O-rutin?.Rutin accounts for 70%-85%of total flavonoids in tartary buckwheat and is mainly distributed in leaves?3%?and seeds?0.8%-1.7%?,regarding as the main quality character and economic indicator for this crop.The biosynthesis pathway of rutin belongs to the downstream of the metabolic pathways of plant flavonoids,which is mainly regulated by key enzymes and transcription factors.Flavonol synthase,the key enzyme to catalyze the synthesis of rutin in plant,could catalyze flavanonols to flavonol,subsequently quercitrin a kind of flavonols would be further glycosylated into rutin.Accordingly,the transcriptional level of flavonol synthase gene could contribute to the synthesis and accumulation of rutin.In this study,"Xiqiao No.2"a tartary buckwheat variety with high flavonoid content,is used as experimental material.Based on the transcriptome data of the tartary buckwheat,three FtFLSs homologous genes were cloned successfully.The expression of FtFLSs and the content of flavonol in tissues of tartary buckwheat were detected by qRT-PCR and HPLC respectively,and the correlation between them was analyzed furtherly.The P_FtFLSstFLSs were cloned by chromosome walking technique,and its basic structure was predicted by bioinformatics.The basic activity and function of P_FtFLSs was analyzed by transient transfection and stable transformation in plant.The expression levels of FtFLSs or GUS driven by P_FtFLSs were investigated under abiotic stresses,which contribute a lot to reveal the molecular regulation mechanism of environmental factors on FtFLSs.The main results of this study were as follows:1.Through PCR technology,three FtFLSs including FtFLS1,FtFLS2 and FtFLS3,were cloned successfully,and their lengths were 2721bp,1069bp and 2074bp,respectively.The gene structure analysis showed that they contained 2,1 and 2 introns,respectively,and all the intron sites fitted with the GT-AG splicing mode.2.Expression level of FtFLSs in different tissues of flowering buckwheat were analyzed.The results showed that FtFLS1 was highly expressed in all tissues,and FtFLS2 was highly expressed in roots and flowers,while FtFLS3 expression was low in all tissues.HPLC analysis showed that the quercetin and rutin were major compounds detected in all tissues,and myricetin was accumulated mainly in leaves,instead a little kaempferol was distributed in all tissue.SPSS analysis showed that the FtFLS1expression had a significant positive correlation with the content of quercetin,myricetin and rutin?P<0.01?,and a significant negative correlation with the kaempferol quantity?P<0.01?.By contrast,the expression of FtFLS2 and FtFLS3 were negatively correlated with the content of quercetin,myricetin and rutin,and positively correlated with the content of kaempferol,in which expression of FtFLS3 always showed a significant correlation?P<0.01?.Under cold or UV-B treatment,FtFLS1 expression was significantly increased?P<0.01?,and the expression of FtFLS2 was significantly reduced?P<0.01?;By contrast,expression of FtFLS3 showed different pattern that is decreased or increased significantly?P<0.01?under cold or UV-B treatment,respectively.Morover,all FtFLSs expression was promoted significantly after the drought treatment?P<0.01?.3.The 5'flanking sequences of FtFLS1,FtFLS2 and FtFLS3 were obtained by chromosome walking,and the lengths are 2479bp,2574bp,and 2046bp,respectively.PLACE and Plant CARE analysis showed that the fragment of FtFLS1,FtFLS2 and FtFLS3,5'flanking sequences within their 1874bp,1728bp and 1836bp,respectively,contained a large number of TATA-Box,CAAT-Box,GC-Box,MBS,ABRE and G-Box cis-acting elements,and so on.The predicted functional 5'flanking sequences of FtFLSs were assembled with reporter gene GUS to plant expression vectors and transiently transformed into tobacco young leaves;the histochemical staining result showed that all the sequences,named as P_FtFLS1,P_FtFLS2 and P_FtFLS3,had the ability to drive expression of GUS.4.After the plant expression vectors of P_FtFLS1,P_FtFLS2 and P_FtFLS3 were transformed into Arabidopsis thaliana respectively,their temporal and spatial specificity,together with their response to environmental factors were analyzed.Histochemical staining and qRT-PCR results showed that GUS2(GUS derived by P_FtFLS2)was highly expressed in roots of Arabidopsis seedling stage,while expression of GUS1(GUS derived by P_FtFLS1)and GUS3(GUS derived by P_FtFLS3)were almost not detected.At the flowering stage,GUS1 and GUS3 expression was increased sharply.Particularly,GUS1 expression was significantly higher than the others?P<0.01?in all tissues.In detail,GUS1 was highly expressed in stems,leaves and flowers of Arabidopsis,while GUS2 was only expressed in roots and flowers,and GUS3 was highly expressed in leaves and flowers.After cold and UV-B treatment on transgenic Arabidopsis,the expression of GUS1 and GUS3 was increased significantly?P<0.01?,and GUS2 expression did not show a significant change.After drought treatment,all the GUSs expression levels were increased significantly?P<0.01?.Taken together,the space-time specificity of FtFLSs promoters could determine that of target genes.Meanwhile,promoter response to the environment change is an crucial way to regulate FtFLSs experssion.In this study,not only some details was clarified about the key steps in rutin synthesis,but also the theoretical guidance was provided for improving rutin synthesis by controlling environmental factors.