Functional Study Of Flavonol Synthase Homologous Genes FtFLS1-5 From Tartary Buckwheat

Tartary buckwheat（Fagopyrum tataricum（L）Gaertn.）is a kind of homology of medicine and food miscellaneous grain crop,and its main quality character and economic index are rutin content.Flavonol synthase（FLS）and its homologous genes are key enzymes and limiting factors for the synthesis and accumulation of Tartary buckwheat.The transcriptional intensity and catalytic properties of the FLS genes encoded products are the important reasons for the difference of species and content of Tartary buckwheat flavonols.In this study,five Tartary buckwheat flavonol synthase gene Ft FLS1-5 were used to analyze the correlation between the temporal and spatial expression patterns of Ft FLS1-5and the content of flavonoids in Tartary buckwheat during the 7 stages.The biological activity of Ft FLS1-5 in Arabidopsis was identified by transgenic technique,and their effects on the expression of flavonoids synthesis related genes and metabolite accumulation in Arabidopsis were analyzed.The recombinant Ft FLS1-5 proteins were isolated and purified by prokaryotic expression affinity chromatography and their catalytic characteristics in vitro were determined by HPLC technique.This study preliminarily clarified the molecular characteristics of Tartary buckwheat Ft FLS1-5,identified Ft FLS1-5activity in Arabidopsis,and clarified the flavonol synthase major gene of Tartary buckwheat flavonols synthesis branch.This study provides theoretical basis and basic materials for molecular breeding of Tartary buckwheat with high rutin in the future.The main results were as follows:1.According to the transcription and genome sequencing results in public data bases,there were five FLS homologous genes in Tartary Buckwheat genome,named Ft FLS1-5.Molecular identification results showed that Ft FLS1 and Ft FLS2 were located on chromosome 7 of tartary buckwheat genome and belonged to tandem repeat sequence,Ft FLS3 was located on chromosome 2,Ft FLS4 was located on chromosome 3,and Ft FLS5was located on chromosome 1.Sequence analysis showed that Ft FLS1,Ft FLS2,Ft FLS3and Ft FLS5 contained three exons and two introns,while Ft FLS4 contained two exons and one intron.Ft FLS1-5 had a 75.7%homology in exon sequence,while the number,size,location,and base sequence of introns varied greatly,indicating that the FLS homology genes had differences in evolution and functional differentiation in Tartary Buckwheat.The phylogenetic tree,basing on the flavonoid related 2-ODD proteins from Tartary Buckwheat,showed that Ft FLS1 and Ft FLS3 had the closest phylogenetic relationship and clustered with typical plant FLS,Ft FLS2 had a relatively close evolutionary relationship with anthocyanin synthase（ANS）,Ft FLS5 and Flavanone 3-hydroxylase（F3H）belonged to one group,and Ft FLS4 was independent of all branches and related to other 2-ODDs.2.In order to understand the expression of Ft FLS1-5 in flavonoid accumulation,the flavonoid contents in 7 stages of tartary buckwheat were detected.Results showed that rutin was the main flavonoids in Tartary Buckwheat and its average content were 1.22%in leaves,46%in flowers,and 1.10%in seeds,while the roots（0.044%）and stems（0.12%）had lower accumulation.In sprout stage,anthocyanins had the highest amount（0.19%）in the stems,however,in the 7 other stages it presented a trace content.The results of q RT-PCR showed that the expression of Ft FLS1-5 had tissue-specific patterns during 7growth stages.The expression of Ft FLS1 increased sharply with the differentiation of reproductive organs,and reached the peak in immature seeds of Stage 5.The expression of Ft FLS2 and Ft FLS4 decreased slightly at maturity.The expression patterns of Ft FLS2 and Ft FLS4 were similar,mainly expressed in roots and stems of Tartary Buckwheat sprouts before the undifferentiated reproductive organs（Stage 1-2）,and in flowers after the reproductive stage.The expression of Ft FLS3 and Ft FLS5 in buckwheat seeds was not significant（P>0.05）,but the expression of Ft FLS3 and Ft FLS5 in all parts of the whole growth period was not significant（P>0.05）.Statistical analysis results show that the expression pattern of Ft FLS1 was positively correlated with total flavonoids and rutin content,it negatively correlated with anthocyanin content in the 7 stages of Tartary buckwheat.There was no significant correlation between Ft FLS2 and Ft FLS4 expression and total flavonoids,rutin and anthocyanin content.The expression of Ft FLS3 and Ft FLS5were positively correlated with total flavonoids and rutin contents,but not with anthocyanin contents.3.The plant expression vector p CAMBIA1301-Ft FLS1-5 were constructed and transformed into Arabidopsis thaliana using the Agrobacterium tumefaciens strain GV3101.Transgenic plants of T3 generation were obtained by screening resistance plate and PCR.Phenotypic identification showed that the transgenic plants bolted earlier than wild-type plants and were taller than wild-type plants before flowering.In transgenic Arabidopsis,the overexpression of Ft FLS1-5 resulted in a significant increase in total flavonoids and rutin content（P<0.01）,the overexpression of Ft FLS1 resulted in a significant increase in both rutin and anthocyanin contents（P<0.05）,the overexpression of Ft FLS5 resulted in a significant decrease in anthocyanin content（P<0.05）,and the overexpression of Ft FLS2-4 had a similar anthocyanin content to that in wild type.In transgenic lines of Ft FLS1,the expression of endogenous flavone synthesis related genes,including At PAL,At C4H,At4CL,At CHS,At CHI,At F3H,At F3’H,At DFR and At ANS,were significantly up-regulated（P<0.05）.In transgenic lines of Ft FLS2,the expression of endogenous At CHS and At FLS were significantly inhibited（P<0.05）.In transgenic lines of Ft FLS3,At PAL,At C4H,and At4C were significantly down-regulated in Arabidopsis with Ft FLS2 overexpression（P<0.05）;At PAL,At C4H,At4C were significantly down-regulated in Arabidopsis with Ft FLS3 overexpression.The expression of L,At CHS,At CHI,At F3’H,At F3H and At FLS genes increased significantly（P<0.05）;the expression of At PAL,At C4H,At4CL,At CHS,At CHI,At F3H and At FLS genes increased significantly（P<0.05）,while the expression of At F3’H,At DFR and At FLS genes was significantly inhibited（P<0.05）;the expression of endogenous flavonoids in Ft FLS5 overexpressed Arabidopsis was significantly inhibited（P<0.05）.The changes of synthesis-related genes were similar to those in Ft FLS4 over-expressed Arabidopsis thaliana.The expressions of At PAL,At C4H,At CHS,At CHI,At F3H and At FLS genes were significantly increased（P<0.05）.,while the expressions of At F3’H,At DFR and At FLS genes were significantly inhibited（P<0.05）.Transgenic results showed that Ft FLS1-5 of Tartary Buckwheat had biological activity in Arabidopsis thaliana,and its translation products could induce the synthesis and accumulation of different flavonoids in Arabidopsis thaliana.4.The recombinant plasmids p ET-30（b）-Ft FLS1-5 were constructed and transformed into the expression strain Escherichia coli BL（DE3）.By IPTG inducing,the recombinant proteins of Ft FLS1-5 were obtained,they showed a molecular weight of 45 k Da and appeared as a soluble form.The affinity chromatography was performed to purify the recombinant Ft FLS1-5 proteins,and then their in vitro activities were determined by HPLC.The results showed that the 5 recombinant proteins could catalyze dihydroquercetin to quercetin.The specific activity of Ft FLS1 was the highest(60.79×10^-3 IU/mg),and that of the other 4 proteins ranged from 2.56 to 17.46×10^-3 IU/mg.Importantly,our data showed that Ft FLS1 and Ft FLS3 could catalyze dihydromountain,however,only Ft FLS1 can catalyze dihydromyricetin to myricetin with a specific activity of 19.11×10^-3 IU/mg.These results strongly suggested that Ft FLS1 might play a leading role in flavonol synthesis and metabolism.In conclusion,there were five FLS homologous genes（Ft FLS1-5）in Tartary Buckwheat genome.Ft FLS1-5 exhibited different gene structures,molecular features and biological functions.The expression pattern of Ft FLS1 showed the highest product correlation in Tartary Buckwheat growth stages,in vitro and in vivo activities of Ft FLS1suggested that it was the main functional gene among Ft FLSs in flavonol synthesis and played a key role in rutin metabolism pathway of Tartary buckwheat.Ft FLS3 might be the functional redundancy and backup gene of Ft FLS1.Ft FLS2,Ft FLS4 and Ft FLS5 might participate in the synthesis of other flavonoids in Tartary buckwheat,and their expression might be induced by internal and external environments.