Cloning And Functional Identification Of Flavanone 3-hydroxylase Gene And Its Promoter From Tartary Buckwheat

Tartary buckwheat（Fagopyrum tataricum）,a kind of cereal rich in nutrients and flavonoids（mainly rutin）,is widely used as a food and medicine for healthcare.Recently,the flavonoid synthesis and metabolism regulation in tartary buckwheat is a hot spot of concern and has been made a significant progress.As the foundation of molecular regulation,almost genes of the key enzymes in flavonoids biosynthesis pathway have been cloned and functioned.However,flavanone 3-hydroxy-lase（F3H）gene located in the centre of flavonoids biosythensis is still unclear about its enzymatic activity,expression regulation and influence on metabolic flux.In this study,FtF3H gene was cloned from tartary buckwheat variety“Xi Qiao No.2”,and its molecular characterizations was analyzed bioinformatically.Then,the promoter of FtF3H gene was cloned,and its response to environmental factors and hormones was checked through transgenic Arabidopsis thaliana.After FtF3H protein was expressed in E.coli,its catalytic activity was qualitatively detected.Furthermore,the effects of FtF3H gene on flavonoids biosynthesis were explored in transgenic Arabidopsis.The main research results are as follows:1.Based on the genomic and transcriptome data,the DNA and ORF sequences of FtF3H gene were successfully cloned from tartary buckwheat.Bioinformatics analysis showed that FtF3H gene DNA sequence was 1768 bp in length,including 2 introns and 3exons.Its ORF sequence was 1104 bp in length,encoding 367 amino acid residues.Its encoding protein FtF3H was classified into Fe（II）-oxygenase superfamily because it had the highly conserved 2OG-Fe II-Oxy and Pcb C classical domains.Moreover,sequence alignment indicated that it was clustered with the other F3Hs from plant.Additionally,the molecular docking analysis revealed that only naringin could be used as the substrate for F3H protein.2.According to the genomic data,a FtF3H promoter sequence,named P_FtF3H,was successfully amplified with 2000 bp in length,P_FtF3Hwas analyzed at Place Care website.The predication results indicated there were not only the basic elements of promoter but also many light response elements and hormone response elements induced by Me JA and ABA in P_FtF3H.Then,the effects of P_FtF3H on transcription activation were evaluated through detecting the GUS report gene expression in response to Me JA,ABA and light treatment in P_FtF3H::GUS transgenic Arabidopsis.The fluorescence quantitative PCR analysis indicated Me JA and ABA had significant inhibitory effect on GUS expression（P<0.05）while light could obviously induce GUS expression（P<0.05）.Generally,the above results were in accordance with the histochemical GUS staining.3.FtF3H expression levels were detected by q-PCR after tartary buckwheat sprouts were treated with Me JA,ABA and light.The results showed that FtF3H expression was remarkably repressed by Me JA and ABA（P<0.05）,and was tending towards stability after arriving at the lowest with 1.5h treatment.On the contrary,light had a prominent induction effect on FtF3H gene expression（P<0.05）,and its expression reached the peak value which was about twice as much as that of the control group 1h later.4.Through prokaryotic expression vector construction for FtF3H ORF,soluble FtF3H protein was expressed successfully in E.coli.Unfortunately,the crude FtF3H protein showed no visibly catalytic activity in vitro.However,overexpression FtF3H could promote the total flavonoids content in transgenic Arabidopsis,which was higher about2.83-fold than that in wild type.The expression analysis of flavonoids biosynthesis related key genes figured out six genes,including At CHS,At CHI,At DFR,At F3’H,At FLS and At ANS,were prominently up-regulated at transcription level（P<0.05）,while At F3H expression was obviously decreased（P<0.05）...