Study On Cloning And Functional Identification Of Key Genes For Biosynthesis Of Flavonoids Of Ampelopsis Megalophylla

Ampelopsis megalophylla Diels et Gilg is a characteristic medicinal plant in Enshi,Hubei province,China.The processed products of its tender leaves and tender stems are locally known as “Mei Cha”.At present,the medicinal resources of A.grossedentata mainly come from the wild,and there are some differences in the quality of the plants from different sources.It is well known that the quality of medicinal plant resources is directly related to the accumulation of active secondary metabolites.Therefore,in order to control the quality of its resources,the biosynthesis pathway of active components should be elucidated from the perspective of molecular biology.At present,the cloning and functional identification of key enzyme genes in biosynthesis of the main active components of A.grossedentata have not been reported.The key genes for the biosynthesis of flavonoids,the active constituents of the plant,have been screened on the foundation of previous transcriptome data.The genes of F3',5'H(flavonoid-3',5'-hydroxylase),LAR(leucoanthocyanidin reductase),F3H(flavanone-3-hydroxylase)were cloned and the functional identification of F3',5'H was also been illustrated.These results provide the basis for elucidating the molecular mechanism of biosynthesis of the active components of A.megalophylla and improve the quality of medicinal plant resources from the genomic level in the future.The specific research contents and results are as following:(1)In this paper,F3',5'H,F3 H and LAR genes were cloned by RT-PCR on the basis of previous studies and the full-length was 1931 bp,1318 bp,1267 bp respectively.Multiple sequence alignment showed that amino acid sequence of F3',5'H had PPGP,AGTDT and FGAGRRICAG domains,F3 H had 2OG-Fe?_Ox domain,and LAR had GXXGXXG motif,RFLP,ICCN,THD domains.Bioinformatics analysis showed that the amino acid sequences encoded by the three genes were similar to them in grapes.They converged in one branch of the phylogenetic tree,suggesting that the catalytic activities of F3',5'H,F3 H and LAR were similar.(2)In this paper,the content of main flavonoids(dihydromyricetin and myricetin)in the leaves,flowers and fruits of A.megalophylla was determined by HPLC.Meanwhile,the expression levels of F3',5'H and F3 H genes were measured by real-time fluorescence quantitative PCR(RT-qPCR).The correlation analysis between gene expression and flavonoids accumulation showed that only F3',5'H was positively correlated with the accumulation of dihydromyricetin and myricetin,which indicated that F3',5'H was the key gene for the dihydromyricetin and myricetin formation.(3)In this paper,the recombinant plasmids of F3',5'H and the prokaryotic expression vector pET30 a were constructed by seamless cloning technology,and the prokaryotic expression of host bacteria BL21(DE3)competent cells were transformed.IPTG was added to induce the expression of fusion proteins.The molecular weight of F3',5'H protein was 54.93 KDa.The enzymatic reaction system constructed in vitro verified that F3',5'H could catalyze dihydroquercetin to dihydromyricetin,indicating that F3',5'H had catalytic activity in vitro.