Cloning,Expression And Characterization Of Key Genes Of GbUFGT And GbFOMT In Flavonoid Biosynthesis Of Ginkgo Biloba L.

Flavonoid,one of the most important plant secondary metabolites,not only plays important roles in the growth,development and defense of plants,but also has many pharmaceutical properties for human being.Ginkgo biloba,a precious relic plant with tenacious vitality for a long history,is native to China,not only is one of the peculiar treasure gymnosperm species,but also has the much important economic,social,ecological and ornamental values.In the leaf-purpose of which,the leaves have been used to extract the active ingredients such as flavonoid,ginkgolides,bilobalid and polyprenols etc.,and maked to a preparation of EGb?Extract of Ginkgo biloba?which were further used to manufacture to the medicines and health care products for treatment of cardiovascular and cerebrovascular diseases such as hypertension,dementia,vertigo,tinnitus,peripheral vascular disorders and peripheral arterial occlusive etc.In Schwabe Pharmaceuticals of Germany the content of flavonoids in EGb761 has been highly required to reach at 24%,so it has been designated as the key international quality index for the active ingredients preparation products of Ginkgo.Glycosylation and O-methylation are two of the most important modification reactions after the basic structure being formed of flavonoid,regulating the characteristics of solubility,regional distribution,chemical stability and biological activity of flavonoid in plant tissues and cells,and playing important roles in flavonoid biological metabolism and the improvement of flavonoid content by biological techniques.At present,many plant UFGT and FOMT genes have been cloned,but there has been no report about ginkgo flavonoid glycosylation and O-methylation.It is the first time to study the ginkgo flavonoid glucosyltransferase and flavonoid O-methyltransferase in this paper.The main contents and results in the present study are as follows:1.The determination of total flavonoid glycoside,quercetin and isorhamnetin contents in ginkgo leaf.The results of contents determination by high-performance liquid chromatography?HPLC?showed that:quercetin,kaempferol and isorhamnetin are the three major constituents of ginkgo flavonoid aglycones,in which the contents of quercetin and kaempferol are high,and that of isorhamnetin is far lower;With March 25th as the initiation of ginkgo leaf bud break,the content of flavonoid glycoside increases gradually in 30-210 days after leaf bud break?DALB?,with 150 and 210 DALB as the 2 peaks,and accumulating rapidly during 30 to 60 DALB and 150 to 210 DALB;The content of isorhamnetin varifies differently from both of quercetin and total flavonoid glycoside accumulating patterns,with little chang during 30 to 90 DALB,and with 120 and 210 DALB as the 2 content peaks,150 DALB as the lowest period.2.The full length cDNA and genomic DNA sequences of flavonoid glycosyltransferases gene were isolated from G.biloba for the first time?designated as GbUFGT;GenBank Accession No.JN640564.2?.GbUFGT was not an interrupted gene,with no intron in its genomic DNA sequence.And the full length of GbUFGT cDNA sequence was 2016 bp long with a 1491 bp long open reading frame?ORF?.The deduced protein of GbUFGT?GenBank Accession No.AEQ33588.2?including 496 amino acids,with predicted molecular weight of 55 kDa,pI of 6.05,instability index???of 57.04,and grand average of hydropathicity of-0.127,classified GbUFGT as unstable and water soluble protein.The results of BLASTn?BLASTp and multi-alignment showed that GbUFGT with a typical conversed plant secondary product glycosyltransferase?PSPG?motif,had high identities to other plant UFGTs.3.The full length cDNA sequence of flavonoid O-methyltransferase gene from G.biloba?designated as GbFOMT;GenBank Accession No.JN222561?was 1397 bp long with a 1089 bp open reading frame?ORF?.And the genomic DNA sequence of GbFOMT gene was 1966 bp long,including three exons and two introns which both obeyed the GT/AG rule.The molecular weitht and pI of the deduced GbFOMT protein?GenBank Accession No.AER35881.1?,including 362 amino acids,were 39.74 kD and 5.90,respectively.And the instability index and grand average of hydropathicity of GbFOMT were 37.50 and-0.043 respectively,classifying GbFOMT as stable and water soluble protein.Sequence alignment showed that the deduced GbFOMT,with a dimerisation domain?pfam08100?in its N-terminus,and O-methyltransferase domain?Methyltransf₂,pfam00891?in its C-terminus,had relatively high identities to other plant OMTs and FOMTs.4.Phylogenetic tree analysis revealed that GbUFGT and GbFOMT shared common evolutionary ancestors with UFGTs and FOMTs from other plants,respectively.GbUFGT is closely related to UFGTs from Vitis vinifera and Prunus persica.Plant FOMTs could be clustered into two groups,in which the first group?group ??uses flavonoid as substrate while most of the second group?group ??uses isoflavonoid as substrate.And GbFOMT belongs to group ?,which uses flavonoids as substrate.5.The secondary structure elements of GbUFGT and GbFOMT both include a-helix,?-sheet and randon coil.3D structure modeling of GbUFGT which belonged to the GT-B fold,showed that the spherical structure of GbUFGT with typical N-domain and C-domain,was similar with that of VvGTl from Vitis vinifera.GbFOMT may be homo-dimeric in ginkgo cells,and the 3D structure of its monomer with obvious N-terminal dimerisation domain and C-domain is similar with caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase?COMT;PDB No.1kyzC?from alfalfa.6.Both GbUFGT and GbFOMT are water soluble proteins,leading to little significant transmembrane helices in ginkgo.WoLF PSORT analysis revealed that GbUFGT might be located in chloroplast and extracellular,while GbFOMT in cytosol of ginkgo cells.7.Gene family analysis indicated that GbUFGT belonged to a multi-gene family,while GbFOMT belonged to a single copy gene or small multi-gene family.The gene copies of GbFOMT was less than that of GbUFGT.8.Semi-quantitative RT-PCR analysis revealed that GbUFGT and GbFOMT expressed at the whole developmental stages of ginkgo leaf,but in different periods when flavonoid glycoside content changed greatly,the expression levels of GbUFGT were similar,which is different from those of GbFOMT.GbFOMT was a developmental stage-specfic gene,in which the transcription levels were significantly correlated with quercetin accumulation rates,suggesting that the expression of GbFOMT might be regulated by quercetin in leaves of G.biloba.9.The in vitro enzyme activity assay by HPLC indicated that GbFOMT enzyme was highly regiospecific,catalyzing methylation only on the 3'-hydroxyl of flavonoid,and the methylation activity was strongly influenced by substituents on C-3 and C-3' positions of flavonoid substrates,classifying GbFOMT as 3'-FOMT.Ions influence assay showed that Mg2+ ions or other metal ions were not necessary for the enzyme activity of GbFOMT.Results of this paper not only provide a new feasibal theory and engineering technology to improve flavonoid contents,but also can be directly applied to select strains which have the high yield of ginkgo flavonoid.Therefore,these results have important roles in research and application.