Molecular Cloning And Primary Functional Analysis On FcIRL In Fagopyrum Cymosum(Trev.)Meisn

Fagopyrum cymosum(Trev.)Meisn,one of perennial herbs of the genus Fagopyrum in the family Polygonaceae,is an important traditional Chinese medicinal materials,with rich nutrition and medicinal values.The active extract of rhizome of F.cymosum is one of main components of many efficient drugs,which has prominent functions in anti-cancer,controlling tumour cell affect and transfering to lung,and anti-inflammatony antibacteria ect..The rearch up to now focuses on analysis of nutrition ingredient and chemical composition,study on pharmacodynamics,and antitumor mechanism.However,the rearches on the key enzymes involved in the secondary metabolism pathway is noly about the cloning characterization of dihydroflavonol-4-reductase gene(DFR)and a transcription regulator gene(P).There hasn't been any report about the others structural genes and regulatory genes.With the purpose of studying more about the medicinal secondary metabolism,we has been successfully cloned and isolated the presumed isoflavone reductase-like gene(FcIRL),analysed the characterization about bioinformatics to forecast its function elementarily,constructed the sense expression vector.Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens LBA4404(containing pC2301-FcIRL)recombinant plasmid were transformed into model plants,tobacoo and petunia.The full-length cDNA of the FcIRL gene was 1217 bp long(accession no.EU116032)and contained a 942 bp open reading frame(ORF)encoding a 313 amino acid protein.Two stop codons(TAG)were found in 5'UTR and a putative polyadenylation signal ATAAA at 24 bp upstream from the polyadenylation site was found in 3' UTR.There is no any intron in the genomic sequence.The FcIRL contained a predicted N-terminal acetylation site(M₁-K₅)and a NADPH-binding motif(G₁₀-G-T-G₁₃-Y-I-G₁₆)in the N-terminal region,a conserved NmrA (nitrogen metabolite repression regulator)domain(V₆-N₂₄₄),multi- phosphorylation sites and one conserved N-glycosylation site(N₂₁₄).The detailed bioinformatics analysis including sequence homology comparison,phylogenetic tree and prediction of advanced structures all suggested that FcIRL belonged to the class of pinoresinol- lariciresinol reductase(PLR)with bioactivity of PLR.The protein PLR is a key enzyme in synthetic pathway of 8-8'-linked lignans,which catalyzes reduction of pinoresinol and lariciresinol into secoisolariciresinol,and is involved in medicinal secondary metabolism and resistance in F.cymosum.We successfully constructed the sense expression vector.Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens LBA4404(containing pC2301-FcIRL) recombinant plasmid were transformed into model plants,tobacoo and petunia.The transgenic lines were identified by resistance screening,GUS histochemical staining ananysis and PCR analysis.All the analysis suggested that the FcIRL has been successfully recombined into the genomes of the two model plants.