Molecular cloning and characterization of a novel isoflavone reductase-like gene (FcIRL) from high flavonoids-producing callus of Fagopyrum cymosum

Lignans are important defensive compounds in plants and have good biological activities protecting human health.  In order to study the medicinal secondary metabolism of Fagopyrum cymosum (Trev.) Meisn, a traditional Chinese medicine with anti-tumor effect, a novel isoflavone reductase-like gene, FcIRL, was cloned using RACE strategy from a cDNA library of high flavonoids-producing callus.  The full-length cDNA of the FcIRL was 1 217 bp (accession no. EU116032), which contained a 942 bp open reading frame (ORF) encoding  a 313 amino acid protein.  Two stop codons (TAG) and a putative polyadenylation signal ATAAA at 24 bp   upstream from the polyadenylation site was found in 5′ and 3′ UTR, separately.  And no intron was found in the genomic sequence yet.  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).  Sequence    homology comparison, phylogenetic analysis and advanced structures prediction all suggested that FcIRL    belonged to the class of pinoresinol-lariciresinol reductase (PLR), which is a key enzyme in synthetic pathway of 8−8′-linked lignans, with function in catalyzing reduction of pinoresinol and lariciresinol into secoisolariciresinol, and medicinal secondary metabolism and resistance in F. cymosum.