Molecular Cloning and Characterization of Isoflavone Synthase Gene from Pueraria lobata

Abstract

Isoflavones are the main active component of kudzu(Pueraria lobata).The first committed step in the production of isoflavones is performed by a cytochrome P450 enzyme,IFS.While IFS genes have been isolated from kudzu,no biological functions have been identified to date.We cloned an IFS gene via RT-PCR,named PlIFS,using kudzu collected from Langxi(Anhui,China)as plant material.The PlIFS contained an open reading frame(ORF)encoding 521 amino acid residues.The amplified PlIFS gene was cloned into pESC-TRP vector,with yeast GAL1 divergent promoters as the control.The constructed recombinant plasmid,named pESC-TRP-PlIFS,was transformed into Saccharomyces cerevisiae strain WAT11 according to the LiAc/ssDNA/PEG method.The PlIFS expressed in yeast was able to catalyze daidzein formation from liquiritigenin,indicating that it had IFS activity.Gene expression pattern analysis revealed that PlIFS was expressed in roots at the highest level,which was consistent with isoflavones accumulation in kudzu plantlets.