Abstract

Stephania cepharantha Hayata is a traditional Chinese medicinal plant with diverse pharmacological activities. Benzylisoquinoline alkaloids (BIAs), including cepharanthine and cycleanine, are the major bioactive constituents in this species. Although previous studies have proposed the biosynthetic pathway of cepharanthine based on genomic data of S. cepharantha and validated one norcoclaurine synthase (NCS) and one 4′- O -methyltransferase (4′OMT) involved in the BIA biosynthesis, the complete biosynthetic pathway of BIAs in this plant and the key enzymes involved remain largely uncharacterized. In this study, we conducted transcriptome analysis of different tissues of S. cepharantha (roots and leaves), complemented by chemical analysis of major BIAs, to discover candidate enzymes involved in BIA biosynthesis. A total of 67,851 unigenes and 110,252 transcripts were assembled from RNA-seq data using the Illumina HiSeq X Ten platform. HPLC analysis showed a significantly higher accumulation of major BIAs in the roots compared to the leaves. Based on the transcriptome data and the BIA distribution, forty BIA-related candidate genes were identified and a putative BIA biosynthetic pathway for S. cepharantha was proposed. Furthermore, two key cytochrome P450 genes, ScCYP80B1 and ScCYP80B2 , were cloned from S. cepharantha and co-expressed with ScCPR1 (also cloned from this plant) in yeast for functional characterization. Sc CYP80B1 showed broad 3′-hydroxylation activity towards both N -methylcoclaurine and coclaurine, while Sc CYP80B2 specifically catalyzes the 3′-hydroxylation of N -methylcoclaurine. The transcriptome analysis of S. cepharantha advances our understanding of the BIA biosynthetic pathway and the functional characterization of the two CYP80B genes provides new genetic elements for the BIA biosynthesis.