Cloning and analysis of β-amyrin synthase gene in Bupleurum chinense

Abstract

Bupleurum chinense DC. is one of the source plants of a well-known crude drug, Chai hu (Radix Bupleuri), producing triterpenoid saponins (saikosaponins) with a wide-spectrum of pharmacological applications. The biosynthesis of triterpenoid saponins involved with the cyclizing of the precursor 2,3-oxidosqualene to produce the first committed triterpene β-amyrin catalyzed by β-amyrin synthase (β-AS), whereafter diverse of triterpenoid saponins was biosynthesized. In addition, 2,3-oxidosqualene could be catalyzed by cycloartenol synthase directing to the synthesis of phytosterol. β-AS was thus defined as an important branch point between primary and secondary metabolisms, and may play a regulating role in the control of triterpenoid saponins biosynthesis. In this study, the promoter and protein-encoding regions of a β-AS gene (designated bcAS1) were isolated by genome walking and PCR from B. chinense. Several important cis-acting elements for gene regulation were identified within the promoter region including light-responsive, hormone-responsive and various other stress-related elements. Approximate 0.8 kb fragment on upstream of ATG start codon of bcAS1 was sub-cloned into pAN580 vector to replace the 35S promoter driving the expression of green fluorescent protein (GFP) gene. The promoter activity was detected by transient expression in onion epidermis cells by the expression of GFP. Approximately 6 kb length of bcAS1 gene was cloned, containing 18 exons and 17 introns. Although a dozen of β-AS cDNA was isolated, seldom the promoter and gene of it was reported. This work was a valuable foundation for further studies on the regulatory role of β-AS in biosynthesis of saikosaponins.