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 b-amyrin catalyzed by b-amyrin synthase (b-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. b-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 b-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, hormoneresponsive 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 b-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 b-AS in biosynthesis of saikosaponins.

Differential Expression of Genes Involved in Saikosaponin Biosynthesis Between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd

“…A previous study indicated that β-AS in B. kaoi and B. chinense exhibits tissue-specific responses to both MeJA and PEG (Lin et al, 2013;Zhang et al, 2016). We have also cloned the promoter of β-AS in B. chinense and evaluated its activity previously (Gao et al, 2015). We found three β-AS genes (BcBAMS98705, BcBAMS12370, and BcBAMS21523) with significantly increased expression in S3 of B. chinense.…”

Transcriptome Level Reveals the Triterpenoid Saponin Biosynthesis Pathway of Bupleurum falcatum L.

“…β-AS is a member of the OSC (oxidosqualene cyclase) family, which acts in the initial step of the biosynthesis of SSs, and the resultant β-amyrin is the backbone of various SS monomers [ 49 ]. β-AS was an important branch point between specialized and primary metabolism and could regulate the metabolic accumulation of triterpenoid saponins.…”

Ginsenosides in Panax genus and their biosynthesis

“…DDSs are uncommon in nature, which result in the unique ginsenoside scaffolds predominant in Panax species, and β -ASs are widespread in plant kingdom such as in Araliaceae 113 , Umbelliferae 114 , Leguminosae 115 and other families 116 , 117 . DDS and β -AS from Panax genus are single product producing enzymes, while some other OSCs are multifunctional enzymes.…”