Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic.Based on previous research,these effects are mediated by a number of active ingredients,especially glycyrrhizic acid(GA).In the present study,a gene encodingβ-amyrin synthase(β-AS)involved in GA biosynthesis in G.uralensis has been cloned and expressed in Saccharomyces cerevisiae.The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene intoβ-amyrin.In fact theβ-AS gene is particularly important in the GA biosynthetic pathway in G.uralensis.The complete sequence of the enzyme was determined and a phylogenetic tree based on theβ-AS gene of G.uralensis and 20 other species was created.This showed that Glycyrrhiza glabra had the closest kinship with G.uralensis.The results of this work will be useful in determining how to improve the efficacy of G.uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.