Molecular characterization and promoter analysis of squalene epoxidase gene from Withania somnifera (L.) Dunal

Abstract

Withania somnifera is a rich reservoir of pharmaceutically active steroidal lactones known as withanolides. The plant is well characterized in terms of its chemistry and pharmacology, but very little is known about the pathway involved in the biosynthesis of withanolides. The present investigation describes the cloning, characterization and expression of squalene epoxidase (SE) gene from W. somnifera. SE (SQE; EC. 1.14.99.7) is one of the rate limiting enzymes in the biosynthesis of triterpenoids, catalyzing the stereospecific epoxidation of squalene to 2,3-oxidosqualene. A full length SE gene (WsSQE) of 1,956 bp was cloned which contained an open reading frame of 1,596 bp, encoding a protein of 531 amino acids with a predicted molecular mass of 57.67 kDa and theoretical PI of 8.48. Full length WsSQE was cloned into pGEX4T-2 vector and expressed in E.coli. Phylogenetic analysis indicated a significant evolutionary relatedness of WsSQE with squalene epoxidases of other plant species and the degree of relatedness with deduced amino acid sequences showed a significant correlation with different plant species. Using genome walking approach, a promoter sequence of 513 bp of WsSQE was isolated which revealed several key cis-regulatory elements known to be involved in various biotic and abiotic plant stresses. Comparative expression analysis of tissue specific WsSQE done by quantitative-PCR demonstrated the highest transcript levels in leaves, as compared to stalk and root tissues. This is the first report of cloning and bacterial expression of SE from W. somnifera and may be of significant interest to understand the regulatory role of SE in the biosynthesis of withanolides.

Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases from Withania somnifera (L.) Dunal

“…Restriction of secondary metabolite biosynthesis to particular tissue(s) in plants is mostly executed by regulating the expression levels of biosynthetic pathway genes. In accordance with this fact, our previous studies have shown elevated expression profile of three upstream genes, namely squalene synthase (WsSQS), squalene epoxidase (WsSQE), and cytochrome P450 reductase 2 (WsCPR2) of withanolide biosynthetic pathway in leaves as compared with other tissues (8,17,50). Additionally, enhanced expression of obtusifoliol-14-demthylase (CYP51) and sterol methyl transferase (SMT-1), which constitute an important part of downstream withanolide biosynthetic pathway, has also been reported in leaves of Ws (51).…”

Biotechnological interventions inWithania somnifera(L.) Dunal

“…This expression pattern of OSC/CS is in correlation with the higher amounts of withanolides in the leaves (Dhar et al, 2014). As discussed above, expression of upstream genes like SQS, SQE, and cytochrome P450 reductase 2 (CPR2) was also found to be higher in leaves as compared to other tissues Rana et al, 2013;Razdan et al, 2013). Additionally, enhanced expression of obtusifoliol-14-demthylase (CYP51) and sterol methyl transferase (SMT-1) that constitute important part of downstream withanolide biosynthetic pathway has also been reported in leaves (Pal et al, 2011).…”

Recent advances in steroidal saponins biosynthesis and in vitro production

“…Besides Panax species and yeast, SE was also characterized in Euphorbia tirucalli (Uchida et al 2007), Withania somnifera (Razdan et al 2013), Arabidopsis (Laranjeira et al 2015), Gynostemma pentaphyllum (Guo et al 2016) etc.…”