title: Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal.
abstract: Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.
authors.affiliation: Plant Biotechnology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi-180001, India.
authors.forename: Wajid Waheed
authors.initials: WW
authors.lastname: Bhat
authors.forename: Surrinder K
authors.initials: SK
authors.lastname: Lattoo
authors.forename: Sumeer
authors.initials: S
authors.lastname: Razdan
authors.forename: Niha
authors.initials: N
authors.lastname: Dhar
authors.forename: Satiander
authors.initials: S
authors.lastname: Rana
authors.forename: Rekha S
authors.initials: RS
authors.lastname: Dhar
authors.forename: Shabnam
authors.initials: S
authors.lastname: Khan
authors.forename: Ram A
authors.initials: RA
authors.lastname: Vishwakarma
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008954:Models, Biological; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014161:Transduction, Genetic; D032341:Withania / Q000235:genetics*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D013186:Farnesyl-Diphosphate Farnesyltransferase