Molecular characterization and promoter analysis of squalene epoxidase gene from Withania somnifera (L.) Dunal.
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.
Plant Biotechnology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi, 180001, India.
Sumeer
S
Razdan
Wajid Waheed
WW
Bhat
Satiander
S
Rana
Niha
N
Dhar
Surrinder K
SK
Lattoo
Rekha S
RS
Dhar
Ram A
RA
Vishwakarma
D000595:Amino Acid Sequence; D001483:Base Sequence; D053898:Biosynthetic Pathways; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D050603:Squalene Monooxygenase / Q000235:genetics*; D040681:Structural Homology, Protein; D032341:Withania / Q000201:enzymology* / Q000235:genetics; D054358:Withanolides / Q000378:metabolism
D010940:Plant Proteins; D054358:Withanolides; D050603:Squalene Monooxygenase