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

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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.

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Acknowledgments

We gratefully acknowledge the financial grant from Council of Scientific and Industrial Research, Government of India, New Delhi under Network Project NWP 0008. IIIM/1491/2012 is the publication contribution number of Indian Institute of Integrative Medicine, Jammu.

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Authors and Affiliations

  1. Plant Biotechnology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi, 180001, India

    Sumeer Razdan, Wajid Waheed Bhat, Satiander Rana, Niha Dhar, Surrinder K. Lattoo & Rekha S. Dhar

  2. Medicinal Chemistry, Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, India

    Ram A. Vishwakarma

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  1. Sumeer Razdan
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  3. Satiander Rana
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Correspondence to Rekha S. Dhar.

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Razdan, S., Bhat, W.W., Rana, S. et al. Molecular characterization and promoter analysis of squalene epoxidase gene from Withania somnifera (L.) Dunal. Mol Biol Rep 40, 905–916 (2013). https://doi.org/10.1007/s11033-012-2131-9

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