Molecular cloning, expression and in-silico characterization of rosmarinic acid synthase from Ocimum tenuiflorum L.
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ORIGINAL ARTICLE
Molecular cloning, expression and in-silico characterization of rosmarinic acid synthase from Ocimum tenuiflorum L. Manish Kumar Suthar1
•
Parul M. Purohit1 • Parmeshwar Lal Saran1
Received: 29 June 2020 / Accepted: 19 August 2020 Ó Society for Plant Biochemistry and Biotechnology 2020
Abstract Rosmarinic acid synthase (RAS) is a control point at which metabolites derived from L-Phenylalanine and L-Tyrosine are channeled for the biosynthesis of rosmarinic acid (RA). Ocimum tenuiflorum or holy basil has been utilized as a source of RA. To acquire a better understanding of the biosynthesis of this metabolite, isolation, and cloning of O. tenuiflorum RAS (OtRAS) cDNA and its heterologous expression in bacterial cells was assessed. The full-length OtRAS (Accession No. MN542659) was 1278 bp in size encoding 425 amino acid long protein product. The subunit molecular mass of recombinant OtRAS was estimated to be * 47.5 kDa with a theoretical pI of 6.35. Sequence analysis indicated OtRAS belongs to BAHD superfamily proteins. Phylogenetic analysis revealed close evolutionary relatedness of OtRAS with RAS proteins reported in other lamiaceae members. Gene expression analysis showed that callus derived from leaves accumulated more transcripts of OtRAS than leaf and stem tissues derived from field-grown plants. OtRAS was up-regulated under flood and cold stress whereas down-regulated under drought and salt conditions. Further, three-dimensional structure of OtRAS showed two pseudo symmetrical domains harboring an active site in between these two domains. OtRAS active site comprised of conserved His41, His152, Trp363, and Lys391 residues. Overall, our results indicated the OtRAS regulation is a mechanism that probably depended on a variety of abiotic stresses. Keywords Rosmarinic acid synthase Ocimum tenuiflorum Cloning Gene expression Abiotic stress Abbreviations RA Rosmarinic acid RAS Rosmarinic acid synthase OtRAS RAS from Ocimum tenuiflorum CbRAS RAS from Coleus blumei PAL Phenylalanine ammonia-lyase C4H Cinnamic acid 4-hydroxylase 4CL 4-Coumaric acid CoA-ligase HPPR Hydroxyphenylpyruvate reductase Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13562-020-00591-8) contains supplementary material, which is available to authorized users. & Manish Kumar Suthar [email protected] Parul M. Purohit [email protected] Parmeshwar Lal Saran [email protected] 1
ICAR-Directorate of Medicinal and Aromatic Plants Research, Boriavi, Anand, Gujarat 387310, India
TAT SOH NTA MS LB IPTG
Tyrosine aminotransferase Shikimate-O-hydroxycinnamoyltransferases Nitrilotriacetic acid Murashige and Skoog Luria–Bertani Isopropyl-b-D-thiogalactopyranoside
Introduction Ocimum tenuiflorum (also known as O. sanctum L., Tulsi, Holy basil) is an annual herbaceous species belonging to the family Lamiaceae, which is well known for its valuable aromatic properties and medicinal values. Ocimum tenuiflorum is considered as Indigenous to the Indian subcontinent and no
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