Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal

Frontiers in Plant Science
Arti SharmaSurrinder K Lattoo

Abstract

Withania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (CYP85A69) from W. somnifera. The full length WsCYP85A69, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, in vitro conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a m/z value of 506 [M+ACN]+. In planta transient over-expression of WsCYP85A69 significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of WsCYP85A69 resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether...Continue Reading

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Datasets Mentioned

BETA
PHT91631.1
MK410296

Methods Mentioned

BETA
enzymatic
electrophoresis
PCR
protein assay
Assay

Software Mentioned

Expasy
MEGA7
Protein Homology / analogy Recognition Engine
GraphPad Prism
WMD3
PLACE
BLASTp
Phyre
MUSCLE
BLASTn

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