Abstract
Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.
Highlights
Withania somnifera (Indian ginseng) is one of the most famous and widely used medicinal plants [1]
Oxidosqualene cyclases (OSC’s) serve as key enzymes in secondary metabolite synthesis by driving the metabolic pool of isoprenoids and converting it to a range ofhigher terpenoid- secondary metabolites via cyclization of 2, 3 oxidosqualene [12,13,14]
Cycloartenol synthases (CAS), an important oxidosqualene cyclases (OCSs) have been cloned from other plant species such as Glycyrrhiza glabra, Oryza sativa licorice, pea etc. and its involvement in terpenoid biosynthesis has been studied [16,17,18,19,20]
Summary
Withania somnifera (Indian ginseng) is one of the most famous and widely used medicinal plants [1]. W. somnifera, commonly known as Ashwagandha, is an ancient medicinal herb that has been used in various pharmacological preparations traditionally. Till several withanolides have been isolated from different parts of W. somnifera including major withanolides i.e. withaferin A, withanone and withanolide A, withanolide D etc [8]. Withanolides are synthesized through isoprenoid pathway via both mevalonate and non-mevalonate pathways [9]. The first committed step in the triterpenoid biosynthesis occurs by cyclization of epoxysqualene, leading to the formation of C30H50O product with 1–5 rings [10]. Plants contain different oxidosqualene cyclases (OSCs) for a single substrate to synthesize different set of sterols and other triterpenoids [11]
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