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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.

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RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera