title: RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.
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.
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.
authors.forename: Smrati
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.
authors.forename: Shilpi
authors.initials: S
authors.lastname: Bansal
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.
authors.forename: Bhawana
authors.initials: B
authors.lastname: Mishra
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.|Center of Innovative and Applied Bioprocessing (CIAB) (A National Institute under Department of Biotechnology, Government of India), C-127, Phase-8, Industrial Area, S.A.S. Nagar, Mohali, Punjab, India.
authors.forename: Rajender Singh
authors.initials: RS
authors.lastname: Sangwan
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.
authors.lastname: Asha
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.
authors.forename: Jyoti Singh
authors.initials: JS
authors.lastname: Jadaun
authors.affiliation: Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow, India.
authors.forename: Neelam S
authors.initials: NS
authors.lastname: Sangwan
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D034622:RNA Interference; D032341:Withania / Q000235:genetics / Q000378:metabolism*; D054358:Withanolides / Q000378:metabolism*
chemical_list: D054358:Withanolides; D019751:Intramolecular Transferases; C021923:cycloartenol synthase