Molecular characterization of DWF1 from Withania somnifera (L.) Dunal: its implications in withanolide biosynthesis

摘要

Present study describes isolation and molecular characterization of DWF1 gene from Withania somnifera and also elucidates its role in withanolide biosynthesis. Using the degenerate-reverse transcriptase and RACE strategy full length cDNA of DWF1 was isolated from W. somnifera and subsequently cloned in pGEX-4T-2 vector and expressed in E. coli. Effect of various abiotic and biotic chemicals such as methyl jasmonate (MeJA), salicylic acid (SA), 2,4-dichlorophenoxyacetic acid (2,4-D) and microbe-derived exogenous elicitor yeast extract (YE) was analyzed to establish any correlation between expression levels of transcript and withanolide accumulation. Effects on the transcript levels of WsDWF1 with each progressing ontogenic stages of W. somnifera was also carried out. Tissue-specific expression studies analyzing expression of WsDWF1 in various tissues such as leaf, root and stalk was also carried out. Full length cDNA of WsDWF1 contained an ORF of 1707 bp and encoded 65.8 kDa protein. Based upon elicitation studies using various abiotic and biotic chemicals such MeJA, SA, 2,4-D and YE, a positive correlation between withanolide accumulation and transcript profile of WsDWF1 was observed. Tissue-specific expression studies suggested higher expression of WsDWF1 in leaves followed by stalk and root tissues. A uniform increase in the transcript levels of WsDWF1 with each progressing developemental stage indicated increased substrate pool for phytosterol biosynthesis. Use of YE a known inhibitor of oxidosqualene cyclases (OSCs), showed correspondence with the increased transcript levels of WsDWF1 and an enhanced withanolide accumulation, possibly by re-routing of metabolite flux towards the downstream phytosterol biosynthesis. Present work describes WsDWF1 as a new player in withanogenesis, which can open up a fresh prospect for pathway engineering of W. somnifera.