Abstract
Scopolamine is a pharmaceutically important tropane alkaloid which is used therapeutically in the form of an anesthetic and antispasmodic drug. The present study demonstrates enhanced scopolamine production from transgenic hairy root clones of Duboisia leichhardtii wherein the expression of quinolinate phosphoribosyl transferase (QPT) gene was silenced using the QPT-RNAi construct under the control of CaMV 35 S promoter. The RNAi hairy roots clones viz. P4, P7, P8, and P12 showed the enhanced synthesis of scopolamine with significant inhibition of nicotine biosynthesis. Optimization of culture duration in combination with methyl jasmonate elicitor in different concentrations (50 µM-200 µM) was carried out. Maximum synthesis of scopolamine had obtained from HR clones P7 (8.84 ± 0.117 mg/gm) on the 30th day of cultivation. Conspicuously, elicitation with wound-associated hormone methyl jasmonate enhanced the yield of scopolamine 2.2 fold (19.344 ± 0.275 mg/gm) compared to the culture lacking the elicitor. The transgenic hairy roots cultures established with RNAi mediated silencing of quinolinate phosphoribosyl transferase gene provides an alternative approach to increase the yield of scopolamine in fulfilling the demand of this secondary metabolite.
Highlights
Duboisia leichhardtii indigenous to Australia and New Caledonia contains pyridine alkaloids and tropane alkaloids
The Quinolinic acid phosphoribosyl transferase (QPT) enzyme catalyzes the nicotinate mononucleotide biosynthesis, which subsequently combines with methylpyrrolinium cation to form nicotine
In the background of this information, in the present study, we have used the RNAi approach to silence QPT gene in the nicotine biosynthesis pathway of Duboisia leichhardtii hairy root culture, so that methylpyrrolinium cation, for the nicotine and tropane alkaloid pathway is diverted towards scopolamine synthesis
Summary
The pyridine alkaloid nicotine was not found in any of the transgenic root lines, while the control hairy roots transformed by A. rhizogenes but lacking QPT-RNAi did produce nicotine (0.354 ± 0.0115 mg/gm) (Fig. 3b) This result depicts that the silencing of the QPT gene enhances the scopolamine biosynthesis. The expression of QPT in all the transgenic lines had downregulated at every growth stage compared to the control hairy roots (Fig. 4) This observation was in line with the expectation that silencing of the QPT gene would divert methylpyrrolinium cation towards enhanced scopolamine synthesis. It had found that treatment with MeJa increased the scopolamine accumulation in hairy root tissues and enhanced the leaching out of scopolamine in the culture medium. In P7 root line secretion of scopolamine content in medium markedly increased by treatment with MeJa relative to untreated control (Fig. 6a). It will bridge the gap in the global demand and supply of scopolamine by up-scaling of established hairy root lines which has the ultimate aim of this study
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