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Plant organs and latex of greater celandine are rich in isoquinolin alkaloids that sanguinarine, a member of benzofenanthridine, is one of the most important metabolites, which its accumulation mainly reported in the roots. Sanguinarine biosynthesis requires seven enzymatic steps from the pre-substance s-reticulin using berberine bridge enzyme (BBE), cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis- N-methyltransferase (TNMT), protopine 6- hydroxylase (P6H), S-cis-N-methylstylopine 14-hydroxylase (MSH) and dihydro benzophenanthridine oxidase (DBOX). The study of biosynthetic pathways and the identification of factors affecting the production of secondary metabolites are important steps in metabolite engineering. Nanoparticles are a new category of elicitors that act as inducers for the production of secondary metabolites. For this purpose, the effect of titanium dioxide nanoparticles was investigated on the expression changes of the celandine sanguinarine biosynthetic pathway genes in a factorial experiment with three factors included titanium dioxide concentrations, spraying period and post-induction time.  Analysis of variance for the expression of STS, P6H, BBE and MSH genes revealed significant differences in titanium dioxide concentrations, post-induction times, spraying period, different organs and their interactions at P≤0.05, 0.01 levels of probability. The accumulation of STS and P6H transcripts in the roots was 2-3 times higher than the leaves and stems, and also the transcripts of the BBE and MSH genes was four times higher in the root than the stem. Totally, the concentration of 500 mg/L titanium dioxide increased the expression of STS, P6H, BBE and MSH genes by 63, 66, 500 and 91%, respectively, relative to control treatment. The 24 and 48 hours post-induction time resulted in maximum expression in all four studied genes in comparison to 72 hours post-induction time, and 500 mg/L titanium dioxide showed the highest expression for STS, P6H and BBE genes, and 1000 mg/L for MSH gene. From the results it can be concluded that titanium dioxide nanoparticles are effective in increasing the production of celandine sanguinarine through the regulation of its biosynthetic pathway genes.

Keywords: Elicitor, Gene expression, Nanoparticles, Sanguinarine

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