Papaver Somniferum Case Study

Papaver somniferum L. a member of Papaveraceae family, is one of the most agronomically and economically important medical crops with powerful pharmacological features (Ziegler and Facchini, 2008). The opium poppy produces numerous secondary metabolites such as benzyl isoquinoline alkaloids (BIAs) including the antimicrobial agent sanguinarine, , the cough suppressant and potential anticancer drug noscapine and vasodilator papaverine (Choe et al., 2011). Beaudoin , Facchini ., 2014).
Noscapine belong to benzylisoquinoline alkaloid produced in opium poppy and other members of the Papaveraceae. Noscapine has been used as a human cough suppressant and recently was shown to possess anticancer activity. Unlike codeine and other opiates, noscapine
somniferum (#3, #7, #9, #12 ecotypes) and P. bracteatum were ground to a fine powder under liquid nitrogen.(ref) Then total RNA was isolated using RNeasy Plant Mini Kit-QIAGEN (Lot No: 142338933) according to manufacturer’s instructions. The quality and quantity of the extracted RNA was determined by a NanoDrop spectrophotometer (BioTek, EPOCH, serial 121004C, USA), and confirmed by agarose gel electrophoresis. After synthesis of cDNA, PCR was conducted and expected results were shown. qRT-PCR was performed using BioRad system with the fluorescent dye SYBR®Green Master Mix 2X (Ampliqon, cat No.: A325402, Denmark) in accordance with the manufacturer’s recommendation. The following program was used to perform qRT-PCR: 1 μL of cDNA as template, 1 μL (10 pmol. μl−1) of each primer, 10 μL SYBR®Green Master Mix 2X in a total volume of 20 μL. qRT-PCR was run at 95 °C for 15 min, 35 cycles at 95 °C 30 s, 58 °C for 30, 72 °C for 15 s. Three technical replicates were performed for each sample. For quantifying transcription levels, the reference actin was used as an internal control. Cycle thresholds (C(t)s) were analyzed using Livak method (2 -ΔΔCt) and relative expression levels were calculated using the Microsoft Excel
After PCR, difference between amplified fragments were shown by gel agarose 1.5%. (fig) According to the gel electrophoresis two samples of studied ecotypes named #3 and #7 displayed polymorphism in promoter region of CYP82Y1, whereas, there was no difference in PsMT1 region. CYP82Y1 have reported as a first committed step in noscapine biosynthesis pathway, moreover variation in promoter region especially close to TSS and core promoter might effect on gene expression significantly. For study of this hypothesis, we study on transcript level and metabolite content in these