بهینه‌سازی کالوس زایی و بررسی میزان بیان ژن‌های فلاونول سنتاز و چالکون ایزومراز در مسیر تولید کوئرستین تحت تأثیر اشعۀ UV-B در کشت کالوس خیار تلخ ( .Momordica charantia L )
محورهای موضوعی : گیاهان داروییزیبا فولادوند 1 , بهمن فاضلی نسب 2
1 - دانشجوی دکتری ژنتیک مولکولی دانشگاه تربیت مدرس
2 - عضو هیات علمی دانشگاه
کلید واژه: توفوردی, کوئرستین, فلاونوئید, کارلا, فنیل‌آمونیالیاز,
چکیده مقاله :
هدف از تحقیق حاضر بهینه‌سازی کالوس زایی و بررسی میزان بیان ژن‌های فلاونول سنتاز و چالکون ایزومراز در مسیر تولید کوئرستین تحت تأثیر اشعۀ UV-B در کشت کالوس خیار تلخ می باشد. غلظت‌های مختلف تنظیم‌کننده‌های رشدBAP و 2-4-D و ریز نمونه جهت تولید کالوس در گیاه خیار تلخ در طرح کاملاً تصادفی با سه تکرار ارزیابی شد. تاثیر اشعۀ UV-B (پنج گروه (شاهد (نور طبیعی)، 5 دقیقه در معرض اشعه (بررسی بیان ژن در 24 و 48 بعد از اعمال تیمار) و 10 دقیقه در معرض اشعه (بررسی بیان ژن در 24 و 48 بعد از اعمال تیمار)) بر بیان ژن‌های مسیر بیوسنتزی کوئرستین از جمله فلاونول‌سنتاز و چالکون‌ایزومراز، میزان فعالیت آنزیم آنتی‌اکسیدانی فنیل‌آمونیالیاز و فلاونوئید در بهترین کالوس های انتخاب شده بررسی شد. مقایسه میانگین داده‌ها نشان داد که بیشترین وزن خشک کالوس در غلظت 5/0 میلی‌گرم در لیتر 2-4-D و 1 میلی‌گرم در لیتر BAP برای ریز نمونه ساقه به دست آمد. بیشترین بیان ژن فلاونول سنتاز و چالکون ایزومراز در تیمار اعمال 10 دقیقه اشعۀ UV-B در زمان 24 ساعت بعد از شروع تیماردهی بدست آمده اما این بیان در 48 ساعت بعد از اعمال تیمار کاهش یافته است. بیشترین بیان فنیل‌آمونیالیاز، کوئرستین و فلاونوئید در زمان 5 دقیقه اعمال تیمار اشعه UV-B در 48 ساعت بعد از اعمال تیمار بدست آمده است. در کل نتایج این تحقیق نشان داد که اشعۀ UV-B در سطح کالوس تاثیر مثبتی بر بیان ژن‌های مسیر بیوسنتز فلاونوئیدها داشته است.
The present study aimed to optimize the callus induction and to determine the expression of flavonol synthase and chalcone isomerase genes in the pathway of quercetin production under UV-B rays in bitter cucumber callus cultivation. In this research was used concentration and combine of plant hormone 2, 4-D and BAP and explant to callus induction in Momordica charantia using complete random design with three replication. Genes expression study of Quercetin biosynthesis pathway such as FLS, CHI (were analyzed by 2-∆∆CT) and antioxidant activity assay of phenylalanine ammonia-lyase (Produced by concentration of cinnamic acid) and flavonoid using best callus were selected and affected by UV-B ray (five group(Control(natural light), 5 minute under UV-B ray(studying of gene expression after 24 and 48 hour) and 10 minute under UV-B (studying of gene expression after 24 and 48 hour)). Analysis of variance was shown that kind and concentration of plant hormone was significant on callus induction. Mean square of data was shown that the most callus dry weight was obtained from 0.5 mg/L 2,4-D and 1 mg/L BAP and stem explant. The highest expression of flavonol synthase and chalcone isomerase (chi) was observed in the 10-min UV-B treatment at 24 h after treatment but decreased at 48 hours after treatment. The highest expression of PAL, quercetin and flavonoid were obtained at 5 minutes UV-B treatment at 48 hours after treatment. Overall, the results showed that UV-B radiation at the callus level had a positive effect on the expression of flavonoids biosynthetic pathway genes.
References
1.Agarwal, M., 2015. Tissue culture of Momordica charantia L.: A review. Journal of Plant Sciences, 3(1-1): 24-32.
2.Agarwal, M. and Kamal, R., 2004. In vitro clonal propagation of Momordica charantia L. Indian Journal of Biotechnology, 3(3): 426-430.
3.Akula, R. and Ravishankar, G.A., 2011. Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav, 6(11): 1720-1731.
4.Azuma, A., Yakushiji, H., Koshita, Y. and Kobayashi, S., 2012. Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions. Planta, 236(4): 1067-1080.
5.Bijami, A., Rezanejad, F. and Sasan, H.A., 2011. The effects of post-harvest UV-B radiation on some antioxidant compounds, PAL activity and total protein contents of ripe red tomato (Lycopersicon esculentum). Iranian Journal of Plant Biology, 2(6): 29-38.
6.Cetin, E.S., 2014. Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures. Biol Res, 47(1): 37.
7.Chang, A., Lim, M.-H., Lee, S.-W., Robb, E.J. and Nazar, R.N., 2008. Tomato phenylalanine ammonia-lyase gene family, highly redundant but strongly underutilized. Journal of Biological Chemistry, 283(48): 33591-33601.
8.Chang, C.-C., Yang, M.-H., Wen, H.-M. and Chern, J.-C., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of food and drug analysis, 10(3).
9.Chupeau, M.-C., Granier, F., Pichon, O., Renou, J.-P., Gaudin, V. and Chupeau, Y., 2013. Characterization of the early events leading to totipotency in an Arabidopsis protoplast liquid culture by temporal transcript profiling. The plant cell, 25(7): 2444-2463.
10.Cragg, G.M. and Newman, D.J., 2013. Natural products: a continuing source of novel drug leads. Biochimica et Biophysica Acta (BBA)-General Subjects, 1830(6): 3670-3695.
11.Daniel, P., Supe, U. and Roymon, M., 2014. A review on phytochemical analysis of Momordica charantia. Int J Adv Pharm Biol Chem, 3(1): 214-220.
12.Fang, E., Froetscher, L., Scheibye-Knudsen, M., Bohr, V. and Ng, T., 2018. Emerging antitumor activities of the bitter melon (Momordica charantia). Current protein & peptide science, 19(1): PMID:29932035.
13.Fazeli-Nasab, B. and Mirzaei, N., 2018. Evaluation of total phenol and flavonoid content in a wide range of local and imported plants. Scientific Journal of Ilam University of Medical Sciences, 26(2): 141-154.
14.Frohnmeyer, H. and Staiger, D., 2003. Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection. Plant physiology, 133(4): 1420-1428.
15.Fujita, A., Goto-Yamamoto, N., Aramaki, I. and Hashizume, K., 2006. Organ-specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins. Bioscience, biotechnology and biochemistry, 70(3): 632-638.
16.Gandhi, P.R., Jayaseelan, C., Kamaraj, C., Rajasree, S.R. and Mary, R.R., 2018. In vitro antimalarial activity of synthesized TiO2 nanoparticles using Momordica charantia leaf extract against Plasmodium falciparum. Journal of Applied Biomedicine, 16(4): 378-386.
17.Guo-li, W., 2008. Establishment of in vitro regeneration system of Momordica charantia L. with different genotype [J]. Journal of Anhui Agricultural Sciences, 36(28): 12125-12127.
18.Huda, A. and Sikdar, B., 2006. In vitro plant production through apical meristem culture of bitter gourd (Momordica charantia L.). Plant Tissue Culture and Biotechnology, 16(1): 31-36.
19.Joseph, B. and Jini, D., 2013. Antidiabetic effects of Momordica charantia (bitter melon) and its medicinal potency. Asian Pacific Journal of Tropical Disease, 3(2): 93-102 (doi: 110.1016/S2222-1808(1013) 60052-60053).
20.Keskin, N., Kunter, B., Yaşı, Ü.K., Işını, U. and İnkübasyon, U., 2009. The effects of callus age, UV irradiation and incubation time on trans-resveratrol production in grapevine callus culture. Tarim Bilimleri Dergisi, 15(1): 9-13.
21.Khatami, F. and Ghanati, F., 2011. Effects of UV irradiation on cell viability, anthocyanin, and flavonoid contents of callus-cultured Malva neglecta cells. ICLST, 3: 202-204.
22.Khatib, A., Perumal, V., Ahmed, Q., Uzir, B. and Murugesu, S., 2017. Low inhibition of alpha-glucosidase and xanthine oxidase activities of ethanol extract of Momordica charantia fruit. Journal of Pharmaceutical Negative Results, 8(1): 20-24.
23.Koyama, K., Ikeda, H., Poudel, P.R. and Goto-Yamamoto, N., 2012. Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape. Phytochemistry, 78: 54-64.
24.Larkindale, J. and Knight, M.R., 2002. Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant physiology, 128(2): 682-695.
25.Liu, M., Cao, B., Zhou, S. and Liu, Y., 2012. Responses of the flavonoid pathway to UV-B radiation stress and the correlation with the lipid antioxidant characteristics in the desert plant Caryopteris mongolica. Acta Ecologica Sinica, 32(3): 150-155.
26.Lopes, A.P., Petenuci, M.E., Galuch, M.B., Schneider, V.V.A., Canesin, E.A. and Visentainer, J.V., 2018. Evaluation of effect of different solvent mixtures on the phenolic compound extraction and antioxidant capacity of bitter melon (Momordicacharantia). Chemical Papers: 1-9.
27. Manye., Y., Zhao., M., Lan., L. and Chen., F., 2004. Establishment of in vitro regeneration system of bitter melon (Momordica charantia L.). Chinese Science and Technology Periodical Database, 10(1): 44-48.
28. Menghini, A., Capuccella, M., Mercati, V., Mancini, L. and Buratta, M., 1993. Flavonoid contents in Passiflora spp. Pharmacological Research, 27: 13-14.
29.Miguel, F. and Alexandre, J. 2017. Estudios sobre regeneración y transformación genética en pepino (Cucumis sativus L.) vía Agrobacterium tumefaciens (thesis), https://doi.org/ 10.4995/Thesis/10251/90405
30.Mwambete, K., 2009. The in vitro antimicrobial activity of fruit and leaf crude extracts of Momordica charantia. A Tanzania medicinal plant. African health sciences, 9(1): 34-39.
31.Nasr, N. and Pirani, G., 2016. A review of anti-oxidant effects of the ethanol extract of the medicinal plant carla (Momordica charantia) cultivated in northern region of Iran (mazandaran-simorgh city). Biological Forum – An International Journal, 8(1): 518-521.
32.Negi, P.S., 2012. Plant extracts for the control of bacterial growth: Efficacy, stability and safety issues for food application. International Journal of Food Microbiology, 156(1): 7-17.
33.Nicholas, K. and Nicholas, H., 1997. GeneDoc: A tool for editing and editing multiple sequence alignments. Distributed by the author.
34.Perez, J.L., Jayaprakasha, G.K., Crosby, K. and Patil, B.S., 2019. Evaluation of bitter melon (Momordica charantia) cultivars grown in Texas and levels of various phytonutrients. J. Sci. Food Agri., 99(1): 379-390.
35.Raish, M., Ahmad, A., Ansari, M.A., Alkharfy, K.M., Aljenoobi, F.I., Jan, B.L., Al-Mohizea, A.M., Khan, A. and Ali, N., 2018. Momordica charantia polysaccharides ameliorate oxidative stress, inflammation, and apoptosis in ethanol-induced gastritis in mucosa through NF-kB signaling pathway inhibition. Int. J. Biol. Macromol., 111: 193-199.
36.Rao, C.V., 2018. Immunomodulatory Effects of Momordica charantia Extract in the Prevention of Oral Cancer. Cancer Prev. Res. (Phila), 11(4): 185-186.
37.Saahithya, R., Sridhar, R., Hemalatha, S. and Sriram, P., 2018. Haematological changes in experimentally induced pancreatic atypical acinar cell tumor in male wistar rats and its alleviation by Momordica charantia. The Pharma Innovation Journal, 7(4): 1054-1105.
38.Seo, S.H., Bai, D.G. and Park, H.Y., 2000. High Frequency Shoot Regeneration from Leaf Explants of Cucumber. Journal of Plant Biotechnology, 2(1): 51-54.
39. Singh, A., Selvi, M.T. and Sharma, R., 1999. Sunlight-induced anthocyanin pigmentation in maize vegetative tissues. Journal of Experimental botany, 50(339): 1619-1625.
40.Sobhanizade, A., Solouki, M. and Fazeli-Nasab, B., 2017. Optimization of callus induction and effects of biological and non-biological elicitors on content of phenol/flavonoid compounds in Nigella sativa under in-vitro conditions. Cell and Tissue Journal, 8(2): 165-184.
41.Tůmová, L. and Tůma, J., 2011. The effect of UV light on isoflavonoid production in Genista tinctoria culture in vitro. Acta Physiologiae Plantarum, 33(2): 635-640.
42.Usman, M., Hussain, Z. and Fatima, B. 2011. Somatic embryogenesis and shoot regeneration induced in cucumber leaves. Pak J Bot, 43(2): 1283-1293.
43.Venkatachalam, P., Jinu, U., Sangeetha, P., Geetha, N. and Sahi, S.V., 2018. High frequency plant regeneration from cotyledonary node explants of Cucumis sativus L. cultivar ‘Green Long’via adventitious shoot organogenesis and assessment of genetic fidelity by RAPD-PCR technology. 3 Biotech, 8(1): 60.
44.Virdi, J., Sivakami, S., Shahani, S., Suthar, A., Banavalikar, M. and Biyani, M., 2003. Antihyperglycemic effects of three extracts from Momordica charantia. J. Ethnopharmacol., 88(1): 107-111.
45.Wach, A., Pyrzyńska, K. and Biesaga, M., 2007. Quercetin content in some food and herbal samples. Food chemistry, 100(2): 699-704.
46.Yan, J.-K., Wu, L.-X., Qiao, Z.-R., Cai, W.-D. and Ma, H., 2019. Effect of different drying methods on the product quality and bioactive polysaccharides of bitter gourd (Momordica charantia L.) slices. Food chemistry, 271: 588-596.
_||_