با همکاری مشترک دانشگاه پیام نور و انجمن بیوتکنولوژی جمهوری اسلامی ایران

نوع مقاله : علمی پژوهشی

نویسندگان

1 گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

2 پژوهشکده بیوتکنولوژی دانشگاه شیراز، شیراز، ایران.‏

چکیده

بیماری‌های گیاهی، به ویژه بیماری‌های حاصل از قارچ‌ها و اٌوومیست‌ها، از چالش‌های عمده‌‌ی کشاورزی مدرن جهانی هستند. الگوهای مولکولی مرتبط با پاتوژن (PAMP) مانند کیتین دیواره‌ی سلولی قارچ‌ها و اٌوومیست‌ها، باعث تحریک و سیگنال‌هایی در گیاه میزبان، بیان ژن‌های R و تولید گونه‌های اکسیژن فعال و طیف وسیعی از متابولیت‌ها می‌شوند. تحریک کیتین منجر به بیان ژن‌های مرتبط با دفاع مانند کیتینازها و درنهایت تخریب کیتین دیواره‌ی سلولی پاتوژن‌ها می‌شود. به‌منظور ارزیابی سطح بیان تعدادی از ژن‌های کیتیناز و اندازه‌گیری فعالیت برخی آنزیم‌های آنتی‌اکسیدان، برگ‌های یک ژنوتیپ سیب‌زمینی متحمل به بیماری به نام جلی، در شرایط آزمایشگاهی با الیگومرهای کیتین تلقیح شد. نتایج پژوهش نشان داد که 48 ساعت پس از تلقیح با کیتین، بیان کلاس‌های مختلف ژن کیتیناز در برگ‌های تیمار شده نسبت به شاهد افزایش معنی‌داری پیدا کرد. ژن‌های کیتیناز کلاس I (با 5/5 برابر افزایش بیان نسبت به شاهد) و ژن‌های کیتیناز کلاس III (با 11/1 برابر افزایش بیان نسبت به شاهد)، به ترتیب بیشترین و کمترین بیان را 48 ساعت پس از تلقیح با کیتین داشتند. با این حال، فعالیت آنزیم‌های آنتی‌اکسیدان کاتالاز و آسکوربات‌پراکسیداز تغییر معنی‌داری نسبت به شاهد نداشتند. این نتیجه نشان می‌دهد که استفاده از تیمار کیتین، مسیرهای سیگنال‌دهی درگیر در بیوسنتز آنزیم‌های آنتی‌اکسیدان را در 48 ساعت پس از تیمار کیتین، القا نمی‌کند و نیز ژن‌های کدکننده کیتینازها را می‌توان با روش‌های مهندسی ژنتیک همسانه‌سازی نمود و در نهایت گیاهان تراریخته‌ی مقاوم به پاتوژن‌ها را تولید کرد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Investigating some antioxidant enzymes activity and changing the expression of some defense chitinase genes of potato (Solanum tuberosum L.) after inoculation with chitin

نویسندگان [English]

  • Maryam Faramarzi Jafar Beiglou 1
  • Farhad Nazarian-Firouzabadi 1
  • Seyed Sajad Sohrabi 1
  • Ali Moghadam 2

1 Department of Production Engineering and Plant Genetics, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.

2 Institute of Biotechnology, Assistant professor in plant biotechnology. Shiraz University, Shiraz. Iran

چکیده [English]

Plant diseases, particularly diseases caused by fungi and oomycetes pose significant challenges in modern agriculture worldwide. Pathogen associated molecular pattern (PAMP) like chitin found in the cell walls of fungi and oomycetes, trigger defence signalling, leading to expression of R-genes and the production of reactive oxygen species (ROS), and accumulation of a wide range of metabolites. Chitin elicitors prompt the expression of defence-related genes such as chitinases, ultimately the resulting in the breakdown of chitin in the pathogen's cell wall. To assess the expression level of certain chitinases in potatoes and the activity of antioxidant enzymes, leaves of a tolerant potato genotype (jelly) was challenged with chitin oligomers in vitro. Result of this study revealed that 48 hours post chitin induction, the expression of different classes of chitinase genes were significantly increased. Class I chitinase (Soltu.DM.10G017450) and class III chitinase (Soltu.DM.11G026160) genes, had respectively the highest (5.5-fold relative to control) and the lowest (1.1-fold relative to control) expression level after 48 hours post chitin inoculation. However, the activities of antioxidant enzymes catalase and ascorbate peroxidase did not change significantly compared to the control. These findings suggest that the application of chitin does not activate the signaling pathways involved in the biosynthesis of antioxidant enzymes 48 hours after chitin treatment. In addition, results of this study may imply that chitinase genes can be cloned by genetic engineering approaches to generate transgenic plants resistant to pathogens.

کلیدواژه‌ها [English]

  • antioxidant enzymes
  • chitinase genes
  • gene expression
  • potato
  • Real-time PCR
Abd Elhamid, M. I., Makboul, H. E., Sedik, M. Z., Ismail, I. M., & Ibrahim, M. A. (2010). Cloning, expression and antifungal activity of an endochitinase gene derived from barley (Hordeum vulgare). Research Journal of Agriculture and Biological Sciences, 6 (3), 356-363. Abeles, F. B., Bosshart, R. P., Forrence, L. E., & Habig, W. (1971). Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiology, 47 (1), 129-134. Aebi, H. (1984). [13] Catalase in vitro. In Methods in enzymology (Vol. 105, pp. 121-126). Academic press. Boller, T., & He, S. Y. (2009). Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science, 324 (5928), 742-744. Bravo, J. M., Campo, S., Murillo, I., Coca, M., & San Segundo, B. (2003). Fungus-and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize. Plant molecular biology, 52, 745-759. ‏ Cao, J., & Tan, X. (2019). Comprehensive analysis of the chitinase family genes in tomato (Solanum lycopersicum). Plants, 8 (3), 52. Chen, R. D., Yu, L. X., Greer, A. F., Cheriti, H., & Tabaeizadeh, Z. (1994). Isolation of an osmotic stress-and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense. Molecular and General Genetics MGG, 245, 195-202. Chen, J., Piao, Y., Liu, Y., Li, X., & Piao, Z. (2018). Genome-wide identification and expression analysis of chitinase gene family in Brassica rapa reveals its role in clubroot resistance. Plant science, 270, 257-267. Cota, I. E., Troncoso-Rojas, R., Sotelo-Mundo, R., Sánchez-Estrada, A., & Tiznado-Hernández, M. E. (2007). Chitinase and β-1, 3-glucanase enzymatic activities in response to infection by Alternaria alternata evaluated in two stages of development in different tomato fruit varieties. Scientia Horticulturae, 112 (1), 42-50. Ellis, J., Dodds, P., & Pryor, T. (2000). Structure, function and evolution of plant disease resistance genes. Current opinion in plant biology, 3 (4), 278-284. Felle, H. H. (2001). pH: signal and messenger in plant cells. Plant biology, 3 (06), 577-591. Gurr, S. J., & Rushton, P. J. (2005). Engineering plants with increased disease resistance: how are we going to express it?. TRENDS in Biotechnology, 23 (6), 283- 290. Gregorova, Z., Kovacik, J., Klejdus, B., Maglovski, M., Kuna, R., Hauptvogel, P., & Matusikova, I. (2015). Drought-induced responses of physiology, metabolites, and PR proteins in Triticum aestivum. Journal of agricultural and food chemistry, 63 (37), 8125-8133. Hashimoto, M., Kisseleva, L., Sawa, S., Furukawa, T., Komatsu, S., & Koshiba, T. (2004). A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant and cell physiology, 45 (5), 550-559. Heller, J., & Tudzynski, P. (2011). Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annual review of phytopathology, 49, 369-390. Hückelhoven, R. (2007). Cell wall–associated mechanisms of disease resistance and susceptibility. Annu. Rev. Phytopathol,45,101-127. Jeffares, D. C., Penkett, C. J., & Bähler, J. (2008). Rapidly regulated genes are intron poor. Trends in genetics, 24 (8), 375-378. Liliane, B. D. A., Sachetto-Martins, G., Contarini, M. G., Sandroni, M., Ferreira, R. D. P., de Lima, V. M., ... & Margis-Pinheiro, M. (1997). Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris. Febs Letters, 419 (1), 69-75. Livak, K.J., & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), 402-408. Moghaddam, G. A., Rezayatmand, Z., Esfahani, M. N., & Khozaei, M. (2019). Genetic defense analysis of tomatoes in response to early blight disease, Alternaria alternata. Plant Physiology and Biochemistry, 142, 500-509. Moradi, N., Rahimian, H., Dehestani, A., & Babaeizad, V. (2016). Cucumber Response to Sphaerotheca fuliginea: Differences in antioxidant enzymes activity and pathogenesis-related gene expression in susceptible and resistant genotypes.‏ J Plant Mol Breed, 4(2), 33-40. Muleta, H. D., & Aga, M. C. (2019). Role of nitrogen on potato production: a review. Journal of Plant Sciences, 7 (2), 36-42.‏ Nakano, Y., & Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and cell physiology, 22 (5), 867-880. Návarová, H., Bernsdorff, F., Döring, A. C., & Zeier, J. (2012). Pipecolic acid, an endogenous mediator of defense amplification and priming, is a critical regulator of inducible plant immunity. The Plant Cell, 24 (12), 5123-5141. Navarro, L., Zipfel, C., Rowland, O., Keller, I., Robatzek, S., Boller, T., & Jones, J. D. (2004). The transcriptional innate immune response to flg22. Interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis. Plant physiology, 135 (2), 1113-1128. Nürnberger, T., Brunner, F., Kemmerling, B., & Piater, L. (2004). Innate immunity in plants and animals: striking similarities and obvious differences. Immunological reviews, 198 (1), 249-266. Pandey, P., Irulappan, V., Bagavathiannan, M. V., & Senthil-Kumar, M. (2017). Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits. Frontiers in plant science, 8, 537. Pavet, V., Olmos, E., Kiddle, G., Mowla, S., Kumar, S., Antoniw, J., ... & Foyer, C. H. (2005). Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis. Plant physiology, 139 (3), 1291-1303. Prasad, K., Bhatnagar-Mathur, P., Waliyar, F., & Sharma, K. K. (2013). Overexpression of a chitinase gene in transgenic peanut confers enhanced resistance to major soil borne and foliar fungal pathogens. Journal of Plant Biochemistry and Biotechnology, 22, 222-233. Que, Y., Su, Y., Guo, J., Wu, Q., & Xu, L. (2014). A global view of transcriptome dynamics during Sporisorium scitamineum challenge in sugarcane by RNA-Seq. PLoS One, 9 (8), e106476. Rawat, S., Ali, S., Mittra, B., & Grover, A. (2017). Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea. Biotechnology reports, 13, 72-79. Roby, D., Toppan, A., & Esquerré-Tugayé, M. T. (1988). Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment. Physiological and molecular plant pathology, 33 (3), 409-417. Saboki Ebrahim, K. U., & Singh, B. (2011). Pathogenesis related (PR) proteins in plant defense mechanism. Sci. Against Microb. Pathog, 2, 1043-1054. Samet, M., Charfeddine, M., Kamoun, L., Nouri-Ellouze, O., Gargouri-Bouzid, R. (2018) Effect of compost tea containing phosphogypsum on potato plant growth and protection against Fusarium solani infection. Environmental Science and Pollution Research 25, 18921-18937. Schutzendubel, A., & Polle, A. (2002). Plant responses to abiotic stresses: heavy metal‐induced oxidative stress and protection by mycorrhization. Journal of experimental botany, 53 (372), 1351-1365. Singh, H. R., Deka, M., & Das, S. (2015). Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum). Functional & integrative genomics, 15, 461-480. Su, Y., Xu, L., Wang, S., Wang, Z., Yang, Y., Chen, Y., & Que, Y. (2015). Identification, phylogeny and transcript of chitinase family genes in sugarcane. Scientific reports, 5 (1), 10708. Tehrani, M. M., Esfahani, M. N., Mousavi, A., Mortezaiinezhad, F., & Azimi, M. H. (2020). Regulation of related genes promoting resistant in Iris against root rot disease, Fusarium oxysporum f. sp. gladioli. Genomics, 112 (5), 3013-3020. Van Loon, L. C., & Van Strien, E. A. (1999). The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and molecular plant pathology, 55 (2), 85-97. van Loon, L. C., Rep, M., & Pieterse, C. M. (2006). Significance of inducible defense-related proteins in infected plants. Annu. Rev. Phytopathol., 44, 135-162. Wessels, J. G. H., & Sietsma, J. H. (1981). Fungal cell walls: a survey. In Plant carbohydrates II: extracellular carbohydrates (pp. 352-394). Berlin, Heidelberg: Springer Berlin Heidelberg. Zhu, M., Kong, C., Zhuang, M., Zhang, Y., Lv, H., Ji, J., ... & Yang, L. (2020). Genome-wide identification and expression analysis of chitin-binding gene family in Brassica oleracea L. reveals its role in different disease resistance. Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J. D., Boller, T., & Felix, G. (2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell, 125 (4), 749-760.