بررسی مولکولی برخی تغییرات فیزیولوژیک در ژنوتیپ های حساس و متحمل ذرت در پاسخ به آلودگی ویروس موزاییک کوتولگی ذرت (Maize Dwarf Mosaic Virus; MDMV)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری بیماری شناسی گیاهی گروه گیاهپزشکی دانشگاه زابل.

2 دانشیار گروه زیست شناسی پردیس علوم دانشگاه یزد.

3 استادیار گروه اصلاح نباتات وبیوتکنولوژی دانشگاه علوم کشاورزی ومنابع طبیعی گرگان.

4 دانشیار گروه گیاهپزشکی دانشگاه علوم کشاورزی ومنابع طبیعی گرگان.

5 دانشیار گروه گیاهپزشکی دانشکده کشاورزی دانشگاه زابل.

چکیده

چکیده
در این تحقیق تغییرات فعالیت آنزیم­های آنتی­اکسیدانی کاتالاز و پراکسیداز به روش طیف سنجی و سطح بیان ژن­های NPR1، FKBP، Chlorophyll a-b binding protein و Metallothionein-like protein به روش واکنش زنجیره­ای پلی­مراز کمی (qRT-PCR) در دو ژنوتیپ متحمل (هیبرید 8) و حساس (SC705) ذرت در پاسخ به بیماری ویروس موزاییک کوتولوگی ذرت مورد ارزیابی قرار گرفت. آزمایش در شرایط گلخانه بصورت مایه­زنی مکانیکی صورت گرفت و نمونه­برداری در زمان­های 0، 1، 9، 24 و 72 ساعت انجام شد. نتایج نشان دهنده­ی افزایش فعالیت پراکسیداز در ژنوتیپ متحمل در کلیه­ی زمان­های نمونه­برداری نسبت به ژنوتیپ حساس بود. بالاترین میزان افزایش در بازه­ی زمانی 24 ساعت بعد از مایه­زنی درمقایسه با شاهد ثبت گردید. این میزان در بازه­ی زمانی 72 ساعت بعد از آلوده­سازی نسبت به گیاهان کنترل کاهش یافت. فعالیت آنزیم کاتالاز در تمام بازه­های زمانی بعد از آلودگی به میزان قابل ملاحظه­ای نسبت به گیاهان کنترل کمتر بود. تغییرات بیان ژن­های NPR1 و MT-LPدر هر دو ژنوتیپ افزیش داشته ولی این افزایش در گیاه متحمل بیشتر بود. میزان بیان ژن FKBPدر ژنوتیپ متحمل از اولین ساعت پس از آلودگی ویروسی افزایش یافت و با سرعت بیشتری نسبت به گیاه حساس کاهش یافت. تغییرات بیان در ژن Chlorophyll a-bدر گیاه حساس کاهش معنی­داری نسبت به گیاهان شاهد داشت. تغییر فعالیت آنزیم­های آنتی اکسیدانی و میزان رونوشت­ ژن­ها در آلودگی ویروسی می­تواند در مطالعات تعامل بین گیاه و ویروس به عنوان مارکر زیستی انتخابی برای مقاومت به بیماری­های ویروسی مورد استفاده قرار گیرد.

 

کلیدواژه‌ها


عنوان مقاله [English]

Molecular Study on Some of Physiological Changes in Susceptible and Tolerant Genotypes of Maize in Response to Maize Dwarf Mosaic Virus Infection

نویسندگان [English]

  • Farveh Sadat Mostafavi Neishaburi 1
  • Seyed kazem Sabbagh 2
  • Ahmad Yamchi 3
  • Saied Nasrollahnejad 4
  • Naser Panjekeh 5
1 Ph. D. Student of Plant Pathology, Department of Plant Protection, Faculty of Agriculture, University of Zabol, Iran.
2 Associate Professor, Department of Biology, Campus of Sciences. Yazd University, Iran.
3 Assistant Professor, Department of Plant Breeding & Biotechnology, Gorgon University of Agricultural Sciences & Natural Resources, Iran.
4 Associate Professor, Department of Plant Protection, Gorgon University of Agricultural Sciences & Natural Resources, Iran.
5 Associate Professor, Department of Plant Protection, Faculty of Agriculture, University of Zabol, Iran.
چکیده [English]

Abstract
In this work, the changes of antioxidant enzymes activity were studied by spectrophotometry method and expression level of NPR1، FKBP، Chlorophyll a-b binding protein andMetallothionein-like protein genes in two tolerant and susceptible genotypes of maize in response to maize dwarf mosaic virus was investigated by qRT-PCR method. Experiment was done in green-house condition by mechanical inoculation and sampling was carried out at different time intervals (0, 1, 9, 24,72) hors post inoculation (hpi). The results showed a significant increase of peroxidase activity in the tolerant genotype at all sampling times when compared to the susceptible genotype. The highest increase of peroxidase enzyme was recorded at 24hpi for both genotypes in comparison to the control plants, but after 72hpi the rate of peroxidase enzyme was reduced. Catalase enzyme activity was significantly reduced at all sampling time after virus inoculation when compared to the control plants. Gene expression analysis showed that the related expression of NPR1 and MT-LP genes were increased in both genotypes but this increase was significant only in the tolerant genotype. The expression level of FKBP gene in the tolerant genotype increased at the early times after virus inoculation and was decreased more rapidly than susceptible cultivar. The expression changes of Chlorophyll a-b binding gene plants significantly reduced in susceptible plants compared to the control plants. Changes in the antioxidant enzyme activity and gene expression could be used as bio-markers for virus-plant interaction studies and the associated markers may be selected for plant resistance to viral disease

کلیدواژه‌ها [English]

  • Keywords: Catalase
  • Gene Expression
  • Maize dwarf mosaic virus
  • Peroxidase
Ashfaq M, Aslam Khan M, Javed N, Mughal SM, Shahid M and Sahi ST, 2010. Effect of urdbean leaf crinkle virus infection on total soluble protein and antioxidant enzymes in blackgram plants. Pakistan journal of Botany 42: 447-454.
Bindschedler LV, Dewdney J, Blee KA, Stone MJ, Tsuneaki, A, Plotnikov J, Denoux C, Hayes T, Gerrish Ch, Davies DR, Ausubel FM and Bolwell GP, 2006. Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant Journal 47: 851–863.

Cao H, Li X and Dong X, 1998. Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proceedings of the National Academy of Sciences 95: 6531-6536.

Cheng Ch, Zhang Y, Zhong Y, Yang J and Yan Sh, 2016. Gene expression changes in leaves of Citrus sinensis (L.) Osbeck infected by Citrus tristeza virus. The Journal of Horticultural Science and Biotechnology 91: 466-475.
Choi D, Kim HM, Yun HK, Park J, Kim WT and Bok SH, 1996. Molecular Cloning of a Metallothionein-Like Gene from Nicofiana glufinosa L. and Its induction by Wounding and Tobacco Mosaic Virus infection. Plant Physiology 11: 353-359.
Clarke SF, Guy PL, Burrit DJ and Jameson PE, 2002. Changes in the activities of antioxidant enzymes in response to virus infection and hormone treatment. Physiologia Plantarum 114(2): 157-164.
Clemente-Moreno MJDíaz-Vivancos PRubio MFernández-García N and Hernández JA, 2013. Chloroplast protection in Plum pox virus-infected peach plants by L-2-oxo-4-thiazolidine-carboxylic acid treatments: effect in the proteome. Plant, Cell Environment 36: 640-654.
Converse RH and Martin RR, 1990. ELISA methods for plant viruses. APS Press. pp. 179-196.
Cueto-Ginzo IA, Serrano L, Sin E, Rodríguez R, Morales JG, Lade SB, Medina V and Achon MA, 2016. Exogenous salicylic acid treatment delays initial infection and counteracts alterations induced by Maize dwarf mosaic virus (MDMV) in the maize proteome. Physiological and Molecular Plant Pathology 96: 47-59.
Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D andVan Breusegem F, 2000. Dual action of the active oxygen species during plant stress responses. Cellular and Molecular Life Sciences57: 779-795.
Deepaka Sh, Niranjan-Raja S, Shailasreea Sh, Kinia RK, Boland W, Shettya HS and Mithofer A, 2007. Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon. Physiological and Molecular Plant Pathology 71: 96-105.
Di Carli M, Villani ME, Bianco L, Lombardi R, Perrotta G, Benvenuto E and Donini M, 2010. Proteomic analysis of the plant-virus interaction in Cucumber mosaic virus (CMV) resistant transgenic tomato. Journal of Proteome Research 9: 5684-5697.
Doubnerova V, Janoskova M, Synkova H, Subr Z, Cerovska N and Ryslava H, 2007. Effect of potato virus y on the activities of antioxidant and anaplerotic enzymes in Nicotiana tabacum l. transgenic plants transformed with the gene for p3 protein. Genetic Applied Plant Pathology 33: 123-140.
Du YY, Wang PC, Chen J and Song CP, 2007. The comprehensive functional analysis of catalase gene family in Arabidopsis thaliana. Journal of Integrative Plant Biology 121: 16-23.
Ford RE, Tosic M, Shukla DD, 2004. Maize dwarf Mosaic Virus. AAB Descriptions of Plant Viruses Online: Description no. 341.
Gandia M, Conesa A, Ancillo G, Gadea J, Forment J, Pallás V and Guerri J, 2007. Transcriptional response of Citrus aurantifolia to infection by Citrus tristeza virus. Virology 367: 298–306.
Galvez-Valdivieso G, Mullineaux PM, 2010. The role of reactive oxygen species in signalling from chloroplasts to the nucleus. Physiologia Plantarum 138: 430-439.
Goncalves LSA, Rodrigues R, Diz MSS, Robaina RR, Do Amaral Junior AT, Carvalho AO and Gomes VM, 2013. Peroxidase is involved in Pepper yellow mosaic virus resistance in Capsicum baccatum var. pendulum. Genetic and molecular research 12: 1411-1420.
Hernandez JA, Talavera JM, Martínez-Gómez P, Dicenta F and Sevilla F, 2001. Response of antioxidative enzymes to plum pox virus in two apricot cultivars. Physiologia Plantarum 111: 313-321.
Lee DH and Lee CB, 2000. Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays. Plant Science 159: 75-85.
Liu Y, Wang G, Wang Z, Yang F, Wu G and Hong N, 2012. Identification of differentially expressed genes in response to infection of a mild Citrus tristeza virus isolate in Citrus aurantifolia by suppression subtractive hybridization. Scientia Horticulturae 134: 144–149.
Luck H, 1974. Methods in Enzymatic Analysis. 2nd Ed. Academic Press. New York.
Ludmerszki E, Almasi A, Racz I, Szigeti Z, Solti A, Olah C and Rudnoy S, 2015. S-methylmethionine contributes to enhanced defense againstMaize dwarf mosaic virus infection in maize. Brazilian Journal of Botany 38(4): 771–782.
Madhusudhan KN, Srikanta BM, Shylaja MD, Prakash HS and ShettyHS, 2008. Changes in antioxidant enzymes, hydrogen peroxide, salicylic acid and oxidative stress in compatible and incompatible host-tobamovirus interaction. Journal of Plant Interactions 4: 157-166.
Maule A, Leh V and Lederer C, 2002. The dialogue between viruses and hosts in compatible interactions. Current Opinion in Plant Biology 5: 279–284.
Mohammadi, M and Kazemi H, 2002. Changes in peroxidase and polyphenol oxidase activities in susceptible and resistant wheat heads inoculated with Fusarium graminearum and induced resistance. Journal of Plant Science 162: 491-498.
Morris K, Mackerness SA, Page T, John CF, Murphy AM, Carr JP and Buchanan-Wollaston V, 2000. Salicylic acid has a role in regulating gene expression during leaf senescence. Plant Journal 23: 677- 685.
Mostafavi Neishaburi FS, Masumi M, Nasrollanejad S, Rahpeyma-Sarvestani N, Izadpanah K. 2015. Analyses of complete nucleotide sequence of iranian isolate of maize dwarf mosaic virus (MDMV) and notes on the origin and evolution of MDMV. Iranian Journal of Plant Pathology 51:69-81.
Neuenschwander, U., Vernooij, B., Friedrich, L, Uknes, S., Kessmann, H. & Ryals, J. (1995). Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?. Plant Journal, 8,227_233.
Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research, 29:2002–2007.
Pechanova O and Pechan T, 2015. Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective. International Journal of Molecular Sciences 16: 28429–28448.
Pineda M, Sajnani C and Baron M, 2010. Changes induced by the Pepper mild mottle tobamoviruson the chloroplast proteome of Nicotiana benthamiana. Photosynthesis Research103: 31.
Reuveni R, 1995. Biochemical markers as tools for screening resistance against plant pathogens, In: Reuveni, R. (Eds.), Novel Approaches to Integrated Pest Management, CRC Press, Boca Raton, FL, pp. 21-45.
Riedle-Bauer M, 2000. Role of reactive oxygen species and antioxidant enzymes in systemic virus infections of plants. Journal of Phytopathology 148:297_302.
Uzarowska A, Dionisio G, Sarholz B, Piepho HP, Xu M, Ingvardsen CR, Wenzel G and Lubberstedt T, 2009. Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling. BMC Plant Biology 9: 15.
WangWW, MaQ, XiangY, ZhuSW and ChengBJ, 2012. Genome-wide analysis of immunophilin FKBP genes and expression patterns in Zea mays. Genetics and Molecular Research 11: 1690-700.
Wu L, Wang S, Chen X, Wang X and Wu L, 2013. Proteomic and Phytohormone Analysis of the Response of Maize (Zea mays L.) Seedlings to Sugarcane Mosaic Virus. PLoS ONE 8: 1-17.
Xu Q, Ni H, Chen Q, Sun F, Zhou T, Lan Y and Zhou Y, 2013. Comparative proteomic analysis reveals the cross-talk between the responses induced by H2O2 and by long-term Rice Black-streaked dwarf virus infection in rice. PLoS ONE 8: 81640.
Yang F, Wang G, Jiang B, Liu YH, Liu Y, Wu GW and Hong N, 2013. Differentially expressed genes and temporal and spatial expression of genes during interactions between Mexican lime (Citrus aurantifolia) and a severe Citrus tristeza virus isolate. Physiological and Molecular Plant Pathology 83: 17–24.
Yang H, Huang Y, Zhi H and Yu D, 2011. Proteomics-based analysis of novel genes involved in response towards soybean mosaic virus infection, Molecular Biology Reports 38: 511-521.
Zare Z, Nasrollahnejad N, Sadeghi Z, Mosavat A, Rzinotaj M. 2016. Effect of maize dwarf mosaic virus in morphological features of 12 hybrids of maize. Thesis in Gorgan University of Agricultural Science and Natural Resources. 120 pp.