تهیۀ گیاه توتون تراژن مصون به طیف گسترده‌ای از سویه‌های ویروس وای سیب‌زمینی

خاطری, هادی; مصاحبی‌محمدی, غلامحسین; وینتر, اشتفان; کوهی‌حبیبی, مینا; دیزجی, اکبر

doi:10.22059/ijpps.2014.53498

تهیۀ گیاه توتون تراژن مصون به طیف گسترده‌ای از سویه‌های ویروس وای سیب‌زمینی

نوع مقاله : مقاله پژوهشی

نویسندگان

¹ گروه گیاه‌پزشکی پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

² بخش ویروس‌های گیاهی DSMZ آلمان، برانشوایگ

10.22059/ijpps.2014.53498

چکیده

ویروس وای سیب‌زمینی (Potato virus Y, PVY) از مهم‌ترین عوامل خسارت‌زا در مزارع توتون مناطق مختلف استان‌های گلستان، مازندران و گیلان است. با توجه به بی‌تأثیری حشره‌کش‌ها در کاهش آلودگی به PVY، شکسته شدن منابع طبیعی مقاومت توسط سویه‌های خاص و مشکلات روش‌های سنتی اصلاحی، به‌کارگیری روش‌های جایگزین ایجاد مقاومت به PVY در توتون با به‌کارگیری مقاومت مشتق‌شده از بیمارگر می‌تواند راهکاری برای کاهش خسارت در نظر گرفته شود. در این پژوهش گیاهان توتون تراژنی تولید شدند که در برابر طیف گسترده‌ای از جدایه‌های PVY از سویه‌های مختلف این ویروس مقاوم‌اند. ناحیه‌ای به اندازۀ ۴۷۲ جفت‌باز از ژنوم یک جدایۀ ایرانی PVY شامل توالی نوکلئوتیدی بخش‌هایی از CP و 3'UTR به منظور تهیۀ یک سازۀ سنجاق‌سری برای مقاومت به PVY به‌کار گرفته شد و تراژن‌سازی توتون رقم Wisconsin 38 با این سازه به کمک اگروباکتریوم انجام گرفت. ۶۱ درصد از گیاهان تراژن T0 حاصل به PVY مقاوم بودند و انتقال مقاومت به نسل بعد در 9 لاین مختلف با استفاده از روش داس الایزا تأیید شد. یکی از این لاین‌ها با ۱۲ جدایۀ مختلف PVY شامل چهار جدایۀ ایرانی و هشت جدایه از کشورهای دیگر تحت آزمایش قرار گرفت و مصونیت آن بر اساس عدم ظهور علائم و نتایج آزمون‌‌های داس الایزا و RT-PCR به تأیید رسید. این پژوهش نشان داد که سازۀ سنجاق‌سری به‌کاررفته دارای کارایی بسیار زیادی برای ایجاد توتون تراژن مصون در مقابل سویه‌های مختلف PVY بوده است.

کلیدواژه‌ها

20.1001.1.20084781.1393.45.2.4.3

عنوان مقاله [English]

Generation of transgenic tobacco plants with immunity against a broad spectrum of Potato virus Y strains

نویسندگان [English]

Hadi Khateri ¹
Gholam-Hosein Mosahebi-Mohammadi ¹
Stephan Winter ²
Mina Koohi-Habibi ¹
Akbar Dizadji ¹

¹ Department of Plant Protection, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

² Plant virus Department, German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany.

چکیده [English]

Potato virus Y (PVY) is one of the most important plant viruses affecting tobacco fields in Golestan, Mazandaran and Guilan provinces (Iran). Given the lack of insecticide impact in reducing infection to PVY, breakdown of the natural resistance resources by certain strains and the problems with traditional breeding methods, the use of alternative methods to generate PVY resistant tobacco by pathogen-derived resistance could be considered as a method for reducing the damages. The objective of this study was to produce transgenic tobacco plants with resistance against a diverse spectrum of PVY isolates from different strains. A 472 bp fragment of the genome of an Iranian PVY isolate including the partial nucleotide sequences of CP and 3'UTR regions was used for constructing a hairpin structure for PVY resistance. This construct was used for Agrobacterium-mediated transformation of tobacco variety Wisconsin 38. Following transformation, 61% of the resulting transgenic plants in T0 were resistant to PVY and the inheritance of resistance to the next generation of nine different lines was confirmed using DAS-ELISA which confirmed the immunity in all tested plants. One of these lines was tested against 12 different PVY isolates, including four Iranian and eight foreign ones, and the immunity against PVY was confirmed based on the lack of symptoms and the results of DAS-ELISA and RT-PCR tests. The results showed that the hairpin construct had a high-performance for generating transgenic tobacco plants with resistance against different PVY strains

کلیدواژه‌ها [English]

hairpin construct
broad-spectrum resistance
Agrobacterium-mediated transformation

مراجع

1. Abedi, H. (1998). Appearance of PVY in tobacco fields of Iran (Regions of Mazandaran and Gorgaan). In: Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Congress, 11-15 Oct., Brighton, England, CORESTA, p. 146.

2. Bau, H. J., Cheng, Y. H., Yu, T. A., Yang, J. S. & Yeh, S. D. (2003). Broad-spectrum resistance to different geographic strains of Papaya ringspot virus in coat protein gene transgenic papaya. Phytopathology, 93 (1), 112-120.

3. Beachy, R. N. (1990). Coat protein mediated resistance in transgenic plants. In: T. P. Pirone and J. G. Shaw (Eds), Viral Genes and Plant Pathogenesis. (Pp. 13-22). New York: Springer.

4. Bhaskar, P. B. & Jiang, J. (2010). Silencing as a tool for transgenic crop improvement. In: C. Kole, C. H. Michler, A. G. Abbott and T. C. Hall (Eds), Transgenic Crop Plants: Principles and Development. (Pp. 187-199). Berlin Heidelberg: Springer-Verlag.

5. Bravo-Almonacid, F., Rudoy, V., Welin, B., Segretin, M. E., Bedogni, M. C., Stolowicz, F., Criscuolo, M., Foti, M., Gomez, M., Lopez, M., Serino, G., Cabral, S., Dos Santos, C., Huarte, M. & Mentaberry, A. (2012). Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina. Transgenic Research, 21 (5), 967–982.

6. Chen, X. M., Liu, J., Xu, L., Jiang, F., Xie, X. Y., Zhu, C. X. & Wen, F. J. (2010). Inhibiting virus infection by RNA interference of the eight functional genes of the Potato Virus Y genome. Journal of Phytopathology, 158 (11-12), 776–784.

7. FAO (2011). FAOSTAT-Agriculture, Production Statistics, Food and Agriculture Organization of the United Nations Statistical Databases.

8. Fatima, T., Rivera-Dom´ınguez, M., Troncoso-Rojas, R., Tiznado-Hern´andez, M. ı.-E., Handa, A. K. & Mattoo, A. K. (2008). Tomato. In: C. Kole and T. C. Hall (Eds), Compendium of Transgenic Crop Plants: Transgenic Vegetable Crops. (Pp. 1-45). Blackwell Publishing Ltd.

9. Gray, S., Whitworth, J., Xu, H. & Singh, R. (2013). The current state (2012) of Potato virus Y (PVY) affecting potato grown in North America. In North American Plant Protection Organization (NAPPO) Science and Technology Documents.

10. Guerineau, F. (1995). Tools for expressing foreign genes in plants. In: H. Jones (Ed), Plant Gene Transfer and Expresssion Protocols. (Pp. 1-32). Totowa, NJ: Humana Press Inc.

11. Hull, R. (2009). Comparative Plant Virology (2nd). Academic Press.

12. Kavosipour, S., Niazi, A., Izadpanah, K., Afsharifar, A. & Yasaie, M. (2012). Induction of resistance to Cucumber mosaic virus (CMV) using hairpin construct of 2b gene. Iranian Journal of Plant Pathology, 48 (2), 65-67. (In Farsi).

13. Lacroix, C., Glais, L., Kerlan, C., Verrier, J. L. & Jacquot, E. (2010). Biological characterization of French Potato virus Y (PVY) isolates collected from PVY-susceptible or -resistant tobacco plants possessing the recessive resistance gene va. Plant Pathology, 59 (6), 1133-1143.

14. Latorre, B. A. & Flores, V. (1985). Strain identification and cross-protection of Potato Virus Y affecting tobacco in Chile. Plant Disease, 69 (11), 930-932.

15. Lawson, C., Kaniewski, W., Haley, L., Rozman, R., Newell, C., Sanders, P. & Tumer, N. E. (1990). Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank. Biotechnology (N Y), 8 (2), 127-134.

16. Lindbo, J. A., Silva-Rosales, L. & Dougherty, W. G. (1993). Pathogen derived resistance to potyviruses: working, but why? Seminars in Virology, 4 (6), 369-379.

17. Mackenzie, A. M., Nolan, M., Wei, K. J., Clements, M. A., Gowanlock, D., Wallace, B. J. & Gibbs, A. J. (1998). Ceratobium mosaic potyvirus: another virus from orchids. Archives of Virology, 143 (5), 903-914.

18. Martin, R. C., Mok, D. W. S., Smets, R., Van Onckelen, H. A. & Mok, M. C. (2001). Development of transgenic tobacco harboring a zeatin O-glucosyltransferase gene from Phaseolus. In Vitro Cellular & Developmental Biology-Plant, 37 (3), 354-360.

19. Missiou, A., Kalantidis, K., Boutla, A., Tzortzakaki, S., Tabler, M. & Tsagris, M. (2004). Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Molecular Breeding, 14 (2), 185-197.

20. Mitter, N., Sulistyowati, E. & Dietzgen, R. G. (2003). Cucumber mosaic virus Infection Transiently Breaks dsRNA-Induced Transgenic Immunity to Potato virus Y in Tobacco. Molecular Plant-Microbe Interactions, 16 (10), 936-944.

21. Mitter, N., Sulistyowati, E., Graham, M. W. & Dietzgen, R. G. (2001). Suppression of gene silencing: a threat to virus-resistant transgenic plants? Trends in Plant Science, 6 (6), 246-247.

22. Pourrahim, R., Ahounmanesh, A., Hashemi, H., Zeynali, S. & Farzadfar, S. H. (2006). Assessment of virus resistance in transgenic Nicotiana tabacum cv. Samsun lines against three Iranian isolates of potato virus Y. Applied Entomology and Phytopathology, 73 (2), 17-38. (In Farsi).

23. Powell-Abel, P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T. & Beachy, R. N. (1986). Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science, 232 (4751), 738-743.

24. Raccah, B. (1986). Nonpersistent viruses: epidemiology and control. Advances in Virus Research, 31, 387-429.

25. Raccah, B. & Fereres, A. (2009). Plant virus transmission by insects. Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd.

26. Sambrook, J. & Russell, D. (2001). Molecular Cloning: A Laboratory Manual (3rd). New York, USA: Cold Spring Harbor Laboratory Press.

27. Schubert, J., Matoušek, J. & Supp, P. (2005). Stability of pathogen-derived Potato virus Y resistance in potato under field conditions and some aspects of their ecological impact. In: J. H. H. Wesseler (Ed), Environmental Costs and Benefits of Transgenic Crops. (Pp. 63-78).

28. Simon-Mateo, C. & Garcia, J. A. (2011). Antiviral strategies in plants based on RNA silencing. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1809 (11-12), 722-731.

29. Smith, N. A., Singh, S. P., Wang, M. B., Stoutjesdijk, P. A., Green, A. G. & Waterhouse, P. M. (2000). Total silencing by intron-spliced hairpin RNAs. Nature, 407 (6802), 319-320.

30. Stark, D. M. & Beachy, R. N. (1989). Protection against potyvirus infection in transgenic plants: evidence for broad spectrum resistance. Nature Biotechnology, 7 (12), 1257-1262.

31. Sudarsono, J., Woloshuk, S., Parry, D., Hellmann, G., Wernsman, E., Lommel, S. & Weissinger, A. (1995). Transgenic burley and flue-cured tobacco with resistance to four necrotic isolates of potato virus Y. Phytopathology, 85, 1493-1506.

32. van der Vlugt, R. A., Ruiter, R. K. & Goldbach, R. (1992). Evidence for sense RNA-mediated protection to PVY^N in tobacco plants transformed with the viral coat protein cistron. Plant Molecular Biology, 20 (4), 631-639.

33. Vancanneyt, G., Schmidt, R., O'Connor-Sanchez, A., Willmitzer, L. & Rocha-Sosa, M. (1990). Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Molecular and General Genetics MGG, 220 (2), 245-250.

34. Vargas, M., Martinez-Garcia, B., Diaz-Ruiz, J. R. & Tenllado, F. (2008). Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids. Virology Journal, 5.

35. Waterhouse, P. M., Graham, M. W. & Wang, M. B. (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences, 95 (23), 13959-13964.

36. Wise, A. A., Liu, Z. & Binns, A. N. (2006). Culture and maintenance of Agrobacterium strains. Methods in Molecular Biology, 343, 3-13.

37. Zhu, J., Zhu, X., Wen, F., Bai, Q., Zhu, C. & Song, Y. (2004). Effect of cDNA fragments in different length derived from potato virus Y coat protein gene on the induction of RNA-mediated virus resistance. Science in China series C Life Sciences, 47 (4), 382-388.