تهیه سازه برای ایجاد مقاومت همزمان به سه ویروس ‏PLRV، ‏PVX‏ و ‏CMV‏ در سیب زمینی از طریق ‏RNA Silencing

نوع مقاله : مقاله پژوهشی

نویسندگان

1 کارشناسی ارشد، دانشکده کشاورزی دانشگاه ارومیه، ارومیه، ایران

2 استاد، دانشکده کشاورزی دانشگاه ارومیه، ارومیه، ایران

3 دانشیار، دانشکده کشاورزی دانشگاه شهید مدنی آذربایجان، تبریز، ایران

چکیده

سیب‌زمینی در جهان از نظر تولید پس از گندم، برنج و ذرت در جایگاه چهارم اهمیت قرار دارد. به دلیل تکثیر رویشی، ویروس‌ها از عوامل بیماریزای مهم در سیب­زمینی محسوب می­شوند. در گیاهان، خاموشی RNA یک ساز و کار دفاعی علیه ویروس‌هاست. تحقیقات زیادی برای ایجاد مقاومت به ویروس‌ها با خاموش کردن ژن­های خاص آنها صورت گرفته است، اما در نهایت به خاطر فعالیتِ پروتئین‌های ویروسیِ ممانعت کننده از خاموشیِ RNA، مقاومت گیاه شکسته شده و ویروس قادر به تکثیر و ایجاد خسارت می‌شود. هدف از این تحقیق ایجاد مقاومت هم‌زمان برای سه ویروس مهم PLRV، PVX و CMV در سیب­زمینی است. قطعاتی از ژن های پروتئین‌های سرکوبگر خاموشی RNA در ویروس‌های فوق، به ترتیب P0، P25 و 2b به کمک PCR با آغازگرهای اختصاصی تکثیر و به هم متصل شدند. قطعه نوترکیب ساخته شده در داخل پلاسمید pFGC5941 در جهت سنس و آنتی­سنس در دو طرف اینترون تحت پروموتر 35S بصورت تولید کننده RNA با ساختار سنجاق‌سری قرار گرفت. قسمت T-DNA پلاسمید حاصل به کمک آگروباکتریوم به سیب­زمینی رقم آگریا انتقال یافت. لاین­های تراریخته به‌دست‌آمده به‌منظور تأیید حضور ترانس­ژن، توسط PCR بررسی شدند و لاین‌های تراریخته انتخاب و تکثیر یافتند. در ادامه تحقیق قرار است مقاومت لاین­های منتخبِ تراریخته به سه ویروس سنجیده شود.

کلیدواژه‌ها

عنوان مقاله [English]

Preparation of a construct to establish simultaneous resistance against three PLRV, ‎PVX and CMV viruses in potato by RNA silencing method

نویسندگان [English]

  • Mona Bordar 1
  • Reza Darvishzadeh 2
  • Maqsoud Pazhouhandeh 3
1 M. Sc. Faculty of Agriculture, Urmia University, Urmia, Iran
2 Professor, Faculty of Agriculture, Urmia University, Urmia, Iran
3 Associate Professor, Faculty of Agriculture, Shahid Madani University, Tabriz, Iran
چکیده [English]

Potato is the world's fourth-largest food crop after maize, wheat, and rice. Potato is seriously affected by both single and mixed viral infections because of vegetative propagation. In plants, RNA silencing is a protective mechanism against viral infections. Some researchers have been used RNA silencing technique to silence viral genes, but ultimately, due to the activity of viral RNA silencing-suppressor proteins, the resistance of plants is broken down and the virus can replicate and make the damage. The purpose of this study was to provide simultaneous resistance to three important viruses including Potato X Virus, Potato Leafroll Virus, and Cucumber Mosaic Virus. From genes corresponding to proteins of suppressoer of RNA silencing in these viruses (P0, P25 and 2b, respectively), a fragment was amplified with specific primers by PCR and ligtaed to each other. The recombinant final fragment was cloned in the form of sense and antisense orientations with an intron between them, in pFGC5941 plasmid under 35S promoter to produce a hairpin RNA after transcription in plant. The T-DNA of recombinant hairpin vector was transformed into potato (cv. Agria) genome by Agrobacterium. The obtained transgenic plants were screened by PCR to confirm the presence of the transgenes. The resistance of selected tansgenic lines will be assessed against three viruses.

کلیدواژه‌ها [English]

  • Cloning
  • disease resistance
  • gene silencing
  • plant virus
  • transgenic plants‎

مراجع

  1. Ai, T., Zhang, L., Gao, Z., Zhu, C. X. & Guo, X. (2011). Highly efficient virus resistance mediated by artificial microRNAs that target the suppressor of PVX and PVY in plants. Plant Biology, 13(2), 304-316.
  2. Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell, 101(3), 235-238.
  3. Baulcombe, D. C., CHIU, M. H., CHEN, I. H. & TSAI, C. H. (2010). The silencing suppressor P25 of Potato Virus X interacts with Argonaute1 and mediates its degradation through the proteasome pathway. Molecular Plant Pathology, 11(5), 641-649.
  4. Bazzini, A. A., Asurmendi, S., Hopp, H. E. & Beachy, R. N. (2006). Tobacco mosaic virus (TMV) and potato virus X (PVX) coat proteins confer heterologous interference to PVX and TMV infection, respectively. Journal of General Virology, 87(4), 1005-1012.
  5. Bortolamiol, D., Pazhouhandeh, M., Marrocco, K., Genschik, P. & Ziegler-Graff, V. (2007). The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing. Current Biology, 17(18), 1615-1621.
  6. Chaube, H. S. & Pundhir, V. S. (2005). Crop diseases and their management. PHI Learning Pvt. Ltd.
  7. Chen, X., Liu, J., Xu, L., Jiang, F., Xie, X., Zhu, C. & Wen, F. (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.
  8. Covey, S. N., Al-Kaff, N. S., Langara, A. & Turner, D. S. (1997). Plants combat infection by gene silencing. Nature, 385(6619), 781-782
  9. Dalakouras, A., Tzanopoulou, M., Tsagris, M., Wassenegger, M. & Kalantidis, K. (2011). Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway. Transgenic Research, 20(2), 293-304.
  10. Dalakouras, A., Wassenegger, M., Dadami, E., Ganopoulos, I., Pappas, M. L. & Papadopoulou, K. (2020). Genetically modified organism-free RNA interference: Exogenous application of RNA molecules in plants. Plant Physiology, 182(1):38-50.
  11. Di Nicola-Negri, E., Brunetti, A., Tavazza, M. & Ilardi, V. (2005). Hairpin RNA-mediated silencing of plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transgenic Research, 14(6), 989-994.
  12. Doyle, J.J. & Doyle, J.L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf Phytochemistry Bulletin, 19,11-15.
  13. Fateri Rezvani, S., Pazhouhandeh, M., Shirzad, A. (2016). Production of potato resistant plant to PVX using an RNA silencing mechanism. Genetic Engineering and Biosafety Journal, 5(1), 1-14.
  14. Feng, D., Liu, X., Meng, K., Liao, L., Wei, X., Xu, H. & Zhu, Z. (2003). Silencing of potato virus X coat protein gene in transgenic tobaccos by codon replacement that confers resistance to PVX infection. Chinese Science Bulletin, 48(15), 1592-1598.
  15. Fletcher, J. D. (2012). A virus survey of New Zealand fresh process and seed potato crops during 201011. New Zealand Plant Protection, 65, 197-203.
  16. Gellért, Á., Nemes, K., Kádár, K., Salánki, K. & Balázs, E. (2012). The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination. Journal of Molecular Graphics and Modelling, 38, 446-454.
  17. González‐Jara, P., Tenllado, F., Martínez‐García, B., Atencio, F. A., Barajas, D., Vargas, M. & Díaz‐Ruíz, J. R. (2004). Host‐dependent differences during synergistic infection by Potyviruses with potato virus X. Molecular Plant Pathology, 5(1), 29-35.
  18. Hameed, A., Iqbal, Z., Asad, S. & Mansoor, S. (2014). Detection of multiple potato viruses in the field suggests synergistic interactions among potato viruses in Pakistan. The Plant Pathology Journal, 30(4), 407.
  19. Hu, Q., Niu, Y., Zhang, K., Liu, Y. & Zhou, X. (2011). Virus-derived transgenes expressing hairpin RNA give immunity to tobacco mosaic virus and cucumber mosaic virus. Virology Journal, 8(1), 41.
  20. Jeffries, C., Barker, H. & Khurana, S. M. P. (2006). Viruses and viroids. In: Handbook of Potato Production, Improvement, and Postharvest Management. Food Products Press, New York, USA, pp. 387-448.
  21. Koizumi, M., Shimotori, Y., Saeki, Y., Hirai, S., Oka, S. I. & Kodama, H. (2017). Effects of the 2b protein of cucumber mosaic virus subgroup IB strain IA on different transgene-induced RNA silencing pathways. Plant Molecular Biology Reporter, 35(2), 265-272.
  22. Lecoq, H., Lemaire, J. M. & Wipf-Scheibel, C. (1991). Control of zucchini yellow mosaic virus in squash by cross protection. Plant Disease, 75(2), 208-211.
  23. Lee, S. N., Choi, S. H., Ryu, K. B., Kim, H. H. & Ryu, K. H. (2011). Molecular and cytogenetic assessment of transgenic hot peppers resistant to cucumber mosaic virus. Horticulture, Environment, and Biotechnology, 52(2), 211-217.
  24. Lin, C. Y., Ku, H. M., Chiang, Y. H., Ho, H. Y., Yu, T. A. & Jan, F. J. (2012). Development of transgenic watermelon resistant to cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct. Transgenic Research, 21(5), 983-993.
  25. Lin, S. S., Wu, H. W., Jan, F. J., Hou, R. F. & Yeh, S. D. (2007). Modifications of the HC-Pro of zucchini yellow mosaic potyvirus for generation of attenuated mutants for cross protection against severe infection. Phytopathology, 97, 287-296.
  26. Mascia, T., Cillo, F., Fanelli, V., Finetti-Sialer, M. M., De Stradis, A., Palukaitis, P. & Gallitelli, D. (2010). Characterization of the interactions between cucumber mosaic virus and potato virus Y in mixed infections in tomato. Molecular Plant-Microbe Interactions, 23(11), 1514-1524.
  27. Matzke, M. A., Matzke, A. J., Pruss, G. J. & Vance, V. B. (2001). RNA-based silencing strategies in plants. Current Opinion in Genetics & Development, 11(2), 221-227.
  28. Miao, B., Chen, W. T., Xie, B. Y. & Yang, G. S. (2016). A novel strategy to enhance resistance to cucumber mosaic virus in tomato by grafting to transgenic rootstocks. Journal of Integrative Agriculture, 15(9), 2040-2048.
  29. Moissiard, G. & Voinnet, O. (2004). Viral suppression of RNA silencing in plants. Molecular Plant Pathology, 5(1), 71-82.
  30. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue Physiologia Plantarum, 15, 473-497.
  31. Pazhouhandeh M., Dieterle M., Marrocco K., Lechner E., Berry B., Brault V., Hemmer O., Kretsch T., Richards K.E., Genschik P. & Ziegler-Graff V. (2006). F-box-like domain in the Polerovirus protein P0 is required for silencing suppressor function. Proceedings of the National Academy of Sciences, 103(6):1994-1999.
  32. Pazhouhandeh, M., Bortolamiol, D., Marrocco, K., Genschik, P. & Ziegler-Graff, V. (2007). The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing. Current Biology, 17(18), 1615-1621.
  33. Ratcliff, F. G., MacFarlane, S. A. & Baulcombe, D. C. (1999). Gene silencing without DNA: RNA-mediated cross-protection between viruses. The Plant Cell, 11(7), 1207-1215.
  34. Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. (1997). A similarity between viral defense and gene silencing in plants. Science, 276(5318), 1558-1560.
  35. Rezazadeh, S., Sohani, M. M. & Rezadoost, M. H. (2017). Analysis of transgenic citrus (Citrus aurantium) plants expressing Citrus Tristeza Virus coat protein gene. Iranian Journal of Horticultural Science, 48, 79-91. (in Farsi).
  36. Sajjadi Fard, M., Pazhouhandeh, M., Shirzad, A., Mohajjel Shoja, H. (2016). Production of tabacco plant resistant to PVX by RNA silencing mechanism. Journal of Applied Researches in Plant Protection, 5(1), 103-115
  37. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular cloning; a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  38. Sharma, V. K., Kushwaha, N., Basu, S., Singh, A. K. & Chakraborty, S. (2015). Identification of siRNA generating hot spots in multiple viral suppressors to generate broad-spectrum antiviral resistance in plants. Physiology and Molecular Biology of Plants, 21(1), 9-18.
  39. Srinivasan, R. & Alvarez, J. M. (2007). Effect of mixed viral infections (potato virus Y–potato leafroll virus) on biology and preference of vectors Myzus persicae and Macrosiphum euphorbiae (Hemiptera: Aphididae). Journal of Economic Entomology, 100(3), 646-655.
  40. Syller, J. (2012). Facilitative and antagonistic interactions between plant viruses in mixed infections. Molecular Plant Pathology, 13(2), 204-216.
  41. Tollenaere, C., Susi, H. & Laine, A. L. (2016). Evolutionary and epidemiological implications of multiple infection in plants. Trends in Plant Science, 21(1), 80-90.
  42. Xie, X., Song, Y., Liu, X., Wang, S., Zhu, C. & Wen, F. (2014). Different target genes and chimeric-gene hairpin structures affect virus resistance mediated by RNA silencing in transgenic tobacco. Biologia Plantarum, 58(3), 575-581.
  43. Zaitlin, M. (1976). Viral cross-protection: More understanding is need. Phytopathology, 66, 382–383.
  44. Zhang, H., Demirer, G. S., Zhang, H., Ye, T., Goh, N. S., Aditham, A. J. & Landry, M. P. (2019). DNA nanostructures coordinate gene silencing in mature plants. Proceedings of the National Academy of Sciences, 116(15), 7543-7548.
  45. Zhao, M. M., An, D. R., Zhao, J., Huang, G. H., He, Z. H. & Chen, J. Y. (2006). Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection. Acta Biochimica et Biophysica Sinica, 38(1), 22-28.
علوم باغبانی ایران
دوره 53، شماره 2
شهریور 1401
صفحه 351-369

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سابقه مقاله

  • تاریخ دریافت: 21 اسفند 1398
  • تاریخ بازنگری: 17 مهر 1400
  • تاریخ پذیرش: 03 آبان 1400

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