آنالیز گیاهان نارنج (Citrus aurantium) تراریخت حامل ژن پروتئین پوششی ویروس تریستیزای مرکبات

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی سابق کارشناسی ارشد، گروه بیوتکنولوژی، دانشکده علوم کشاورزی، دانشگاه گیلان، رشت، ایران

2 دانشیار، گروه بیوتکنولوژی، دانشکده علوم کشاورزی، دانشگاه گیلان، رشت، ایران

چکیده

درخت نارنج دارای ویژگی‌های با ارزشِ یک پایه ایده‌آل در مرکبات است اما، این گیاه به بیماری ویروس تریستزای مرکبات (CTV) به شدت حساس است. بر این اساس، بذور نارنج در شرایط in vitro کشت و به مدت 4 هفته درتاریکی و10 روز در روشنایی رشد کردند. ریز نمونه ها از اپی‌کوتیل وهیپوکوتیل تهیه و با استفاده از Agrobacterium tumefaciens نژاد EHA105 حامل وکتور خاموشی pFGC5941 و بخشی از ژن کدکننده پوشش پروتئینی CTV، به مدت 3روز هم‌کشت شدند. سپس ریزنمونه‌ها به محیط کشت انتخابی حاوی علفکش بستا و ترکیبی از تنظیم کننده‌های رشد BAP و NAA منتقل شدند. در اولین غربالگری تعدادی برگ از گیاهچه‌های تراریخت احتمالی در محیطهای MS مایع و همچنین MS جامد حاوی غلظت‌های مختلف علفکش بستا منتقل شدند. تعدادی از قطعات برگی در محیط انتخابی به رنگ سبز باقی ماندند و برگ گیاهان شاهد و غیرتراریخت‌ها سفید رنگ شدند. در مرحله بعد واکنش PCR با آغازگرهای اختصاصی ژن‌های CTV و BAR در میان گیاهان باقی مانده از غربالگری اولیه انجام و برخی از باندها توالی یابی شدند. تعداد نسخه‌های تراژن CTV با استفاده از تکنیک quantitative Real-Time در تعدادی از گیاهچه‌های نارنج محاسبه و تعداد آن‌ها بین 4-1 نسخه در ژنوم تعیین شد. تکثیر ویروس مطالعه و تست الایزا نشان داد که ویروس در گیاهان تراریخت تکثیر نشده است. در این تحقیق، روش‌های آسان و اقتصادی  برای غربالگری اجرا شد که با استفاده از آن‌ها تمایز درست گیاهان تراریخت در مقابل غیرتراریخت نارنج امکان‌پذیر شد.

کلیدواژه‌ها

عنوان مقاله [English]

Analysis of transgenic citrus (Citrus aurantium L.) plants expressing Citrus Tristeza Virus coat protein gene

نویسندگان [English]

  • Sakineh Rezazadeh 1
  • Mohammad Mehdi Sohani 2
  • Mohammad Hossein Rezadoost 1
1 Former M. Sc. Student, Department of Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran
2 Assocaite Professor, Department of Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran
چکیده [English]

Due to high quality of fruits, resistance to various pathogens and abiotic stress, Citrus aurantium is widely used and considered as the most favorable rootstock worldwide. Genetic engineering approaches such as pathogen-derived resistance (PDR), is a common practice in citrus breeding. Analysis of transgenic plants requires reliable and quick methods for early screening of T0 generation. In a PDR approach, a mosaic gene from Shiraz CTV strains was cloned and transferred into sour orange. Forty putative transformed shoots were isolated on selective medium, which were acclimatized and transferred to growth room. In the first screening method, leaf assay for Basta resistance was performed on liquid as well as solid selective medium. To further analyze the putative transgenic seedlings, PCR using CTV and BAR gene specific primers were performed and some of the PCR products were randomly chosen for sequencing. The transgene copy number(s) in individual genotypes was resolved using real-time PCR technique. Finally, the functionality of the transgene was provided using ELISA test, which confirmed that CTV amplification suppressed in five tested seedlings. In this experiment, the simple and economical screening methods were set up, which made unambiguous discrimination between transgenic and non-transgenic citruspossible.

کلیدواژه‌ها [English]

  • ELISA test
  • Herbicide resistance
  • Pathogen-derived resistance
  • Transgene copy number

مراجع

  1. Baulcombe, D. C. (1996). Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell, 8, 1833-1844.
  2. Beltra´n, J., Jaimes, H., Echeverry, M., Ladino, Y. & Lo´pez, D. (2009) Quantitative analysis of transgenes in cassava plants using real-time PCR technology. In Vitro Cellular & Developmental Biology, 45, 48-56.
  3. Chabirand, A., Anthoine, G., Fax, A. & Laurenson, L. (2016) Quality assurance for molecular testing in plant health In: N. B., Fera, J. T. Fera, R. M. Fera (Ed), Molecular Methods in Plant Disease Diagnostics Principles and Protocols, pp. 173-193.
  4. Converse, R. H. & Martin, R. R. (1990). ELISA methods for plant viruses. In: R.O. Hampton, E.M. Ball, S.H. De Boer (Ed), Serological methods for detection and identification of viral and bacterial plant pathogens. (pp.179-196) APS Press, USA.
  5. Costa, L. D., Vaccari, I., Mandolini, M. & Martinelli, L. (2009). Elaboration of a Reliable strategy based on real-time PCR to characterize genetically modified plantlets and to evaluate the efficiency of a marker gene removal in grape (Vitis spp.). Journal of Agricultural and Food Chemistry, 57(7), 2668-77.
  6. Doshi, K. M., Eudes, F., Laroche, A. & Gaudet, D. (2007). Anthocyanin expression in marker free transgenic wheat and triticale embryos. In Vitro Cellular & Developmental Biology - Plant, 43, 429-435.
  7. Craig, W., Gargano, D., Scotti, N., Nguyen, T. T., Lao, N. T., Kavanagh, T. A., Dix, P. J. & Cardi, T. (2005). Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG mediated transformation of protoplasts. Plant Cell Reports, 24, 603-611.
  8. Dellaporta, S. L., Wood, J. & Hicks, J. B. (1983). A plant DNA minipreparation: version II. Plant Molecular Biology Reporter, 1, 19-21.
  9. FAOSTAT database results. (2007). http://apps.fao.org/ lim500/nph-wrap.pl
  10. Fuchs, M. & Gonsalves, D. (1995). Resistance of transgenic hybrid squash ZW-20 expressing the coat protein genes of zucchini yellow mosaic virus and watermelon mosaic virus to mixed infections of both potyviruses. Nature Biotechnology, 13, 1466-1473.
  11. Grosser, J. W. & Gmitter, F. G. (2005). Applications of somatic hybridization and cybridization in crop improvement, with citrus as a model. In Vitro Cellular and Developmental Biology-Plant, 41, 220-225.
  12. Huang, Y., Yin, X., Zhu, C., Wang, W., Grierson, D., Xu, C. & Chen, K. (2011), Standard Addition Quantitative Real-Time PCR (SAQPCR): A Novel Approach for Determination of Transgene Copy Number Avoiding PCR Efficiency Estimation. PLOS, 8(1), e53489.
  13. Iyer, L. M., Kumpatla, S. P., Chandrasekharan, M. B. & Hall, T. C. (2000). Transgene silencing in monocots. Plant Molecular Biology, 43, 323-346.
  14. James, V. A., Avart, C., Worland, B., Snape, J. W. & Vain, P. (2002). The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theoretical and Applied Genetics, 104, 553-561.
  15. Kariola, T., Brader. G., Li, J. & Palva, E. T. (2005). Chlorophyllase 1, a damage control enzyme, affects the balance between defense pathways in plants. Plant Cell, 17, 282-294.
  16. Mason, G., Provero, P., Vaira, A. M. & Accotto, G. P. (2002). Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnology, 2, 20.
  17. Miyao, A., Tanaka, K., Murata, K., Sawaki, H., Takeda, S. et al. (2003) Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell, 15, 1771-1780.
  18. Murakami, T., Anzai, H., Imai, S., Saoh, A., Nagaoka, K. & Thompson, C. J. (1986). The bialophos biosynthetic genes of Streptomyces hygroscopicus: molecular cloning and characterization of gene cluster. Molecular Genetics and Genomics, 205, 42-50.
  19. Mendes-Da-Gloria, F. J., Mourao Filho, F. A. A. & Mendes, B. M. J. (2000). Caipira sweet orange rangpur lime: a somatic hybrid with potential for use as rootstock in the Brazilian citrus industry. Genetics and Molecular Biology, 23, 661-665.
  20. Miki, B. & McHugh, S. (2004). Selectable marker genes in transgenic plants: applications, alternatives and biosafety, Journal of Biotechnology, 107, 193-232.
  21. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-479.
  22. Omar, A. A., Dekkers, M. G. H., Graham, J. H. & Grosser, J. W. (2008). Estimation of transgene copy number in transformed citrus plants by quantitative multiplex real-time PCR. Biotechnology Progress, 24, 1241-1248.
  23. Rezadoost, M. H., Sohani M. M., Hatamzadeh A. & Mirzaii M. R. (2013). In vitro regeneration of sour orange (Citrus aurantium L.) via direct organogenesis, Plant Knowledge Journal, 150-156.
  24. Rocha-Pe˜na, M. A., Lee, R. F., Lastra, R., Niblett, C. L., Ochoa-Corona, F. M., Garnsey, S. M. & Yokomi, R. K. (1995). Citrus tristeza virus and its aphid vector Toxoptera citricida: threats to citrus production in the Caribbean and Central, and North America. Plant Disease, 79, 437-443.
  25. Roistacher, C.N. (1991). Graft-transmissible diseases of citrus: Handbook for detection and diagnosis. Food & Agriculture Organisation.
  26. Scorza, R., Ravelonandro, M., Callahan, A. M., Cordts, J. M., Fuchs, M., Dunez, J. & Gonsalves, D. (1994). Transgenic plums (Prunus domestica L.) express the Plum pox virus coat protein gene. Plant Cell Report, 14, 18-22.
  27. Schmidt, M. A. & Parrott, W. A. (2001). Quantitative detection of transgenes in soybean (Glycine max (L.) Merrill) and peanut (Arachis hypogaea L.) by real-time PCR. Plant Cell Reports, 20, 422-428.
  28. Shou, H., Frame, B., Whitham, S. & Wang, K. (2004). Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Molecular Breeding, 13(2), 201-208.
  29. Sohani, M. M., Rezadoost, M. H., Zamani, A. H., Mirzae, M. R. & Afsharifar, A. R. (2015). High efficiency Agrobacterium-mediated transformation of sour orange (Citrus aurantium L.) using gene encoding Citrus Tristeza Virus coat protein. Journal of Applied Horticulture, 17(2), 109-114.
  30. Sundar, I. K. & Sakthivel, N. (2008). Advances in selectable marker genes for plant transformation, Journal of Plant Physiology, 165, 1698-1716.
  31. Subr, Z., Novakova, S. & Drahovska, H. (2006). Detection of transgene copy number by analysis of the T1 generation of tobacco plants with introduced P3 gene of potato virus A. Acta Virologica, 50, 135-138.
  32. Tan, S., Evans, R. & Singh, B. (2006). Herbicidal inhibitors of amino acid biosynthesis and herbicide- tolerant crops. Amino Acids, 30, 195-204.
  33. Tricoli, D., Carney, K. J., Russell, P. F., McMaster, J. R., Groff, D. W., Hadden, K. C., Himmel, P. T., Hubbard, J. P., Boeshore, M. L. & Quemada, H. D. (1995). Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to Cucumber Mosaic Virus, watermelon mosaic virus 2 and zucchini yellow mosaic virus. Nature Biotechnology, 13, 1458-1465.
  34. Tang, G., Galili, G. & Zhuang, X. (2007). RNAi and microRNA: breakthrough technologies for the improvement of plant nutritional value and metabolic engineering. Metabolomics,3, 357-369.
  35. Weng, H., Pan, A., Yang, L., Zhang, C. & Liu, Z. (2004). Estimating number of transgene copies intransgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Molecular Biology Reporter, 22, 289-300.
  36. Weigel D. & Glazebrook, J. (2002). Arabidopsis: A Laboratory Manual by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  37. Wong, M. M. & Medrano's, J. F. (2005). Real-time PCR for mRNA quantification, Biotechnogy Techniques, 39, 75-85.
  38. Yi, C. X., Zhang, J., Chan, K. M., Liu, X. K. & Hong, Y. (2008). Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum). Analytical Biochemistry, 375, 150-152.

 

علوم باغبانی ایران
دوره 48، ویژه نامه
مهر 1396
صفحه 79-91

فایل ها

سابقه مقاله

  • تاریخ دریافت: 29 اردیبهشت 1395
  • تاریخ بازنگری: 17 شهریور 1395
  • تاریخ پذیرش: 03 مهر 1395

هم رسانی

ارجاع به این مقاله

آمار