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
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