مقایسه بیان موقت پروتیین HDA19 در برگ‌های Nicotiana tabacum و Nicotiana bentamiana

نوع مقاله : علمی پژوهشی

نویسندگان

1 دانشجوی دکتری گروه اصلاح نباتات و بیوتکنولوژی، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

2 دانشیار گروه اصلاح‌نباتات و بیوتکنولوژی، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

3 موسسه بیوشیمی بیماری‌های گیاهی، مرکز هلم هولتز مونیخ، مرکز تحقیق آلمان برای سلامت محیطی، نوهربرگ، 85764، آلمان

4 استادیار گروه اصلاح نباتات و بیوتکنولوژی، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

چکیده

اخیراً بیان موقت ژن به‌منظور فراهم سازی روش‌های بسیار سریع ارزیابی بافت‌های گیاهی به‌عنوان یک منبع تولید پروتیین در مقایسه با روش وقت گیر و پرهزینه تراریختی پایدار گیاهان گسترش یافته است. مطالعه حاضر بیان موقت پروتیین HDA19 را در دو گونه از گیاه tobacco (Nicotiana tabacum وNicotiana bentamiana) نشان می‌دهد. برای این منظور پرایمرهای اختصاصی برای واکنش زنجیره ای پلی مراز طراحی و برای همسانه سازی ژن HDA19 در وکتور بیانی pB2GW7 به‌کار رفتند. سازه نوترکیب به Agrobacterium tumefaciens strain GV3101 انتقال یافت سپس برای تراریختی موقت گیاهان tobacco از طریق اگروباکتریوم استفاده شد. در نهایت وجود ژن هدف در لاین‌های تراریخت اگروباکتریوم از طریق کلونی PCR تأیید گردید. از طرفی بیان پروتیین در لاین‌های تراریخت توسط روش دات بلات و الایزا تأیید شد. اگرچه روش دات بلات و SDS-PAGE حاکی از تراریختی هر دو رقم بود ولی روش الایزا تولید پروتیین نوترکیب در گیاه تراریخت Nicotiana tabacum را تایید کرد که 400 میکروگرم در گرم وزن بافت تر برگ گیاه بود. با توجه به یافته‌های فوق این گیاه میزبان مناسبی برای تولید HDA19 نوترکیب، نماینده‌ای از پروتیین‌های هیستون داستیلاز، نسبت به Nicotiana bentamiana می‌باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Comparison of Transient HDA19 Protein Expression in Leaves of Nicotiana tabacum and Nicotiana bentamiana

نویسندگان [English]

  • Maryam Jamshidnia 1
  • Sayed Kamal Kazemitabar 2
  • Christian Lindermayr 3
  • Hamid Najafi Zarini 4
1 Ph.D. Student, Department of Plant Breeding & Biotechnology, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran
2 Associate Professor, Department of Plant Breeding & Biotechnology, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran
3 Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, 85764, Germany
4 Assistant Professor, Department of Plant Breeding & Biotechnology, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran
چکیده [English]

Recently, transient gene expression has been developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. This study reports the expression of HDA19 gene in two species of tobacco plants (Nicotiana tabacum and Nicotiana bentamiana) by means of transient transformation. Specific primers were designed and used for PCR amplification and cloning of HDA19 gene in the plant expression vector pB2GW7. The recombinant construct was transferred into Agrobacterium tumefaciens strain GV3101, and was used for Agrobacterium mediated transformation of tobacco plants. The presence of the desired gene in transgenic lines was confirmed through colony PCR. The expression of the protein in transgenic lines was confirmed by immune-dot blot assay and ELISA. Although the transformation of the two species was confirmed by immune-dot blot assay and SDS-PAGE, recombinant protein production in Nicotiana tabacum plants was confirmed by ELISA and it was estimated 400 µg per gram wet weight of tobacco leaves. According to the results, this species is the appropriate host for the production of recombinant HDA19, one of the histone deacetylases, rather than Nicotiana bentamiana.

کلیدواژه‌ها [English]

  • KEYWORDS: HDA19؛ Tobacco؛ Recombinant protein
  • Nicotiana tabacum
  • Nicotiana bentamiana

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