فاکتورهای تاثیرگذار بر کارایی روش آگرواینجکشن برای بیان سیستمیک ژن خارجی در گیاه ذرت به‌واسطه ویروس موزائیک نیشکر

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشگاه زابل

2 گروه بیوتکنولوژی، دانشکده کشاورزی، دانشگاه زابل

3 دانشیار، گروه مهندسی بیوتکنولوژی، دانشکده کشاورزی، دانشگاه شهید باهنر کرمان

4 دانشیار دانشکده کشاورزی دانشگاه زابل

5 Boyce Thompson Institute for Plant Research

چکیده

هدف: ذرت (Zea mays L.) یکی از مهمترین گیاهان زراعی در سراسر دنیا و از گیاهان مدل خانواده غلات است. استقرار و بهینه‌سازی یک سیستم بیان موقت ژن موفق در ذرت علاوه بر قابلیت استفاده در پروژه‌های خاموشی ژن یا تولید پروتئین‌های نوترکیب، می‌تواند موجب تسهیل مطالعات عملکرد ژن در این گیاه شود. پژوهش حاضر با هدف بهینه‌سازی سیستم بیان سیستمیک ژن به‌واسطه آگروباکتریوم در گیاه ذرت با استفاده از یک ناقل مبتنی بر ویروس موزائیک نیشکر انجام پذیرفت.
مواد و روش‌ها: برای این منظور از ژنوم ویروسی نوترکیب که در آن توالی رمزکننده پروتئین فلورسنت سبز (GFP)، در امتداد چهارچوب قرائت آزاد ویروس و در ناحیه بین توالی P1 و HC-Pro ویروس درج شده بود، استفاده شد. برای انتقال ژنوم ویروسی نوترکیب به گیاهان ذرت، از روش تزریق مستقیم آگروباکتریومی (آگرواینجکشن) به نواحی مریستمی گره کولئوپتیلی گیاهچه استفاده شد. کارایی دو سویه EHA105 و GV3101 باکتری Agrobacterium tumefaciens در انتقال ژنوم نوترکیب ویروس به سه واریته ذرت Iochief، Golden Bantam (ذرت شیرین) و B73 (ذرت دندان اسبی) در دو مرحله رشدی (گیاهچه‌های سه و هفت روزه) مورد مقایسه قرار گرفت. پس از ظهور علائم موزائیک ویروسی در گیاهان تلقیح شده، بیان ژن خارجی GFP با استفاده از میکروسکوپ فلورسنت کانفوکال و همچنین RT-PCR در این گیاهان در مقایسه با گیاهان شاهد ارزیابی شد.
نتایج: ‏ نتایج حاصل از RT-PCR و میکروسکوپ فلورسنت کانفوکال، نشان داد که سویه GV3101 در مقایسه با سویه EHA105 به لحاظ آماری عملکرد بالاتری در انتقال ناقل نوترکیب به گیاهان ذرت دارد. همچنین در مقایسه واریته‌های مختلف ذرت، این روش در بیان پروتئین فلورسنت سبز در ذرت واریته گلدن‌بانتام به‌طور بسیار معنی‌داری موفق عمل نمود. اگرچه در مورد سویه EHA105 درصد گیاهچه‌های سه روزه ذرت شیرین گلدن‌بانتام که به‌طور موفق ناقل محتوی ژن خارجی‌ را دریافت و بیان نمودند بالاتر بود، اما در مورد سویه GV3101 تفاوت معنی‌داری میان گیاهچه‌های سه و هفت روزه مشاهده نشد.
نتیجه‌گیری: ذرت رقم گلدن‌بانتام و آگروباکتریوم سویه GV3101 در مراحل پیش از دوبرگی به‌عنوان یک سیستم مدل بهینه، سریع و کارآمد برای پروژه‌های تحقیقاتی بررسی عملکرد ژن و حوزه‌های پژوهشی مرتبط پیشنهاد می‌گردد.

کلیدواژه‌ها


عنوان مقاله [English]

Factors influencing the efficiency of an agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize

نویسندگان [English]

  • Mahdieh Sadeghian 1
  • Mahmood Solouki 2
  • jafar zolala 3
  • abbasali emamjomeh 4
  • Georg Jander 5
1 University of Zabol
2 Department of Biotechnology, Faculty of Agriculture, University of Zabol
3 Associate professor, Department of Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran
4 Associate professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Zabol, Iran.
5 Boyce Thompson Institute for Plant Research
چکیده [English]

Objective
Maize (Zea mays L.) is a key cereal crop throughout the world and important model for plant genetics and biology. Establishment of an efficient transient gene expression system in maize facilitates plant functional genomics projects using gene silencing or heterologous protein overexpression strategies. The present study was aimed at optimizing an Agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize.
Materials and methods
Recombinant DNA encoding sugarcane mosaic virus (SCMV) containing the coding sequence of green fluorescent protein between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons, in‐frame with the viral open reading frame, was introduced into the meristematic tissue, above the coleoptilar node, of maize seedlings via a direct Agroinoculation procedure. The efficiency of Agrobacterium tumefaciens strains EHA105 and GV3101 in delivering the recombinant vector into three and seven-day old seedlings of three maize varieties, including sweet corn (Iochief and Golden Bantam varieties) and dent corn (inbred line B73), was examined. Expression of GFP transgene in symptomatic Agroinoculated plants was assessed by confocal fluorescent microscopy and RT-PCR in comparison with controls.
Results
Results of RT-PCR and confocal fluorescent microscopy revealed that: 1) A. tumefaciens GV3101 is significantly more successful in delivering the recombinant SCMV-based vector into maize plants than EHA105, 2) the percentage of GFP-expressing Golden Bantam plants is significantly higher than two other maize varieties, and 3) The effectiveness of growth stage of maize seedlings on the percentage of GFP expressing Agroinjected plants depends upon the interactions between Agrobacterium strain and maize genotype.
Conclusions
In conclusion, a combination of the Golden Bantam maize variety and A. tumefaciens strain GV3101 for direct Agroinjection of the SCMV-based vector into seedlings at the early two-leaf stage could be a fast and efficient system for investigating gene functions in maize.

کلیدواژه‌ها [English]

  • : Green Fluorescent Protein
  • Agroinjection
  • In-planta expression system
  • Viral vectors
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