بهینه سازی بیان موقت ژن در گیاه گوجه فرنگی (Solanum lycopersicum)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 1- دانشجوی دکتری بیوتکنولوژی کشاورزی، دانشکده کشاورزی دانشگاه کردستان، سنندج، ایران

2 دانشیار، گروه تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران

3 استاد گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

4 استادیار پژوهشی موسسه تحقیقات کشاورزی دیم، سازمان تحقیقات، آموزش و ترویج کشاورزی (ARREO)، مراغه، ایران

چکیده

چکیده
هدف: هدف از این تحقیق بهره‌گیری از گیاه گوجه فرنگی رقم کال‌جی به عنوان یک گیاه مدل برای انتقال موقت جهت تولید ترکیبات دارویی و صنعتی است.
مواد و روش‌ها: به منظور بررسی انتقال موقت ژن، سه غلظت اگرو باکترویوم در OD600 (4/0، 6/0 و 8/0)، سه زمان پس از تلقیح (4، 7 و 10 روز) و دو حالت با حضور ژن p19 و بدون p19 به عنوان فاکتورهای مؤثر بر بیان موقت ژن انتخاب شدند. ناقلین مورد استفاده در این تحقیق pXK2FS7 و pCAMBIA1304 به ترتیب حاوی ژن‌های GFP و P19 بودند. برای انتقال موقت ژن GFP، برگ گیاهچه‌های 6 برگی با استفاده از روش اگرواینفیلتریشن تلقیح شدند. بدین صورت که غلظت‌های مختلف از اگروباکتریوم حاوی ناقل pXK2FS7 و ناقل P19 با یکدیگر مخلوط شدند و بیان ژن GFP بررسی گردید. همه‌ برگ‌های گیاهان گوجه فرنگی در مرحله مناسب رشدی توسط سرنگ انسولین بدون سوزن در سطح زیرین برگ تزریق شدند. به منظور تایید تراریختی و انتقال ژن استخراج RNA، ساخت cDNA و میزان بیان ژن GFP با استفاده از روش Realtime PCR انجام شد و نهایتا مشاهده بیان پروتئین GFP با میکروسکوپ فلورئوسانس صورت گرفت.
نتایج: نتایج نشان داد که غلظت باکتری، تعداد روزهای پس از تلقیح و بیان ژن p19 (پروتئین سرکوب‌کننده خاموشی ژن) بر بیان ژن GFP اثرات معنی‌داری دارند. به طوری که حضور ژن p19 به عنوان سرکوبگر سیستم خاموشی ژن در مقایسه با عدم حضور در همه حالات باعث بیان بیشتر ژن GFP گردید. همچنین مشخص شد که رقت OD600 6/0 اگروباکتریوم مناسب‌ترین غلظت برای اگرواینفیلتریشن GFP است. به علاوه در 7 روز پس از تلقیح، بالاترین میزان رونویسی ژن GFP بدست آمد. بهترین ترکیب به منظور حصول حداکثر میزان رونویسی ژن GFP، حضور ژن p19، غلظت 6/0 OD600 اگروباکتریوم و جمع آوری نمونه های برگی 7روز بعد از تلقیح می باشد.
نتیجه‌گیری: نتایج حاصل از این تحقیق می‌تواند در مطالعات مقدماتی و یا بهینه‌سازی تولید پروتئین‌های نوترکیب در برگ‌های گوجه فرنگی مورد استفاده قرار گیرد.

کلیدواژه‌ها

عنوان مقاله [English]

Optimization of transient gene expression in tomato (Solanum lycopersicum L.)

نویسندگان [English]

  • Masomeh Nasiri Mogadam 1
  • Asad Maroufi 2
  • Mokhtar Jalali Javaran 3
  • Yousef Sharafi 4
1 PhD student of Plant Biotechnology, Department of Agronomy, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
2 Associate Professor of Agricultural Biotechnology, Department of Plant production and Genetics, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
3 Professor of Molecular Genetics and Genetics Engineering, Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
4 Assistant Professor Dryland Agricultural Research Institute, Agricultural Research, Education and Extension Organization (ARREO), Maragheh, Iran
چکیده [English]

Abstract
Objective
The aim of this study is to optimize tomato as a suitable plant system for transient gene transformation for the production of pharmaceutical and industrial compounds.
Materials and methods
In order to investigate transient gene expression, three concentrations of Agrobacterium in OD600 (0.4, 0.6 and 0.8), three times after inoculation (4, 7 and 10 days) and two conditions with the presence of p19 gene and without p19 were selected as factors affecting the gene expression. The vectors used in this research pXK2FS7 and pCAMBIA1304 contained GFP and P19 genes, respectively. For transient GFP transformation, leaves of seedlings were inoculated using agroinfiltration method. In this way, different concentrations of Agrobacterium containing pXK2FS7 vector and P19 vector were mixed together and the expression of GFP gene was investigated. All the leaves of tomato plants at the appropriate stage were injected by insulin syringe without needle on the lower surface of the leaf. In order to confirm the transfection of the GFP and its expression, RNA extraction, cDNA synthesis and the analysis of expression by Real-Time PCR were performed and the expression of the GFP protein with a fluorescence microscope was finally detected.
Results
The results showed that the concentration of Agrobacteria, the number of days after inoculation, and the expression of the p19 had significant effects on the GFP expression. The expression of p19 as a suppressor of gene silencing mechanism led to higher GFP expression in comparison with the absence of p19. Additionally, it was found that dilution of 0.6OD600 Agrobacterium was the most suitable concentration for high expression of GFP agroinfiltration. Furthermore, the highest level of GFP expression was obtained 7 days after leaf inoculation. The best combination for maximum GFP expression at transcript level obtained at 0.6 OD600, 7 days after inoculation and expression of p19.
Conclusions
The results of this research can be used in preliminary studies or optimization of recombinant protein production in tomato leaves.

کلیدواژه‌ها [English]

  • Agroinfiltration
  • GFP
  • recombinant proteins
  • p19

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مجله بیوتکنولوژی کشاورزی
دوره 15، شماره 4
آذر 1402
صفحه 1-22

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