تأثیر بیان ژن P19 بر افزایش میزان رونویسی و تولید پروتئین نوترکیب پلاسمینوژن بافتی انسانی (rtPA) در گیاه توتون (Nicotiana benthamiana)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه ژنتیک و به‌نژادی گیاهی، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

2 گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

3 تهران- گیشا- دانشگاه تربیت مدرس- دانشکده کشاورزی- گروه اصلاح نباتات و بیوتکنولوژی

چکیده

هدف: فعال‌کننده پلاسمینوژن بافتی (rtPA) یکی از مهم‌ترین داروها در درمان بیمارهای قلبی است. این دارو به‌صورت پروتئین نوترکیب در سیستم‌های بیانی تولید می‌شود که هزینه‌های تولید بسیار بالایی دارد. سیستم بیان موقت به‌دلیل بیان زیاد، سرعت ‌بالا، هزینه پایین و عدم تأثیرپذیری مکانی جهت بیان پروتئین بسیار مناسب می‌باشد. با این ‌وجود مشخص ‌شده است که خاموشی پس از رونویسی حاصل از کمپلکس RISC بر میزان بیان پروتئین نوترکیب تأثیر می‌گذارد. یکی از مهم‌ترین سرکوب کننده‏های خاموشی RNA شناخته شده در گیاهان، پروتئین P19 می‏باشد که از طریق میل ترکیبی زیادی که با siRNA دو رشته‏ای دارد به آن متصل می‌گردد و آن را تجزیه می‌کند و مانع خاموشی ژن می‌گردد. هدف از مطالعه حاضر بررسی تأثیر بیان هم‌زمان ژن P19 بر بیان ژن فعال‌کننده پلاسمینوژن بافتی (rtPA) در سطح رونویسی و پروتئین در گیاه توتون Nicotiana benthamiana بود.
مواد و روش‌ها: در تحقیق حاضر میزان بیان ژن rtPA در سطح رونویسی و پروتئین مورد بررسی قرار گرفت. در این تحقیق از تزریق هم‌زمان اگروباکتریوم حاوی ناقل دوتایی pCAMBIA1304-rtPA و اگروباکتریوم حاوی ناقل pCAMBIA1304-P19 در مقایسه با اگروباکتریوم حاوی تنها ناقل بیانی pCAMBIA1304-rtPA استفاده شد. نمونه­های برگی در روزهای 4، 7 و 10 روز پس از تلقیح با آگروباکتریوم تهیه شدند. سپس میزان رونویسی و پروتئین با استفاده از آزمون ReaTime PCR و الایزا محاسبه شد.
نتایج: نتایج آزمون Real Time PCR حاکی از افزایش 34 درصد میزان رونویسی ژن rtPA در حضور P19 نسبت به شاهد بود. بیشترین میزان رونویسی از ژن‌های P19 و rtPA باگذشت چهار روز از تلقیح گیاهان با اگروباکتریوم حاصل شد. نتایج الایزا نشان داد که میزان بیان پروتئین rtPA در حضور ژن P19 در روز هفتم و دهم پس از تلقیح به ترتیب 89 و 84 میکروگرم بر گرم وزن‌تر برگ بود که در مقایسه با شاهد به‌ترتیب به‌میزان 12 و 15 درصد بیشتر بود.
نتیجه‌گیری کلی: نتایج نشان داد که کاربرد P19 علاوه بر سرکوب خاموشی ژن، می‌تواند برای دست‌یابی به بیان در سطح بالا مؤثر باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Effect of P19 gene expression on transcription rate and production of recombinant human tissue plasminogen (rtPA) in tobacco (nicotiana benthamiana)

نویسندگان [English]

  • Yousef Sharafi 1
  • Mokhtar Jalali Javaran 2
  • Mohammad sadegh Sabet 3
1 Department of Genetics and Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
2 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
3
چکیده [English]

Objective
Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Post-transcriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana.
 
 
Material and methods
To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests.
 
Results
The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 µg.g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively.
 
 Conclusion
The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

کلیدواژه‌ها [English]

  • Agrobacterium
  • Transient Expression
  • Molecular Farming
  • Silencing Suppressor
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