ایجاد توتون تراریخت تولیدکننده لاکاز قارچی با استفاده از یک پروموتر دارای بیان ریشه-ویژه

نوع مقاله : علمی پژوهشی

نویسندگان

1 استادیار

2 دانشجوی دکتری فیزیولوژی گیاهی، دانشگاه پیام نور تهران شرق،ایران

3 گروه زیست شناسی، دانشگاه پیام نور تهران شرق، تهران، ایران

4 گروه زیست شناسی، دانشکده زیست شناسی، دانشگاه دامغان، دامغان، ایران

چکیده

لاکازها گروهی از گلیکوپروتئین‏ها با ویژگی پلیفنلاکسیدازی هستند که می‌توانند ترکیبات گوناگونی را اکسید نمایند. این گروه آنزیمی در گیاهان، قارچ‌ها، حشرات و باکتری‌ها بیان می‌شوند و دارای نقش‌های مهمی در فعالیت‌های زیستی و کاربردهای گوناگونی در صنایع غذایی، دارویی، نساجی و حتی نظامی می‌باشند. با توجه به ساختار این آنزیم و این‌که تشکیلات بیان پروتئین در گیاهان قادر به تولید کامل پروتئین‌ها ازجمله گلیکوپروتئین‏ها می‌باشند به جهت کاربرد در پالایش زیستی، سازه‌ای طراحی گردید که لاکاز II قارچی تحت کنترل پیشرانه باکتریایی ویژه بیان در ریشه مانوپین سنتاز و تشدیدکننده ترجمه Tobacco Etch Virus در گیاه توتون بیان شود. افزودن سیگنال پپتید اندوکیتیناز اسیدی Q توتون به ابتدای آنزیم باعث ترشح آن به فضای آپوپلاستی می‌شود. با استفاده از واکنش زنجیره‌ای پلیمراز، تراریختی گیاهان توتون توسط ژن لاکاز به‌وسیله آگروباکتریوم تأیید گردید. نتایج نیمهکمی بیان ترانسکریپتومیک لاکاز در ریشه و برگ تراریخت‏های مفروض نشانگر بیان متمایز آن در ریشه و برگ بود که ناشی از کارکرد متمایز پیشرانه باکتریایی مانوپین سنتاز می‌باشد. نتیجه وسترن بلاتینگ تولید پروتئین بالغ در ریشه را تأیید کرد. وجود این پروتئین به‌صورت تک سایز نشانه عملکرد صحیح سیگنال پپتید و ترشح آن به فضای آپوپلاستی در ریشه توتون می‌باشد. با توجه به کاربرد این گیاهان جهت استخراج آنزیم یا پالایش زیستی می‌توان تراریخت‏های موردنظر را بر اساس میزان پروتئین و فعالیت آنزیمی غربالگری و گزینش نمود.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Generation of fungal laccase producing transgenic tobacco plants using a root-specific expression promoter

نویسندگان [English]

  • Seyed Javad Davarpanah 1
  • Majid Dana 2
  • Gholamreza Bakhshi Khaniki 3
  • Amir Abbas Mokhtarieh 4
1 Research Assistant Professor
2 Department of Biology, Faculty of Science, Payam Nour University, Tehran, Iran
3 Department of Biology, Faculty of Science, Payam Nour University, Tehran, Iran
4 Department of Biology, School of Biology, Damghan University, Damghan, Iran
چکیده [English]

Laccases are a group of glycoproteins which can oxidize a wide range of compounds with various biological activities and industrial applications in food and beverage, pharmaceutical, textile, and even military-related industries. Regarding this enzyme structure and the ability of plant protein production machinery for protein glycosylation, a construct consisting of fungal laccaseII under control of root-specific mannopine synthase promoter and tobacco etch virus translation enhancer was designed for tobacco transformation to be used in phytoremediation. N-terminal addition of acidic tobacco endochitinase Q to Laccase directs its apoplastic secretion. Putative laccase agrobacterium-mediated transformants were confirmed using polymerase chain reaction. Semi-quantitative PCR of roots and leaves of putative transformants showed differential expression of the laccase gene at the transcriptomic level resulting from the differential function of bacterial mannopine synthase promoter. Western blotting results confirmed production of mature protein in roots which also confirms the correct function of signal peptide and secretion of this enzyme into the apoplastic space of roots. Regarding their application for protein production or phytoremediation transgenics of interest should be screened based on protein concentration and enzyme activity.

کلیدواژه‌ها [English]

  • Laccase
  • Mannopine synthase
  • Rhizosecretion
  • Tobacco
  • Transformation

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