طراحی، ساخت و انتقال سازه‌های ژنی جهت خاموش‌سازی ژن‌های F3′5′H و F3′H در انگور رقم شیراز

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی سابق دکتری و استاد، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

2 دانشیار پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران

3 پژوهشگر و استاد مؤسسۀ CSIRO، ادلاید، استرالیا

چکیده

به‌منظور مطالعۀ عملکرد ژن‏های F3´5´H و F3´H وبرهمکنش آن‌ها در مسیر بیوسنتز فلاونوئیدها در انگور، از طریق طراحی و ساخت سازه‌های مناسب ایجاد‌کنندۀ ihpRNA، لاین‌های تراریخت حاوی سازۀ خاموش‌کنندۀ ژن F3´H وسازۀ خاموش‏کنندۀ هر دو ژن F3´5´H و F3´H ایجاد شدند. به این منظور‌ قطعاتی از دو ژن انتخاب و به‌صورت تکرارهای معکوس در ناقل pHANNIBAL درج و با بهره‌گیری از سیستم ناقل دوگانه، هر یک از سازه‌های تهیه‌شده وارد ناقل p27 mod GFP 4a و به اگروباکتری منتقل شد. کالوس‌های جنین‌زای حاصل از کشت پرچم، توسط کشت توأم با اگروباکتری تراریخت شدند. بیان ژن GFP در کالوس‌های جنین‌زا، پنج روز پس از تلقیح با میکروسکوپ مشاهده شد. در‌نهایت از کشت کالوس‌های جنین‌زای دارای سازۀ خاموشی ژن  F3′Hوسازۀ خاموشی هم‌زمانF3′H و F3′5′H بهترتیب 42 و 34 لاین باززایی شدند که بر‌اساس واکنش زنجیره‌ای پلیمراز، 36 لاین برای سازۀ خاموشی ژن F3′H و 27 لاین برای سازۀ خاموشی هم‌زمانF3′H و F3′5′H، مثبت ارزیابی شدند که این نتیجه نشان از کارایی بالای روش باززایی و تراریختی استفاده‌شده بود

کلیدواژه‌ها


عنوان مقاله [English]

Designing, construction and transformation of silencing constructs for F3´H and F3´5´H genes in grapevine cv. Shiraz

نویسندگان [English]

  • Maryam Pejman mehr 1
  • Ali Ebadi 1
  • Amir Moosavi 2
  • Debra McDavid 3
  • Amanda R. Walker 3
1 Post Graduate Student and Professor, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
2 . Associate Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
3 . Researcher and Professor in CSIRO Plant Industries, Adelaide, Australia
چکیده [English]

abstract
 In order to study F3´5´H and F3´H function and their intraction in the grapevine flavonoids biosynthetic pathway, designing and constraction of suitable constructs encoding ihpRNA was performed and transgenic lines containing silencing construct of F3´H and simultaneous silencing construct of F3´5´H and F3´H were created. For this purpose, fragments were selected from both genes and inserted into pHANNIBAL vector as inverted repeats. Using binary vector system, each construct inserted into p27 mod GFP 4a vector and were used for Agrobacterium transformation. Embryogenic calli, drived from anther culture, were co-cultivated with Agrobacterium for transformation. Expression of GFP gene was detected in transgenic embryogenic calli, 5 days after inoculation using microscope. Finally, 42 and 34 lines were generated from single (F3´H) and simultaneous silencing (F3´5´H and F3´H) cultures, respectively. PCR analysis confirmed 36 single (F3´H) silencing line and 27 simultaneous silencing (F3´5´H and F3´H) lines, showing high efficacy of regeneration and transformation methods.

کلیدواژه‌ها [English]

  • Vitis vinifera
  • gene construct
  • silencing
  • F3´H
  • F3´5´H
 
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