طراحی و ساخت سازه اختصاصی کلرو‌پلاستی گیاه برنج حاوی ژن‌های بتائین آلدهید دهیدروژناز(badh) و فلاودکسین(fld) جهت ایجاد مقاومت به تنش‌های غیر‌زنده

نوع مقاله : علمی پژوهشی

نویسندگان

1 دانشیار بخش کشت بافت و انتقال ژن، پژوهشکده بیوتکنولوژی کشاورزی ایران، کرج

2 دانشجوی کارشناسی‌ارشد بیوتکنولوژی کشاورزی دانشگاه زابل، زابل

3 دانشیار گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه زابل، زابل

4 استادیار بخش کشت بافت و انتقال ژن، پژوهشکده بیوتکنولوژی کشاورزی ایران، کرج

چکیده

تنش‌های غیر‌زیستی از جمله خشکی و شوری اولین عامل کاهش محصول در دنیا است. در این تحقیق ژن بتائین آلدهید دهیدروژناز (badh) هم به عنوان یک نشانگر غیر‌آنتی‌بیوتیکی و هم به عنوان یکی از ژن‌های کاندید در تحمل به تنش‌های غیر‌زیستی به همراه ژن فلاودکسین (fld) در ساخت ناقل کلروپلاستی برای گیاه برنج مورد استفاده قرار گرفت. بدین منظور ابتدا دو قسمت از یک ناحیه اختصاصی هدف‌گیری کننده به پلاستوم برنج (FR) با افزودن جایگاه آنزیمی مناسب جهت ورود کاست-های ژنی به مرکز آن، در دو مرحله به گونه‌ای با استفاده از PCR از ژنوم کلروپلاستی برنج جدا‌سازی شد که امکان اتصال مجدد آنها ضمن کلون‌سازی ایجاد گردد. سپس طراحی کاست ژنی برای ژن های fld و badh تحت نواحی تنظیمی کلروپلاستی انجام شد، به طوری‌که ژن fld همراه با rbcl 5'UTR و ژن badh همراه با T7gene10 5'UTR به صورت پلی‌سیسترونی تحت پیشبر قوی کلروپلاستی Prrn و پایانبر rbcl 3'UTR کلون‌سازی شدند. سرانجام کاست کامل دو ژنی fld/badh به همراه نواحی تنظیمی از ناقل نوترکیب حاصل موسوم به pBF جدا و در مرکز ناحیه هدف‌گیری کننده به کلروپلاست برنج در دو جهت کلون‌سازی گردید. دو ناقل مختص کلروپلاست حاصل موسوم به pFrFB(-) و pFrFB(+) پتانسیل الحاق هدفدار ژن‌های مقاومت به شوری و خشکی را با دو جهت‌گیری مختلف نسبت به نواحی داخلی ژنوم کلروپلاستی گیاه برنج دارا بوده و قادرند با هدف ایجاد مقاومت بالا به شوری، خشکی و سرما در انتقال ژن به کلروپلاست گیاه برنج با استفاده از تفنگ ژنی مورد استفاده قرار گیرند.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Design and Construction of Specific Chloroplast Vectors for Rice Plants Containing Betaine Aldehyde Dehydrogenase (badh) and Flavodoxin (fld) Genes for Resistance to Abiotic Stress

نویسندگان [English]

  • Masoud Tohidfar 1
  • Ebrahimi Ghorishi 2
  • Bartali Fakheri 3
  • Motahareh Mohsenpour 4
1 Associate Professor, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran.
2 M.Sc. student of Department of Agronomy and Plant Breeding, Zabol University, Zabol, Iran.
3 Associate Professor, Department of Agronomy and Plant Breeding, Zabol University, Zabol, Iran
4 Assistant Professor, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran.
چکیده [English]

Abiotic stresses such as drought and salinity are the first factor of YIELD decrease in the world. In this study, betaine aldehyde dehydrogenase gene (badh) is used as both an abiotic stress marker gene and one of the abiotic stress tolerance candidate genes along with Flavodoxin (fld) in construction of chloroplast vector for rice. Thereby, adding the appropriate enzyme role to let gene cassette enter to the center. The first two parts of a specific target areaof Plastom rice (FR) the rice genome using PCR that they can be re-attached in cloning. Then, the gene cassetes were designed for fld and badh genes in regulatory regions of the chloroplast. So that the fld genes and with rbcl 5'UTR and badh genes with T7gene10 5'UTR were cloned the strong chloroplastsic promoter Prrn and the terminator rbcl 3'UTR. Finally, The complete two-gene cassettes fld/badh with the regulatory regions is separated from targeting chloroplast metabolism of rice in two sides cloned. The two specific chloroplast vector called pFrFB(-) and pFrFB(+) are potential to be attached to gene that are resistant to drought and salinity targeted with two different orientations relative to the inner regions of the chloroplast genome of rice plants and are able to be the goal of creating high resistance to salinity, drought and chill in transferring genes to use the chloroplast of rice plant via gene gun.

کلیدواژه‌ها [English]

  • Abiotic Stress
  • betaine aldehyde dehydrogenase
  • Flavodoxin
  • Cloning
  • Vector chloroplastid

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