با همکاری مشترک دانشگاه پیام نور و انجمن بیوتکنولوژی جمهوری اسلامی ایران

نوع مقاله : علمی پژوهشی

نویسندگان

1 دانشجو دکتری بیوتکنولوژی کشاورزی، پژوهشگاه بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران.

2 استادیار بیوتکنولوژی کشاورزی، پژوهشگاه بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران.

3 دانشیار زیست‌شناسی، پژوهشگاه بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران.

چکیده

گیاه دانه‌روغنی گلرنگ از گیاهان بومی ایران است که میزان اسید اولئیک (18:1) آن در تمامی ارقام ایرانی پایین است. روغن‌های دارای سطوح اسید اولئیک بالاتر از 50 درصد به علت وجود یک پیوند دوگانه در اسیداولئیک نسبت به اسیدهای چرب دارای دو یا چند پیوند دوگانه، دارای پایداری اکسیداتیو در برابر حرارت هستند. به‌کارگیری روش‌های مهندسی ژنتیک و ویرایش ژنومی دستیابی به دانه‌های روغنی با اولئیک اسید بالا را ممکن ساخته است. در این پژوهش که به منظور افزایش میزان اسید اولئیک در گیاه گلرنک انجام شد، دو توالی RNAی راهنما برای هدف گیری ژن Fatty Acid Desaturase 2 (FAD2-1) از دو ناحیه طراحی شد که محل هدف گیری آنها درون ناحیه کد کننده این ژن با فاصله 640 جفت باز از یکدیگر قرار داشت. توالی‌های راهنما به‌همراه ژن Cas9 (بهینه‌سازی کدونی شده) در ناحیه T-DNAی سازه اگروباکتریومی کلون‌سازی و به روش In-planta به غوزه گیاه گلرنگ منتقل شد. بذور حاصل کشت و گیاهان حاصل پس از بذرگیری در نسل بعد برای تغییر پروفایل اسیدهای چرب غربال شدند. نتایج نشان داد میزان اسیداولئیک در بذر یکی از لاین‌ها که دارای چهار تغییر اسیدآمینه به طور همزمان بود به طور میانگین به 14/53 درصد رسیده است. این در حالی است که میزان اسید اولئیک در گیاه شاهد به طور میانگین 62/11 درصد اندازه‌گیری شد. نتایج نشان داد در نسل در حال تفرق، تغییر پروفایل اسید چرب در لاین دارای تغییر اسیدآمینه به‌صورت هموزایگوس اتفاق افتاده و گیاهان هتروزایگوس پروفایل روغن مشابه گیاهان شاهد دارند. همچنین نتایج این پژوهش می‌تواند نشان‌دهنده امکان ایجاد افزایش در میزان اسید اولئیک در دانه‌های روغنی با تغییر توالی آنزیم FAD-2 و بدون غیرفعال‌سازی کامل ژن باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Investigating the increase of oleic acid in safflower plant by changes in Fatty Acid Desaturase-2 gene after using CRISPR-Cas9 method

نویسندگان [English]

  • Mohamad Amin Neycee 1
  • Motahhareh Mohsenpour 2
  • Hassan Rahnama 3

1 Ph.D. Student in Agriculture Biotechnology, Agricultural Biotechnology Research Institute of Iran, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.

2 Assistant Professor, Agricultural Biotechnology Research Institute of Iran, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.

3 Associate Professor, Agricultural Biotechnology Research Institute of Iran, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.

چکیده [English]

Safflower with low oleic acid content is one of the native plants of Iran. Generally,, high oleic acid oils have more oxidative stability than the oils with high linoleic acids . Genome editing technology enable us to obtain oilseeds with high oleic acid. In this research, two guide RNA sequences were designed to target of Fatty Acid Desaturase 2 (FAD2-1) gene, which were located within the coding region and at a distance of 640 base pairs from each other. The guide sequences along with the codon optimized Cas9 gene were cloned in the T-DNA region of the Agrobacterium construct and transferred to the safflower by the In-planta method. The resulting seeds were cultivated and the plants were screened to track changes in the fatty acid profile of the seeds. The results showed that the amount of oleic acid in the seeds of one of the lines reached 53.14% on average. This line had four amino acid changes (L66F, N204D, S236A and I238V) at the same time. This is while the amount of oleic acid in the control plant was measured as 11.62% on average. The results showed that in the segregating generation, the change in fatty acid profile occurred in the line with homozygous amino acid change, and the heterozygous plants have the same oil profile as the control plants. Also, the results of this research can indicate the possibility of increasing the amount of oleic acid in oilseeds by changing the FAD2 enzyme sequence and without gene knockout.

کلیدواژه‌ها [English]

  • Oleic acid
  • Safflower
  • CRISPR-Cas9
  • Fatty Acid Desaturase
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