بهینه‌سازی روش استخراجRNA از بافت پوست شاخه درختان خانواده مخروطیان

نوع مقاله: مقاله علمی - پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد، اصلاح نباتات، دانشگاه یاسوج، یاسوج

2 نویسنده مسئول مکاتبات، استادیار، دانشکده کشاورزی، دانشگاه یاسوج، یاسوج پست الکترونیک: Masoumiasl@yu.ac.ir

3 استادیار، پژوهشکده بیوتکنولوژی کشاورزی ایران، کرج

4 استادیار، دانشکده کشاورزی، دانشگاه شهید چمران، اهواز

5 عضو شرکت دانش بنیان زیست فناوران نوین گیتی ژن، تهران

چکیده

فرآیند استخراج RNA از بافت­های سخت چوبی و به­طور خاص درختان خانواده مخروطیان به‎دلیل داشتن غلظت­های بالایی از ترکیبات فنولیک، پلی­ساکاریدها، متابولیت­های ثانویه و لیگنین، بسیار مشکل است و تا به‎حال هیچ­گونه کیت تجاری برای این منظور ساخته نشده است. در این مطالعه مشکلات فوق برطرف گردیده و روشی سریع، ساده و اقتصادی برای استخراج RNA از بافت ساقه گونه‌های Cedrus deodara و Abies grandis که هر دو از خانواده مخروطیان می­باشند، ارائه شده است. نسبت جذب نوری در طول موج‌های 260 به 280 و 260 به 230 نانومتر، به‎ترتیب 88/1 و 1/1 برای گونه  Cedrus deodara و 8/1 و 9/0 برای گونه Abies grandis به‎دست آمد. تغییرات صورت گرفته در جهت بهینه‌سازی استخراج RNA نیز شامل استفاده از تیمار پیش‌گرما روی بافر استخراج، کاهش دور سانتریفیوژ حذف آلودگی DNA و حذف DEPC می‌باشد. ساخت cDNA با استفاده از RNA استخراج شده با موفقیت انجام و واکنش RT-PCR باندهای مورد انتظار را تولید نمود. لذا به­نظر می­رسد این روش برای استخراج RNA از بافت­های چوبی مناسب است.

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