تولیدات گیاهی

تولیدات گیاهی

مقایسه سه روش مختلف استخراج RNA ژنومی از پوست و گوشت میوه پرتقال تامسون ناول (Citrus sinensis L.)

نوع مقاله : علمی پژوهشی - اصلاح نباتات

نویسندگان
فاطمه احمدی 1
محمد مهدی شریفانی 2
امیر موسوی 3
نگین اخلاقی امیری 4
مصطفی خوشحال سرمست 5
خلیل زینلی نژاد 6
1 دانشجوی دکتری علوم و مهندسی باغبانی، دانشکده تولید گیاهی، دانشگاه کشاورزی و منابع طبیعی گرگان، گرگان، ایران.
2 دانشیار گروه مهندسی علوم باغبانی و فضای سبز، دانشکده تولید گیاهی، دانشگاه کشاورزی و منابع طبیعی گرگان، گرگان ، ایران.
3 دانشیار گروه پژوهشی زیست فناوری مولکولی گیاهی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران.
4 استادیار بخش تحقیقات علوم زراعی و باغی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی استان مازندران، ساری، ایران.
5 دانشیار گروه مهندسی علوم باغبانی و فضای سبز، دانشکده تولید گیاهی، دانشگاه کشاورزی و منابع طبیعی گرگان، گرگان، ایران.
6 استادیار گروه اصلاح نباتات و بیوتکنولوژی، دانشکده تولید گیاهی، دانشگاه کشاورزی و منابع طبیعی گرگان، گرگان، ایران.
10.22055/ppd.2023.41200.2043
چکیده
پژوهش حاضر به منظور مقایسه روش­های مختلف استخراج  RNAکل با کیفیت بالا در پرتقال تامسون ناول می­ باشد. مرکبات جز یکی از میوه­ های استراتژیک در ایران محسوب می­گردد و یافتن روش مناسب و کم­هزینه برای کارهای اجرایی و مولکولی حائز اهمیت است. مواد گیاهی مورد استفاده برای استخراج RNA کل از دو قسمت مختلف پوست (شامل فلاودو و آلبدو) و گوشت پرتقال تامسون ناول از ایستگاه تحقیقات باغبانی قائم­شهر، مازندران در سال 1398 جمع ­آوری شدند. میوه­ ها بعد از نمونه ­گیری، در نیتروژن مایع به حالت فریز شده قرار گرفتند و تا زمان استفاده در منفی 80 درجه سانتی­گراد نگهداری شدند. به منظور تجزیه و تحلیل مولکولی، نمونه­ها به آزمایشگاه گروه باغبانی گرگان منتقل شدند. و برای مطالعه از سه روش شامل: تریازول، دنازیست غیر­ستونی و RNX-PlusTM  استخراج RNA کل ارزیابی گردید.  به­ منظور نشان دادن کیفیت و کمیت RNA، بر روی ژل آگارز 1% و با استفاده از اسپکتوفتومتر نانو­درآپ در طول موج 230، 260 و 280 تعیین گردید. نتایج با استفاده از طرح کاملا تصادفی در سه تکرار و آزمون دانکن در سطح معنی­دار 5 درصد و با استفاده از نرم افزار SAS 9.0 مورد تجزیه و تحلیل قرار گرفت. نتایج وجود باندهای ریبوزومی S28 و S18 مجزا و واضح روی ژل الکتروفورز نمایش می­دهد و یکپارچگی RNA با باندهای ریبوزومی شارپ بدست آمد. همچنین با استفاده از این روش­ها نسبت جذب 280/260، بین 94/1- 80/1 بیانگر این است که، نمونه­ها بدون آلودگی پروتئین در RNA استخراج شده بودند. و در نسبت جذب 230/260، بین 14/2- 0/2 نمونه­ها بدون آلودگی پلی­ساکارید و فنل نشان ­داده شد. در مقایسه با سایر روش­ها، در روش تریازول اختلاف معنی­داری مشاهده شد. این روش به طور موفقیت آمیزی باعث جداسازی RNA از دو بخش میوه پرتقال شد. به­طورکلی نمونه­های جذب 9/1 تا 2 بیشترین خلوص RNA استخراجی دارند و متوسط غلظت  RNA در روش تریازول به ترتیب در پوست پرتقال 90/379 (نانو گرم بر میکرو لیتر) و گوشت پرتقال 15/352 (نانو گرم بر میکرو لیتر) داشتند. و کمترین غلظت در روش دنازسیت غیر­ستونی به میزان 10/263 (نانو گرم بر میکرو لیتر) در گوشت میوه محاسبه گردید. روش تریازول یکی از بهترین­ها برای استخراج RNA کل باکیفیت می­باشد. در ارتباط با این روش بیشترین غلظت و خلوص RNA در نمونه ­های مختلف (پوست و گوشت) پرتقال نشان داد. به طور کلی در نمونه­ های پوست (فلاودو و آلبدو) غلظت RNA بیشتری بدست آمد.
 
کلیدواژه‌ها
استخراج
اسید نوکلئیک
پرتقال
پوست
گوشت
موضوعات

عنوان مقاله English

Comparison of three different genomic RNA extraction methods from peel and pulp of Thomson navel orange fruit (Citrus sinensis L.)

نویسندگان English

Fatemeh Ahmadi 1
Mohamad Mehdi Sharifani 2
Amir Mosavi 3
Negin Akhlaghi Amiri 4
Mostafa Khoshhal sarmast 5
Khalil Zaynali Nezhad 6
1 PhD Student, Department of Horticultural Science and Engineering, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.
2 Associate Professor, Department of Horticultural Science and Landscape Engineering, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran
3 Associate Professor, Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
4 Assistant Professor, Department of Horticulture Crops Research, Mazandaran Agricultural and Natural Resources Research and Education Center, AREEO, Sari, Iran.
5 Associate Professor, Department of Horticultural Science and Landscape Engineering, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.
6 Assistant Professor, Department of Plant Breeding & Biotechnology, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.
چکیده English

Introduction
Efficient genomic RNA extraction is a basic requirement of molecular biology and the primary step in the study of gene expression. Woody plants are problematic due to the presence of polyphenolics, polysaccharides and other compounds that bind or co-precipitate with the RNA. The present study is to compare different methods of extracting high-quality total RNA in Thomson navel orange. Citrus is one of the most strategic fruits of Iran, finding an efficient and low-cost method is important for executive and molecular works.
 
Materials and Methods
Plant materials used to extract total RNA were collected from two different parts of Thomson navel orange peel (consisting of both flavedo and albedo) and pulp from Ghaemshahr Horticultural Research Station, Mazandaran in 2020. Fruits were frozen in liquid nitrogen after sampling and were stored at -80°C until used. These samples were transferred to the laboratory department of horticultural science and engineering, Gorgan where the samples were applied to analyze the molecular expriment. For this study three methods including of: Trizol, Non-column Denazist and RNX-PlusTM were evaluated for total RNA extraction. In order to show the quality and quantity of RNA, were assessed on 1% agarose gel and using a nano­drop spectrophotometer with 230, 260 and 280 wavelength. Results were analyzed using a completely randomized design with three replicates, Duncan test with a significance level of 0.05 using SAS 9.0 software.
 
Results and Discussion
The result showed a distinct 28S and 18S rRNA bands visible on an agarose gel. The RNA integrity was assessed by the sharpness of ribosomal RNA bands. Also by using this protocols, the absorbance ratio at 260/280 was ranged between 1.80 – 1.93 which indicated there were no protein in the extracted RNA. A ratio A260/ A280 ratio from 2.0 to 2.14 indicated a lack of polysaccharides and phenol contamination. Trizol method, which was significantly different than compared to other methods. This protocol was successfully applied to isolate total RNA from the organ of two section fruit. Generally, the samples with a ratio in the range of ~ 1.9 to 2 revealed high purity of the extracted RNA using Trizol and the average concentration of RNA ranged from 379.90 (ng/µl) of orange peel and 352.15 (ng/µl) of orange pulp, respectively.  And concentration was observed in Non-column Denazist method which treated with the lowest concentration (263.10 ng/µl) of orange pulp.
 
Conclusion
The Trizol method was the best choice for extraction of high-quality total RNA. In this respect method represented the highest RNA yield and purity from various samples (peel and pulp) of citrus. However, generally peel samples (flavedo and albedo) produced high concentration total RNA.

کلیدواژه‌ها English

Citrus sinensis
extraction
peel
pulp
ribonucleic acid
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تولیدات گیاهی
دوره 46، شماره 1
بهار 1402
صفحه 1-10