شناسایی و تحلیل بیوانفورماتیکی ژن‌های ضد میکروبی دیفنسین از شش ژنوتیپ گیاه عدس

نوع مقاله : علمی پژوهشی

نویسندگان

1 استادیار، گروه زراعت و اصلاح‌نباتات، واحد خرم‌آباد، دانشگاه آزاد اسلامی، خرم‌آباد، ایران،

2 محقق، باشگاه پژوهشگران جوان و نخبگان، واحد خرم‌آباد، دانشگاه آزاد اسلامی، خرم‌آباد، ایران

3 استادیار، گروه کشاورزی، واحد کنگاور، دانشگاه آزاد اسلامی ، کنگاور، ایران

چکیده

دیفنسین‌های گیاهی، خانواده‌ای بزرگ از پپتیدهای ضد میکروبی غنی از سیستئین هستند. این پپتیدها، مولکول‌هایی با جرم مولکولی 5 تا 7 کیلودالتون و دارای هشت اسید آمینه سیستئین محافظت شده هستند که در ایجاد پیوندهای دی‌سولفیدی نقش دارند. در این پژوهش برخی اعضاء خانواده ژنی دیفنسین در ژنوتیپ‌های مختلف گیاه عدس جداسازی و بررسی شدند. ابتدا، تمام توالی‌های کد کننده‌ی ژن‌های دیفنسین گیاهان دو لپه‌ای از بانک ژن دریافت شد. توالی‌های کد کننده‌ی دریافت شده، در کتابخانه EST گیاه عدس همردیف و نتایج حاصل با هم مخلوط و سر هم‌بندی شدند. کانتیگ‌ها و سینگلتون‌های حاصل از سرهم‌بندی با استفاده از ابزار BLASTn، علیه پایگاه داده nr همردیف شدند. از کانتیگ‌ها و سینگلتون‌های دارای چارچوب خوانش باز کامل دیفنسین برای طراحی آغازگرها استفاده شد. در مرحله‌ی بعدی، از ژنوتیپ‌های گچساران، محلی، Filip2003-2L، Filip2003-9L، Filip2005-2L و Filip2006-10L گیاه عدس استخراج DNA صورت گرفت. در نهایت، توالی ژنومی دیفنسین‌ها با استفاده از واکنش PCR تکثیر شد و پس از همسانه‌سازی در ناقل pTZ57R/T مورد توالی‌یابی قرار گرفت. نتایج، شباهت کامل را در دیفنسین‌های جداسازی شده از ژنوتیپ‌های مورد بررسی نشان داد. ژن‌های شناسایی شده همچنین دارای اینترون‌هایی با طول متغیر بودند که ساختاری محافظت شده داشتند و حاوی عناصر تنظیمی برای پاسخ به عوامل و شرایط مختلف بودند. در این پژوهش برای اولین بار، توالی ژنومی سه ژن از ژن‌های خانواده دیفنسین از ژنوتیپ‌های مختلف گیاه عدس جداسازی و ساختار و ویژگی‌های آن‌ها مشخص شد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Identification and bioinformatics analysis of defensin antimicrobial genes from six lentil genotypes

نویسندگان [English]

  • Reza Mir Derikvand 1
  • Seyyed Mohsen Sohrabi 2
  • Kamran Samiei 3
1 Assistant Professor, Department of Agronomy and Plant Breeding, Khorramabad Branch, Islamic Azad University, Khorramabad, Iran
2 Researcher, Young Researchers and Elite Club, Khorramabad branch, Islamic Azad University, Khorramabad, Iran
3 Assistant Professor Department of Agronomy and Plant Breeding, Kangavar Branch, Islamic Azad University, Iran
چکیده [English]

Plant defensins are a large family of cysteine-rich antimicrobial peptides. These peptides are small molecules with molecular mass between 5 and 7kD and eight conserved cysteines that are involved in disulfide bond formation. In the present study, some members of defensin gene family were isolated and characterized from lentil genotypes. All coding sequences of the defensin genes of dicotyledon plants were retrieved from GenBank. The retrieved coding sequences were aligned against lentil EST library and the resulted sequences were pooled and assembled. The resulted contigs and singletons were aligned against GenBank nr database using BLASTn tool. The contigs and singletons with the full open reading frame were used to primer design. In the next step, DNA extraction was performed from Gachsaran, Mahali, Filip2003-2L, Filip2003-9L, Filip2005-2L and Filip2006-10L genotypes. Finally, by using PCR, genomic sequence of defensins were amplified, cloned into pTZ57R/T plasmid and sequenced. The results showed complete identity among the isolated defensins from all genotypes. The identified genes also contain conserved introns with variable length that comprised of regulatory elements for response to different factors and conditions. For first time in this study, genomic sequences of three members of defensin gene family were isolated from lentil genotypes and structures and features of them were determined.

کلیدواژه‌ها [English]

  • Antimicrobial peptides
  • Biotechnology
  • Defensins
  • Gene isolation
  • Lentil

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