جمع‌آوری و تعیین ویژگی جدایه‌های ایرانی ویروس چندوجهی هسته‌ای کرم غوزه پنبه Helicoverpa armigera Single Nucleopolyhedro virus (HearSNPV) بر مبنای ژن پلی‌هدرین (polh)

نوع مقاله : مقاله پژوهشی


1 گروه گیاه‌پزشکی، دانشکده علوم و مهندسی کشاورزی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج.

2 عضو هیات علمی دانشگاه تهران

3 استادیار پژوهش، موسسه تحقیقات ثبت و گواهی بذر و نهال، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران

4 دانشگاه شهید مدنی آذربایجان

5 عضو هیات علمی

6 بخش تحقیقات آنفلونزا و سایر بیماریهای تنفسی انستیتو پاستور ایران، تهران


استفاده از روش‌های مبتنی بر اسیدنوکلئیک در شناسایی ویروس‌ها به دلیل ساختار کوچک ژنومی آن‌ها و تفاوت‌های جزئی در سطح نوکلئوتید بین جدایه‌ها یا استرین‌ها، از اهمیت زیادی برخوردار است. در پژوهش حاضر، لاروهای مرده و/یا بیمار از مزارع گوجه‌فرنگی مناطق مختلف جغرافیایی ایران جمع‌آوری و برای آلودگی باکولوویروسیHelicoverpa armigera Single nucleopolyhedrovirus (HearSNPV) با روش بهینه‌سازی شده بر اساس سدیم دودسیل سولفات برای استخراج OBs، بررسی شدند. واکنش زنجیره‌ای پلی‌مراز (PCR) با استفاده از دو جفت آغازگر اختصاصی (Polh-a, Polh-b) طراحی شده بر اساس ناحیه حفاظت شده ژن پلی‌هدرین(Polh) برای 26 جدایه ثبت شده از ویروس Hear/H. zea-SNPV انجام یافت. آلودگی به HearSNPVبا تکثیر باندهای مونومورفیک 370 و 790 نوکلئوتیدی در 28 لارو تایید شد. همسانه‌سازی و تعیین توالی نوکلئوتیدی باندهای تکثیر شده، تعلق آن‌ها به ژن Polhویروس چندوجهی‌هسته‌ای گونه‌هایarmigera H. و H. zea با شباهت بیش از 6/99 درصد را نشان داد. تجزیه و تحلیل تبارزایی جدایه‌های ایرانی HearSNPV همراه با سایر جدایه‌های ثبت شده از این ویروس در دنیا، جدایه‌های ایرانی را در گروه II آلفاباکولوویروس‌ها طبقه‌بندی و کارایی بیش‌تر روش توسعه داده شده را اثبات کرد. پژوهش حاضر اولین گزارش از معرفی و شناسایی مولکولی جدایه‌های ایرانی HearSNPV با به کارگیری تکنینک PCR و DNA ویروس از لاروهای با هویت نامشخص بیمارگر، می‌باشد. با توجه به حصول اطمینان از موثر و مفید بودن این روش در غربال‌گری سریع لاروهای حشرات از نظر آلودگی به این ویروس و بررسی تبارزایش، بهره‌مندی از آن در پژوهش‌های آتی توصیه می‌گردد.


عنوان مقاله [English]

Collecting and partial characterization of Iranian Helicoverpa armigera Single NucleopolyhedroVirus isolates based on polh gene

نویسندگان [English]

  • Raheleh Shahbazi 1
  • masoud Naderpour 3
  • Ali Mehrvar 4
  • Akbar Dizadji 5
1 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
3 Research Assistant Professor, Seed and Plant Improvement Institute, Education and Extension Organization, Karaj, Iran
4 Department of Plant Protection, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran
5 پردیس کشاورزی و منابع طبیعی دانشگاه تهران- گروه گیاهپزشکی
6 Pasteur Institute, Tehran, Iran
چکیده [English]

Nucleic acid-based diagnostic approaches are significant methods in detection and identification of viruses due to their small genome structures and minimal nucleotide differences among virus isolates or strains. In this study, dead or diseased larvae of Helicoverpa sp. were collected from tomato fields in distinct geographic areas in Iran and baculovirus infection was assayed by occlusion body extraction using an optimized Sodium Dodecyl Sulphate method. Two sets of specific polymerase chain reaction (PCR) primer pairs (Pol-a and Pol-b) were designed based on the conserved polyhedrin gene region of 26 fully sequenced HearSNPV/HzNPV isolates. Accordingly, infection by HearSNPV was confirmed in 28 out of 34 tested larvae by PCR amplification of monomorphic fragments of about 370 and 790 nucleotides, respectively. Cloning and sequencing of fragments showed that they belong to the corresponding polyhedron gene from nucleopolyhedriviruses of H. arimegra and H. zea species with 99.6% sequence identity. The phylogeny trees developed for Iranian isolates based on their polyhedron gene sequences showed that they all belong to the Group II Alphabaculovirus and formed one clade with other HearSNPV isolates. This, further confirmed the efficiency of the developed method for baculovirus detection and characterization. To our knowledge, this is the first report of introduction and molecular characterization of some HearSNPV isolates, circulating in these pests in Iran, using a robust DNA-based approach. The excellent efficacy of this method in virus detection makes it a valuable tool for rapid screening of insect cadavers for HearNPV infection, phylogenic studies and virus monitoring in bioinsecticides application.

کلیدواژه‌ها [English]

  • nucleopolyhedrovirus
  • polymerase chain reaction
  • Helicoverpa sp
  • polh gene
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