همسانه سازی، تنوع ژنتیکی ژن رمزکننده پروتئین پوششی و تعیین زیرگروه های نژادی جدایه‌های ویروس موزائیک خیار در مزارع طالبی شهرستان ورامین

منطقه ی ورامین واقع در جنوب تهران یکی از مراکز مهم تولید خربزه و طالبی است. اخیرا تغییر در جمعیت ویروس های کدوئیان گزارش شده است. از این رو، به منظور بررسی تنوع ژنتیکی ویروس موزائیک خیار و بررسی تغییر احتمالی جمعیت این ویروس در مزارع طالبی مناطق ورامین، پیشوا و پاکدشت، 132 نمونه ی برگی طی سالهای 1393 و 1394 با علایم مشکوک به آلودگی ویروسی شامل موزاییک، پیسک و بدشکلی برگ و نیز کوتولگی گیاه جمع آوری شد. آلودگی 59 نمونه از کل نمونه ها (69/44 درصد) به cmv توسط آزمون الایزا تعیین شد که نشان دهنده میزان آلودگی بالای گیاهان طالبی نمونه‌برداری شده از مزارع مورد مطالعه است. از نمونه های الایزا مثبت، آر‌آن‌ای کل استخراج و ژن پروتئین پوششی به همراه بخشی هایی از اطراف آن طی rtpcr تکثیر شد. برای تعیین زیرگروه های جدایه های جمع‌آوری شده، محصولات rtpcr با استفاده از آنزیم mspi (hpaii) برش داده شدند که در اثر آن قطعاتی به اندازه های 335 و 537 جفت باز تولید شدند. بر این اساس جدایه های جمع آوری شده در این مطالعه در گروه i قرار گرفتند. ژن cp در چهار جدایه که بر اساس فاصله مکانی انتخاب شده بودند به حامل ptg19t متصل و در میزبانescherichia coli همسانه سازی شد. نتایج تعیین توالی، وجود جایگاه برشی آنزیم mspi را در موقعیت یاد شده تایید کرد. چهار جدایه ی تعیین توالی شده در این پژوهش، 99100 درصد در سطح اسید های نوکلئیک و همچنین 100 درصد در سطح آمینواسیدی با یکدیگر تشابه داشتند. میزان تشابه این جدایه ها با جدایه های از قبل گزارش شده از ایران به ترتیب 93100 و 98100 درصد در سطح نوکلئوتیدی و آمینواسیدی دیده شد. نتایج فیلوژنی در سطح نوکلئوتیدی و آمینواسیدی نیز نتایج rtpcrrflp را تایید نمود و مشخص شد که جدایه های cmv در مزارع طالبی ورامین متعلق به زیرگروه ai و جدایه هایی با شدت بیماری زایی شدید هستند.

Cloning, Genetic Diversity of Coat Protein Gene and Detection of Subgroups of Cucumber mosaic virus Strains from Cantaloupe in Varamin

Introduction: Varamin is one of the major producer regions of cantaloupes which is located in southern Tehran. Cucumber mosaic virus (CMV) is the type species of viral genus Cucumovirus in the family Bromoviridae and is causal agent of economically important losses in cucurbits. The CMV virions are isometric in shape and the genome is composed of three singlestranded positive sense RNAs. Each of the genomic RNAs 1 and 2 encodes a single large ORF and RNA2 harbors an additional smaller ORF, 2b, involved in celltocell movement and posttranscriptional gene silencing. The RNA3 has two ORFs, 3a and 3b, encoding a movement protein and coat protein, respectively. Each of genomic RNAs has a Caplike and tRNAlike structures at its 5ʹ and 3 ' ends, respectively. The virus is transmitted by different species of aphids in a nonpersistent manner and is easily transmissible to the indicator hosts by mechanical inoculation. CMV is reported from several cucurbitaceae plants in Iran. Isolates of CMV are classified in different groups or subgroups based on different methods especially serology, RTPCRRFLP, nucleotide sequencing and phylogeny which are useful data for decision making or choosing the strategy during the development of CMVresistant varieties and producing transgenic plants by RNA silencing strategy.;Materials and Methods: During 20142016, a total number of 132 leaf samples were collected from cantaloupes showing viral symptoms such as mosaic, mottling, leaf deformation and plant stunting. These samples were subjected to double antibody sandwich enzyme linked immunosorbent assay (DASELISA) with specific antibodies (DSMZ, Germany) to detect CMV based on the manufactures’ instructions. Total RNA was extracted from ELISA positive samples as explained elsewhere. Two microliters of total RNA used as template in RTPCR to amplify a 870 base pair (bp) fragment of partial CMV genomic RNA3 including the coat protein gene and its flanking regions using specific primers CMVCPf (5 'GCTTCTCCGCGAG3 ') and CMVCPr (5 'GCCGTAAGCTGGATGGAC3 ') corresponded to 1149–1161 nts and 1998–2015 of CMV strain Q, respectively. In order to address the subgroups of CMV isolates, RTPCR products were digested by MspI (HpaII) restriction enzyme and electrophoresis was done on 1.5% agarose gel. We expected the presence of the bands with 335 and 537 bp in size for the subgroup I and 393 and 559 bp for subgroup II isolates of CMV. Then, based on the geographical distance of collected CMV isolates, the CP gene from four isolates were ligated into pTG19T, an AT cloning vector, and the recombinant plasmids were introduced in Escherichia coli. The selected plasmids were extracted via alkaline lyses method and selected recombinant plasmids were sequenced by Macrogen, South Korea. The sequencing data were aligned with those of previously reported CMV isolates. The alignment and pairwise sequencing to estimate similarity between nucleotide sequences were done with BioEdit software. The Phylogeny Inference package (Phylip) version 3.65 was used to draw phylogeny trees.;Results and Discussion: 59 out of 132 samples (%44.69) positively reacted with CMV specific antibodies in DASELISA. Expected DNA fragments of about 870 bp were successfully amplified via RTPCR from the ELISA positive samples. in RTPCRRFLP for addressing the CMV subgroups, all MspI digested RTPCR products from the tested samples produced two DNA bands of 537 and 335 bp and it was concluded that these isolates belong to the CMV subgroup I. Sequencing data of selected CMV CP clones showed 99100% and 100% similarity between tested isolates at the nucleotide and amino acid levels, respectively. These isolates shared 93100% and 97100% similarity with previously reported CMV isolates from Iran at nucleic acid and amino acid levels, respectively. The reconstructed phylogenetic tree, reconfirmed the results of RTPCRRFLP, and Varamin isolates were placed with few other Iranian CMV isolates including E12, Cues, D13, F13, Sh44, Csu and Cud which were collected from nonmelon hosts. Isolate LORF, the only CMV isolate previously reported from melons, belongs to the IB subgroup of CMV. This is the first report of CMV subgroup I isolates from melons.  ;Conclusion: The RTPCRRFLP and sequencing data confirmed the presence of subgroup IA of CMV isolates in Varamin, Pishva and Pakdasht cantaloupe fields whose express severe symptoms in melons. However, the change in Zucchini yellow mosaic virus has been reported from these area, the same isolate of CMV were distributed in the entire region. Unlike Zucchini yellow mosaic virus populations which has been changed in the Varamin, Pakdasht and Pishva regions, it seems that there is just one dominant CMV isolate widespread in these regions.