ارزیابی دو رویکرد غیرترایخت‌سازی در راه‌اندازی تداخل آران‌ای بر علیه ویروس پیچیدگی برگ زرد گوجه‌فرنگی

نوع مقاله : علمی پژوهشی -انگلیسی

نویسنده

استادیار گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه شهید چمران اهواز، اهواز، ایران

چکیده

سویه شدید ویروس پیچیدگی برگ زرد گوجه­‌فرنگی (Tomato yellow leaf curl viru, TYLCV-IL) یک بگوموویروس تک­‌بخشی مخرب با گسترش جهانی است. دو روش غیرتراریخت­‌سازی، واکسیناسیون آران­‌ایِ دولا و کاربرد سازه سنجاق‌­سری بیان­‌کننده آران‌­ایِ دولا، به منظور القای تداخل آران‌­ای بر علیه ویروس پیچیدگی برگ زرد گوجه­‌فرنگی مورد استفاده قرار گرفت. بررسی پروفایل آراِن‌­اِی­‌های کوچک مداخله‌­گر مشتق شده از ژنوم ویروس، دو نقطه داغ شامل قسمت­‌هایی از توالی ژن­‌های کدکننده پروتئین پوششی و پروتئین همراه با همانندسازی را در راه­‌اندازی خاموشی ژن پس از ترانویسی مشخص نمود. آراِن­‌اِی‌­های دولای مشتق شده از این توالی‌­ها در شرایط درون‌­شیشه‌­ای تولید شدند و همزمان و یا سه روز قبل از مایه­‌زنی همسانه عفونت‌­زای ویروس، بر روی سطح برگ گیاهان توتون مورد استفاده قرار گرفتند. تمام گیاهان علایم مشخص آلودگی را در زمان 21 روز پس از مایه­‌ز­نی نشان دادند و قطعات ژنومی ویروس در گیاهان دارای علایم توسط واکنش زنجیره‌­ای پلی‌­مراز تکثیر شد. علی­‌رغم حرکت سیستمیک آران‌­ای‌­های دولایِ مورد استفاده در گیاهان توتون، واکنش زنجیره‌­ای پلی­‌مراز در زمان حقیقی، هیچ تفاوت آماری معناداری در سطح تجمع ژنوم ویروس در بین تیمارهای مختلف نشان نداد. در رویکرد دوم، کاربرد سازه سنجاق­‌سری بیان­‌کننده آران‌­ایِ دولا، بروز علایم آلودگی را تا زمان 21 روز پس از مایه‌زنی سرکوب کرد. همچنین ژنوم ویروس در گیاهان فاقد علایم ردیابی نشد که نشان­‌دهنده قطع همانندسازی ژنوم ویروس است. این نتایج، کارآیی متمایز دو روش القای تداخل آراِن­‌اِی را علیه یک گونه مهم بگوموویروس نشان می‌­دهد. نقش عوامل مختلف تأثیرگذار بر کارآیی تداخل آراِن­‌اِی علیه ویروس پیچیدگی برگ زرد گوجه‌­فرنگی مورد بحث قرار گرفته است.

کلیدواژه‌ها

عنوان مقاله [English]

Evaluation of Two Nontransformative Approaches in Triggering RNAi against Tomato Yellow Leaf Curl Virus

نویسنده [English]

  • S. Tabein
Assistant Professor, Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
چکیده [English]

Tomato yellow leaf curl virus-Israel (TYLCV-IL) is a worldwide destructive monopartite begomovirus. Two nontransformative methods, namely the dsRNA (double-stranded RNA) vaccination and the hairpin construct expressing dsRNA were applied to induce RNA interference (RNAi) against TYLCV-IL infection. Analysis of TYLCV-IL-derived small interfering RNAs profile revealed two hot spots triggering post-transcriptional gene silencing, including a part of the coat protein (CP) and the replication-associated protein (Rep) genes. CP and Rep derived dsRNAs were produced in vitro and applied simultaneously or three days before agroinoculation of TYLCV-IL by rubbing on the leaf surface of Nicotiana benthamiana. All tested plants showed symptoms of typical infection at 21 days' post-inoculation (dpi), and viral genomic fragments were amplified in symptomatic plants by PCR as expected. Despite the systemic movement of applied dsRNAs in N. benthamiana plants, quantitative PCR showed no statistically significant difference between TYLCV-IL genome accumulation in virus infection alone or at the presence of dsRNA molecules. In contrast, the hairpin RNA construct (hpRep) triggered a high resistance against TYLCV-IL infection in tomato plants by targeting the same Rep sequence. HpRep construct suppressed expression of symptoms up to 21 dpi. Moreover, no virus genome was detected in symptomless plants indicating disruption of TYLCV-IL replication. These results indicate the distinct efficiency of two methods of RNAi induction against an important begomovirus species, TYLCV-IL. The role of different factors on the RNAi efficiency against TYLCV-IL has been discussed.

کلیدواژه‌ها [English]

  • TYLCV-IL
  • dsRNA vaccination
  • hairpin construct
  • RNA interference

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 © 2022 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/).
گیاه پزشکی
دوره 44، شماره 4
دی 1400
صفحه 77-88

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