ساخت همسانه عفونت‌زای ویروئید‌های مرکبات و اثبات بیماری‌زایی آنها

نوع مقاله : مقاله کامل پژوهشی

نویسندگان

گروه گیاه‌پزشکی، بخش بیماری شناسی گیاهی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران.

چکیده

ویروئیدها آر ان ا های بیماری‌زا در گیاهان هستند که توانایی رمز کردن پروتئین را ندارند. تاکنون هفت ویروئید از چهار جنس خانواده Pospiviroidaeاز مرکبات گزارش شده‌اند.با ساخت همسانه عفونت­زای ویروئید­های مرکبات امکان سنجش بیماری­زایی آنها فراهم شد. از آر ان ا کل استخراج‌شده از نهال پرتقال خونی مورو ژنوم کامل ویروئیدهای CEVd، HSVd، CBCVd، CDVd، CVdV و CBLVd در واکنش RT-PCR تکثیر و توالی‌یابی شد. سپس ژنوم کامل هر ویروئید بطور جداگانه در ناقل دوتایی pBin62SK تحت کنترل پیشبر s35 ویروس موزائیک گل‌کلم (Cauliflower mosaic virus, CaMV) قرار گرفت. بیماری­زایی ویروئیدها در گیاهان خیار(Cucumis sativus)، گوجه‌فرنگی (Solanum lycopersicum) و نارنج سه برگ (Poncirus trifoliata) از طریق مایه‌زنی اگروباکتریوم حاوی سازه‌های عفونت‌زای آنها انجام شد. سه هفته پس از مایه‌زنی حضور ویروئیدها در برگ­های جدید گیاهان مایه‌زنی‌شده با آزمون RT-PCR با استفاده از آغازگرهای اختصاصی و هیبریداسیون نقطه ای با کاوشگر اختصاصی هر ویروئید تائید شد. پنج هفته پس از مایه‌زنی، علائم ریزبرگی، پیسک و روخمشی توسط CVdV و CBCVd، پیسک توسط CDVd و علایم روخمشی و بدشکلی برگ بوسیله CEVd، HSVd و CBLVd در برگ­های گوجه فرنگی ظاهر شدند. مایه‌زنی مکانیکی عصاره آلوده به گیاهان سالم نیز علائم مشابهی را تولید کرد. ردیابی ویروئید در برگ­های جدید گیاهان مایه‌زنی‌شده بیانگر عفونت‌زایی سازه ساخته شده است. همسانه‌های عفونت‌زای CEVd، HSVd و CBCVd پیش­تر ساخته شده است ولی این اولین گزارش از ساخت همسانه عفونت‌زای ویروئیدهای CDVd، CBLVd وCVdV است.

کلیدواژه‌ها


عنوان مقاله [English]

Construction of infectious clones and demonstration of pathogenicity of citrus viroids

نویسندگان [English]

  • L. Ebrahimi-moghadam
  • M. Zakiaghl
  • B. Jafarpour
  • M. Mehrvar
چکیده [English]

Viroids are pathogenic RNAs in plants without protein encoding capacity. Seven viroids belonging to four genera in the Pospiviroidae family have been reported from citrus species. By constructing the infectious clones of citrus viroids, it was possible to carried out their pathogenicity test. Full-length genome of CEVd, HSVd, CBCVd, CDVd, CVdV and CBLVd was separately amplified in RT-PCR using total RNA extracted from a Moro blood orange plant and resulting PCR products were sequenced. The full-length genome of each viroid was inserted under the control of cauliflower mosaic virus 35S promoter in a pBin62SK binary vector. Pathogenicity of viroids was tested by agroinoculation of their infectious clones to cucumber (Cucumis sativus), tomato (Solanum lycopersicum) and trifoliate orange (Poncirus trifoliata). Three weeks after inoculation, viroids were detected in newly grown leaves of inoculated plants by RT-PCR using specific primers and by dot blot hybridization using viroid specific probes. Five weeks after inoculation, symptoms including little leaf, mottling and epinasty by CVdV and CBCVd, mottling by CDVd, and epinasty and leaf deformation by CEVd, HSVd and CBLVd in tomato leaves were observed. Mechanical inoculation of infected sap of infected tomato plants induced similar symptoms in healthy tomato plants. Identification of the viroids in newly grown leaves of inoculated plants indicated the infectiousness of the generated constructs. Infectious clones of CEVd, HSVd and CBCVd were previously made, but it is the first report of the construction of the infectious clone for CDVd, CBLVd and CVdV.

کلیدواژه‌ها [English]

  • Viroid
  • citrus
  • infectious clone
  • pathogenicity
 
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