بررسی بیان ژن‌های دخیل در مقاومت درخت‌ سیب به شانکر اروپایی با استفاده از آنالیز داده‌های ترانسکریپتوم

نوع مقاله : مقاله پژوهشی

نویسندگان

1 نویسنده مسئول: استادیار، گروه تنوع زیستی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی، دانشگاه تحصیلات تکمیلی و فناوری پیشرفته کرمان، کرمان، ایران

2 استاد، گروه اصلاح نباتات، دانشگاه علوم کشاورزی سوئد، کیخانستاد، سوئد

چکیده

چکیده
هدف: یکی از مخرب ترین بیماری‌های درختان میوه، بیماری شانکر اروپایی با عامل قارچ بیماری زای Neonectria ditissima، در کشورهای تولید کننده سیب با هوای خنک می‌باشد. از آنجایی که کنترل این بیماری به دلیل حضور قارچ بیماری زا در طول کل سال دشوار است، این تحقیق به منظور بررسی الگوی بیان ژن‌های پاسخ دهنده به این عامل بیماری زا در سیب انجام شد که می‌تواند درک بهتر از تعامل بین میزبان و پاتوژن در جهت بهبود استراتژی‌های مدیریتی فراهم کند.
مواد و روش ها: بدین منظور رقم نیمه مقاوم سیب ("جاناتان"، منشا نیویورک) با سوسپانسیون قارچ تلقیح داده شد و نمونه‌های شاهد و تلقیح داده شده برای استخراج RNA و توالی یابی در سه نقطه زمانی 5، 15 و 30 روز پس از تلقیح برداشت شدند. پس از کنترل کیفی و کمی RNA کل استخراج شده، توالی‌یابی کل ژنوم به صورت دو سویه توسط شرکت ایلومینا و دستگاه‌ توالی‌یاب Hiseq2000 انجام شد. کنترل کیفیت داده‌ها توسط نرم افزارFastQC صورت پذیرفت. سپس خوانش‌ها با استفاده از نرم افزارTopHat2 با ژنوم مرجع سیب نقشه‌یابی شدند. نرمال‌سازی و تجزیه و تحلیل ژن‌های با بیان متفاوت با نرم افزار DESeq2 و آنالیز غنی‌سازی مسیرهای DEGs با نرم افزار KEGG انجام شد.
یافته ها: تجزیه و تحلیل‌های غنی‌سازی GO و KEGG در ژن‌های دارای بیان افتراقی (Differentially expressed genes, DEGs) ، تعدادی ژن مرتبط با پاسخ دفاعی را شناسایی نمود. در رقم سیب تلقیح ‌شده با N. ditissima، تغییرات قابل‌توجهی در ژن‌های مرتبط با دفاع و ژن‌های دخیل در سم‌زدایی، پراکسیداز و متابولیسم فنیل پروپانوئید مشاهده شد. بالاترین سطح بیان ژن‌های مرتبط با دفاع، 30 روز پس از تلقیح با N. ditissima دیده شد. این موضوع می‌تواند بیانگر این باشد که پاتوژن برای ایجاد آلودگی نیاز به زمان دارد و به سرعت نمی‌تواند در بافت گیاه گسترش پیدا کند.
نتیجه گیری: ژن‌های شناسایی شده درگیر در بیماری ‌زایی N. ditissima دخیل در لیگنین شدن، سم‌زدایی، فسفوریلاسیون و دفاع پاتوژن بوده و منبعی ارزشمند در تحقیقات ژنتیکی هستند و به ما این امکان را می‌دهد تا تعامل پاتوژن با گیاه میزبان را بهتر درک کنیم و می توانند در برنامه‌های اصلاحی کنترل این بیماری مورد استفاده قرار گیرند.

کلیدواژه‌ها


عنوان مقاله [English]

Identification of genes associated with tolerance to apple canker by genome-wide transcriptome analysis

نویسندگان [English]

  • Marjan Ghasemkhanni 1
  • Hilde Nybom 2
1 Corresponding author. Assistant Professor, Department of Biodiversity, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
2 Professor, Department of Plant Breeding, Balsgård, Swedish University of Agricultural Sciences, Kristianstad, Sweden
چکیده [English]

Objective
Neonectria ditissima, the causal agent of fruit tree canker, is one of most destructive diseases of apple trees in producing countries with cool weather. Since it is difficult to control this disease due to the presence of the pathogenic fungus throughout the year, for better understanding of this host–pathogen interaction in order to improve management strategies, a transcriptional analysis of apple gene expression in response to N. ditissima was conducted.
Materials and methods
In the present study, we evaluated transcriptome responses to N. ditissima by selecting the partially resistant cultivar “Jonathan”. The leaf scars of apple trees were artificially inoculated with suspension of N. ditissima and water as control. Then the samples were taken for RNA extraction at three different time points, 5, 15, and 30 days after inoculation. The quality and quantity of the extracted RNA were checked and total RNA was sequenced using Illumina paired-end sequencing and Hiseq2000 sequencer. The quality of the sequence data was done by FastQC software. Then the reads were mapped to apple reference genome by TopHat2 software. Normalization and differential expression analysis of genes were performed with DESeq2. Enrichment analysis of DEGs pathways was done through KEGG software.
Results
Based on GO enrichment and KEGG pathway analyses, it was found that some of the defense response genes were differentially expressed between control and treatment groups. The data provides evidence that apple cultivars inoculated with N. ditissima exhibit significant upregulation of defense-related genes and genes involved in detoxification, peroxidase related reactions, phenylpropanoid metabolism. The highest expression level of genes related to defense was observed 30 days after inoculation. It shows that the pathogen needs time to cause infection and cannot spread quickly in the plant tissue.
Conclusion
Identification of candidate genes involved in pathogenicity of N. ditissima are involved in lignification, detoxification, phosphorylation and pathogen defense. They are a valuable resource in genetic research and allow us to better understand interaction of fungus and the apple defense system, and may assists in apple canker breeding programs.

کلیدواژه‌ها [English]

  • Defense response
  • Next-generation sequencing (NGS)
  • Malus × domestica
  • RNA-Seq
جعفری احمدآبادی سید علی اصغر، عسکری­همت حشمت­اله، محمدآبادی محمدرضا (1402) تاثیر شاهدانه بر بیان ژن DLK1 در بافت‌ قلب بره‌های کرمانی. مجله بیوتکنولوژی کشاورزی، 15(1)، 217-234.
شکری سمیرا، خضری امین، محمدآبادی محمدرضا، خیرالدین حمید (1402) بررسی بیان ژن MYH7  در بافت‌های ران، دست و راسته بره‌های پرواری نژاد کرمانی. مجله بیوتکنولوژی کشاورزی، 15(2)، 217-236.
محمدآبادی محمدرضا، گلکار افروز، عسکری حصنی مجید (1402) اثر رازیانه (Foeniculum vulgare) بر بیان ژن فاکتور 1 رشد شبه انسولین در بافت شکمبه گوسفند کرمانی. مجله بیوتکنولوژی کشاورزی، 15(4)، 239-256.
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