ارزیابی پاسخ به تنش خشکی در سطح رونویسی در برنج با استفاده از فراتحلیل داده‌های RNA-seq

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری بیوتکنولوژی کشاورزی، گروه اصلاح نباتات و بیوتکنولوژی، دانشکده کشاورزی، دانشگاه شهرکرد، شهرکرد، ایران.

2 استاد، گروه اصلاح نباتات و بیوتکنولوژی، دانشکده کشاورزی، دانشگاه شهرکرد، شهرکرد، ایران.

3 استادیار، گروه اصلاح نباتات و بیوتکنولوژی، دانشکده کشاورزی، دانشگاه شهرکرد، شهرکرد، ایران.

4 دانشیار، گروه زیست‌شناسی، دانشکده علوم، دانشگاه رازی کرمانشاه، کرمانشاه، ایران.

5 استادیار ، دانشکده پزشکی دانشگاه کالیفرنیا، ریورساید، کالیفرنیا، آمریکا

چکیده

هدف: درک مکانیسم‌های مولکولی پاسخ به تنش از جمله تنش خشکی می‌تواند به‌طور قابل‌توجهی علم اصلاح مولکولی گیاهان را ارتقا بخشد. مطالعات ترنسکریپتومی می‌تواند حجم زیادی از اطلاعات را در دسترس محققان قرار دهد. ادغام چنین اطلاعاتی از منابع مختلف از طریق روش‌های آماری پیشرفته مانند فراتحلیل فرصت جدیدی برای غلبه بر پیچیدگی بیولوژیکی، شناسایی ژن‌های با بیان متفاوت (DEGs) و کسب نتایج قابل اعتمادتر، فراهم می‌آورد. مطالعه حاضر با هدف شناسایی DEGها در پاسخ به تنش خشکی با استفاده از داده‌های ترنسکریپتومی از طریق فراتحلیل داده‌های RNA-seq انجام شد.
مواد و روش‌ها: داده‌های RNA-seq از پایگاه داده EMBL-EBI دانلود شد و پس از پیش پردازش، نقشه‌یابی خوانش‌های با کیفیت بر روی ژنوم مرجع برنج با نرم افزار STAR صورت گرفت. تغییرات بیان ژن‌ها با استفاده از پکیج edgeR به‌صورت جداگانه برای هر مجموعه داده بررسی و سپس از خروجی حاصله برای فراتحلیل با استفاده از پکیج metaRNAseq استفاده شد. ژن‌های با بیان متفاوت و معنادار در پاسخ به تنش، از نظر عمکرد زیستی، مسیرهای زیستی درگیر و برهم‌کنش پروتئینی بررسی شد و در نهایت ژن‌های کلیدی در پاسخ به تنش خشکی تعیین گردید.
نتایج: مطابق نتایج فراتحلیل 6607 ژن با متوسط بیان log2FC≥|1| و FDR≤0.05 شناسایی شد که به ترتیب 3313 و 3294 از آن‌ها تحت تنش، بیانشان افزایش و کاهش یافته است. از این تعداد 162 ژن درآنالیز‌های انفرادی به‌عنوان DEG تعیین نشده بودند و تنها از طریق فراتحلیل شناسایی شدند، که این امر نشان‌دهنده قدرت آماری این روش در شناسایی ژن‌های جدید است. نتایج بررسی عملکردی DEGها نشان‌دهنده القای مسیرهای متابولیکی مختلف از جمله بیوسنتز متابولیت‌های ثانویه و اسیدهای آمینه، متابولیسم کربوهیدرات‌ها و مسیر انتقال پیام فیتوهورمون‌ها تحت تنش است. بررسی برهم‌کنش پروتئینی و شناسایی ژن‌های کلیدی نیز حاکی از نقش آن‌ها در پاسخ به تنش، فعالیت اکسیدوردوکتازی و متابولیسم اسید آمینه بود.
نتیجه‌گیری: این مطالعه می‌تواند درک ما را از مکانیسم‌های مولکولی پاسخ برنج به تنش‌ خشکی افزایش دهد و همچنین در شناسایی ژن‌های کلیدی و جدید، حتی به‌عنوان نشانگرهای مولکولی در راستای بهبود تحمل به تنش‌ خشکی در برنامه‌های اصلاحی برنج مفید باشد.

کلیدواژه‌ها

عنوان مقاله [English]

Evaluation of Transcription Response to Drought Stress in Rice Using RNA-seq Meta-analysis

نویسندگان [English]

  • Shima Karami 1
  • Behrouz Shiran 2
  • Rudabeh Ravash 3
  • Hossein Fallahi 4
  • Arghavan Alisolitani 5
1 Ph.D. Student, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran.
2 Professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran.
3 Assistant Professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran.
4 Assistant Professor, Department of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran.
5 Assistant Professor, University of California Riverside School of Medicine, Riverside, California, USA.
چکیده [English]

Objective
Understanding the molecular mechanisms of response to stress such as drought can significantly improve the science of plant molecular breeding. Transcriptomic studies can make a large amount of information available to researchers. Integrating such data from different sources through advanced statistical methods such as meta-analysis provides a new opportunity to overcome biological complexity, identify differentially expressed genes (DEGs), and obtain more reliable results. The present study aimed to identify DEGs in response to drought stress using transcriptomic data through a meta-analysis of RNA-seq data.
Materials and methods
RNA-seq data were downloaded from the EMBL-EBI database and after preprocessing, high-quality reads were mapped on the rice reference genome with the STAR software. Differential expression genes were evaluated separately for each dataset using the edgeR package. The outputs were used for meta-analysis using the metaRNAseq package. Genes with different and significant expressions in response to drought stress were examined for functional enrichment, biological pathways, and protein interaction. Finally, hub genes were identified.
Results
According to the meta-analysis results, 6607 differential expression genes with average log2FC≥|1| And FDR≤0.05 were detected. 3313 and 3294 of them were regulated up and down, respectively, and 162 genes were not identified as DEG in individual analyzes and were identified only by meta-analysis, which shows the statistical power of this method in identifying new genes. The results of functional enrichment of DEGs indicate the induction of various metabolic pathways under stress including biosynthesis of secondary metabolites and amino acids, carbohydrate metabolism, and plant hormone signal transduction. Investigation of protein interaction and identification of hub genes also showed their role in stress response, oxidoreductase activity, and amino acid metabolism.
Conclusions
This study can increase our understanding of the molecular mechanisms of rice response to drought stress and be useful in identifying key and new genes, even as molecular markers to improve drought stress tolerance in rice breeding programs.

کلیدواژه‌ها [English]

  • Drought stress
  • RNA-seq
  • Meta-analysis
  • Rice

مراجع

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مجله بیوتکنولوژی کشاورزی
دوره 14، شماره 4
دی 1401
صفحه 221-246

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