تولیدات گیاهی

تولیدات گیاهی

شناسایی و پیش‌بینی عملکرد ‌RNAهای طویل غیرکدکننده در پروفایل بیانی ریشه گیاه لوبیا ‌(.Phaseolus vulgaris L) تحت تنش شوری

نوع مقاله : علمی پژوهشی - بیوتکنولوژی گیاهی

نویسندگان
ستاره میرزاوند
احمد اسماعیلی
سید سجاد سهرابی
گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه لرستان، خرم آباد، ایران
10.22055/ppd.2025.49829.2278
چکیده
لوبیای معمولی یکی از مهم‌ترین گیاهان زراعی خانواده لگوم‌ها است که به‌دلیل ارزش غذایی بالا و توانایی تثبیت نیتروژن، نقش کلیدی در تأمین امنیت غذایی ایفا می‌کند. این گیاه منبعی غنی از پروتئین، آهن، روی و سایر ریزمغذی‌هاست، اما عملکرد آن به‌شدت تحت تأثیر تنش‌های محیطی، به‌ویژه شوری قرار دارد. شوری خاک با ایجاد اختلال در جذب آب و عناصر غذایی، تجمع یون‌های سمی و القای تنش اکسیداتیو، رشد و نمو گیاه را مختل می‌کند. در سال‌های اخیر، RNAهای طویل غیرکدکننده (lncRNAها) به‌عنوان تنظیم‌کننده‌های مهم در واکنش گیاهان به انواع تنش‌ها شناخته شده‌اند، اما عملکرد آن‌ها در لوبیای معمولی تحت شرایط شوری هنوز به‌طور کامل مورد بررسی قرار نگرفته است. این پژوهش در سال ۱۴۰۲ در دانشکدۀ کشاورزی دانشگاه لرستان با هدف شناسایی و تحلیل‌lncRNA های مرتبط با تنش شوری در ریشه لوبیای معمولی انجام شد. برای این منظور، از داده‌های RNA-Seq مطالعات پیشین استفاده گردید. خوانش‌های پردازش‌شده با ژنوم مرجع لوبیا هم‌تراز شده و توالی‌های فاقد ظرفیت کدکنندگی با استفاده از ابزارهای CPC2، PLncPRO و BLAST تحلیل و پالایش شدند. سپس، ژن‌های هدف lncRNAها با ابزار LncTar شناسایی و بیان افتراقی آن‌ها با نرم‌افزار DESeq2 تحلیل شد. علاوه بر این، موقعیت زیرسلولی این توالی‌ها و مسیرهای زیستی مرتبط با آن‌ها با بهره‌گیری از ابزارهای تخصصی پیش‌بینی گردید. برای اعتبارسنجی نتایج، از روش real-time PCR استفاده شد. برای این منظور، گیاهان لوبیا در شرایط گلخانه‌ای تحت تیمار شوری (150 میلی‌مولار کلرید سدیم) قرار گرفتند. پس از استخراج RNA از بافت ریشه گیاهان شاهد و تیمارشده، بیان نسبی ژن‌های منتخب lncRNA مورد ارزیابی قرار گرفت. در مجموع، ۲۰۳۱ توالی lncRNA با اطمینان بالا از بافت ریشه شناسایی شد که بخشی از آن‌ها برای نخستین بار در این گونه گزارش می‌شوند. مقایسه این توالی‌ها با پایگاه‌هایGreeNC ، PLncDB  و CANTATA نشان داد که ۶۲۰ توالی دارای شباهت معنی دار با داده‌های پیشین هستند، در حالی که مابقی احتمالاً اختصاصی لوبیا محسوب می‌شوند. نتایج تحلیل‌های بیوانفورماتیکی نشان داد که این‌lncRNA ها در مسیرهای زیستی متنوعی از جمله بیوسنتز ترکیبات ثانویه، مسیر سیگنال‌دهی MAPK، واکنش به هورمونABA ، سنتز ترپنوئیدها و متابولیسم اسید لینولئیک نقش دارند. از میان توالی‌های شناسایی‌شده، سه lncRNA شامل R_lncRNA1، R_lncRNA2 و R_lncRNA3 تغییرات معنی‌دار بیانی در واکنش به شوری نشان دادند. نتایج qRT-PCR این یافته‌ها را تأیید کرد. بررسی مکان‌یابی زیرسلولی نیز نشان داد که R_lncRNA2 در هسته و دو توالی دیگر در سیتوپلاسم حضور دارند؛ و این تفاوت می‌تواند بیانگر نقش‌های تنظیمی متفاوت آن‌ها باشد. همچنین، تحلیل غنی‌سازی GO نشان داد که این lncRNAها در فرآیندهایی مانند تنظیم رونویسی، تاخوردگی پروتئین، متابولیسم ترپن‌ها و واکنش به گونه‌های اکسیژن واکنش گر دخیل هستند. یافته‌های این پژوهش نقش کلیدی lncRNAها را در واکنش لوبیای معمولی به تنش شوری نشان می‌دهد. این مولکول‌ها از طریق تنظیم مسیرهای سیگنال‌دهی، تولید متابولیت‌های دفاعی و برهمکنش با ژن‌های عملکردی، به افزایش تحمل گیاه در برابر شوری کمک می‌کنند. بنابراین، lncRNAها می‌توانند به‌عنوان اهداف بالقوه برای اصلاح ژنتیکی و کاربردهای زیست‌فناوری به‌منظور توسعۀ ارقام متحمل به تنش‌های محیطی مورد توجه قرار گیرند. این پژوهش گامی مؤثر در جهت درک عمیق‌تر شبکه‌های تنظیمی lncRNA در گیاهان زراعی تحت تنش شوری به‌شمار می‌آید.
کلیدواژه‌ها
بیان افتراقی
تنش اکسیداتیو
متابولیسم ثانویه
مسیرهای سیگنال‌دهی
مکان‌یابی زیرسلولی
موضوعات

عنوان مقاله English

Identification and functional prediction of long non-coding RNAs in the expression profile of common bean (Phaseolus vulgaris L.) under salt stress conditions

نویسندگان English

Setareh Mirzavand
Ahmad Ismaili
Seyed Sajad Sohrabi
Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
چکیده English

Introduction
Common bean (Phaseolus vulgaris) is one of the most essential legumes crops worldwide due to its high nutritional value and significant contribution to food security. It is a primary source of plant-based proteins, as well as essential micronutrients such as iron, zinc, thiamine, and folic acid. Despite these advantages, the productivity of common bean is severely affected by environmental stressors, especially salinity. Soil salinization, an increasingly prevalent issue driven by climate change, poor irrigation practices, and improper drainage, hampers plant growth by disrupting water uptake, causing ion toxicity, and inducing oxidative stress. Plants respond to salt stress through complex molecular and physiological mechanisms, including hormonal signaling, transcriptional regulation, and metabolic reprogramming. In recent years, long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression involved in diverse biological processes, yet their specific roles under salt stress in Phaseolus vulgaris remain largely unexplored.
Materials and Methods
This study was conducted in 2023 at the Faculty of Agriculture, Lorestan University, with the aim of identifying and characterizing lncRNAs involved in salinity stress responses in the roots of common bean. For this purpose, the sequenced RNA data from previous studies were used in this study, and then, high-quality reads were mapped to the Phaseolus vulgaris reference genome. Candidate lncRNAs were identified based on transcript length, coding potential, and filtering out known protein-coding RNAs, rRNAs, and tRNAs. Differential expression analysis was conducted using DESeq2. Functional annotation of target genes was performed using KEGG and GO enrichment analysis, and subcellular localization of lncRNAs was predicted. For validation the relative expression levels of selected lncRNAs, real-time PCR was performed. For this, the experiment was conducted under controlled greenhouse conditions and seedlings were subjected to 150 mM NaCl salinity. Root samples from control and treatment were collected (72 hours after final salt application) and after RNA extraction, finally were used for evaluation of relative gene expression of lncRNAs.
Results and Discussion
In this study, a total of 2,031 high-confidence lncRNA sequences were identified from the root tissues of common bean, some of which are reported for the first time in this species. Comparison of these sequences with the GreeNC, PLncDB, and CANTATA databases revealed that 620 lncRNAs showed significant similarity to previously reported data, while the others are likely specific to common bean and represent novel discoveries. Bioinformatic analyses indicated that these lncRNAs are involved in diverse biological pathways, including secondary metabolite biosynthesis, MAPK signaling, ABA response, terpenoid synthesis, and linoleic acid metabolism. Among the identified transcripts, three lncRNAs R_lncRNA1, R_lncRNA2, and R_lncRNA3 displayed significant differential expression under salt stress conditions. qRT-PCR analysis validated the RNA-Seq results and confirmed that the expression of these lncRNAs changed significantly in response to salinity. Subcellular localization analysis showed that R_lncRNA2 was localized in the nucleus, whereas the other two were found in the cytoplasm, suggesting clear differences in their potential regulatory roles. Moreover, GO enrichment analysis demonstrated that these lncRNAs are associated with processes such as transcriptional regulation, protein folding, terpenoid metabolism, and responses to reactive oxygen species. Overall, these findings highlight the importance of lncRNAs in the molecular networks that govern salt stress responses in common bean.
Conclusion
This study provides the first comprehensive identification and functional prediction of lncRNAs involved in salt stress response in Phaseolus vulgaris. The findings demonstrate that lncRNAs play essential roles in regulating complex stress response networks, mainly by modulating metabolic and signaling pathways. These insights contribute to a deeper understanding of molecular mechanisms underlying salt tolerance and open new avenues for the development of salt-resilient legume cultivars. 

کلیدواژه‌ها English

Differential expression
Oxidative stress
Secondary metabolism
Signal transduction pathways
Subcellular localization
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تولیدات گیاهی
دوره 49، شماره 1
بهار 1405
صفحه 85-101