شناسایی تعدادی از ژن‌های با الگوی بیان متفاوت بین ارقام مقاوم و حساس گندم در پاسخ به تنش خشکی با استفاده از روش متاآنالیز

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد بیوتکنولوژی دانشکده کشاورزی، دانشگاه شهرکرد

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

3 شهرکرد-دانشگاه شهرکرد- دانشکده کشاورزی- گروه اصلاح نباتات و بیوتکنولوژی

4 دانشیار، گروه زراعت و اصلاح نباتات دانشکده کشاورزی، دانشگاه شیراز

چکیده

هدف:گندم از مهمترین منابع غذایی بشر می‌باشد که در تولید این محصول، تنش‌های غیرزیستی مانند خشکی، شوری، دما و غیره دخیل می‌باشند. از این رو شناسایی و ارزیابی ژن‌های دخیل در مقاومت به تنش‌ها در این گیاه از اهمیت بالایی برخوردار است. این تحقیق با هدف شناسایی ژن‌های پاسخ دهنده به خشکی و بررسی تفاوت بیان آنها در گندم حساس و مقاوم به خشکی، انجام شد.
مواد و روش‌ها: از بین داده‌های موجود در سایت NCBI GEO در سال 2018 داده‌های ریزآرایه مورد نظر جمع‌آوری شدند. کتابخانه‌های تهیه شده دارای ارقام مقاوم و حساس به خشکی، تحت دو شرایط تنش و کنترل بودند. با استفاده از متاآنالیز، ژن‌های تغییر بیان‌یافته تحت شرایط تنش برای هر گروه از ارقام شناسایی شد. از ژن‌های تغییر بیان یافته معنی‌دار شناسایی شده، ژن دئوکسی ژنز کاهش‌دهنده شبه‌پروتئین aci (ARD) برای تایید آزمایشگاهی مورد ارزیابی قرار گرفت. نمونه‌برداری از برگ‌ها در مرحله 4 برگی و در سه زمان 2، 4 و 7 روز بعد از اعمال تنش همراه با نمونه‌ی کنترل، با سه تکرار بیولوژیکی انجام گرفت. استخراج RNA از نمونه‌های برگی با استفاده از محلول استخراج RNx™-plus و واکنش Real time PCR با استفاده از آغازگرهای اختصاصی ژن ARD انجام گرفت.
نتایج: در بین پروب‌های تغییر بیان‌یافته، به ترتیب 658 و 1561پروب ایدی به طور اختصاصی در ارقام مقاوم و حساس، در شرایط تنش افزایش بیان داشتند. ژن‌های فاکتورهای رونویسی Myb3، ethylene responsive 5a، MIKC-type MADS-boxWM24B و salinity inducible ERF4 در ارقام مقاوم و فاکتورهای رونویسی WRKY15 ، MADS-box TaAGL8، WRKY39 و Myb در ارقام حساس دارای افزایش بیان معنی‌داری بودند. با کمک متاآنالیز ژن‌ها و فاکتورهای رونویسی، با بیان متفاوت بین ارقام حساس و مقاوم گندم شناسایی شدند و از این نتایج، می‌توان برای شناسایی ارقام مقاوم و حساس گندم نسبت به تنش خشکی استفاده کرد. نتایج Real time PCR ژن ARD برای 2 رقم گندم مقاوم به خشکی 6o4 و رقم حساس به تنش خشکی Sundor همسو با نتایج متاآنالیز انجام شده بود و نتیجه‌ی متاآنالیز را تایید کرد.  
نتیجه‌گیری: با توجه به بررسی رفتار بیانی این ژن و دیگر نتایج بدست آمده متاآنالیز در این مطالعه ، می‌توان گامی موثر در پیش‌بینی مقاومت و حساسیت به خشکی در ارقام مختلف گندم برداشت.

کلیدواژه‌ها


عنوان مقاله [English]

Identification of a number of genes with different expression patterns between resistant and susceptible wheat cultivars in response to drought stress by using meta-analysis method

نویسندگان [English]

  • Sahar Shojaee 1
  • Rudabeh Ravash 2
  • Behrouz Shiran 3
  • Esmael Ebrahimi 4
1 Student of MSc in Biotechnology, Faculty of Agriculture, Shahrekord University
2 Assistant Professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Shahrekord University, Shahrekod, Iran.
3 Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Shahrekord University
4 Associate Professor, Department of Agriculture and Plant Breeding, Faculty of Agriculture, Shiraz University, Shiraz, Iran
چکیده [English]

Objective
The wheat is one of the most important sources of human food. In production of this product abiotic stresses such as drought, salinity, temperature, etc., involved in cultivation. Therefore, identification and evaluation of genes involved in stress resistance in this plant is very importance. Aim of this study was identifying responsive genes to drought and their expression variation in, resistant and susceptible wheat.
 
Materials and methods
 The data available on the NCBI GEO microarray data were collected in 2018. The libraries were belonging to drought resistant and drought susceptible wheat under stress and control conditions. Using meta-analysis, altered genes expressed under stress conditions were identified for each group of cultivars. Significantly expressed altered genes were identified, the aci-reductone-dioxygenase-like protein (ARD) gene was evaluated for laboratory confirmation. Leaf sampling was performed in 4-leaf stage at three times 2, 4 and 7 days after stress with a control sample with three biological replications. RNA extraction from leaf samples was performed using RNx ™ -plus extraction solution and Real time PCR reaction using specific primers of d ARD gene.
 
Result
 Among differential expressed genes of resistant cultivars, transcription factors genes were Myb3, ethylene responsive 5a, MIKC-type MADS-box WM24B and salinity inducible ERF4 and in sensitive cultivars, transcription factors such as WRKY15, MADS-box TaAGL8, WRKY39 and Myb have increased expression. By identifying the genes and transcription factors mentioned and changing their expression, wheat cultivars can tolerate drought stress. Real time PCR results of ARD Gene for 6o4 and Sundor, drought resistant and drought susceptible wheat varieties was consistent with the results of the meta-analysis and it confirmed the result.
 
Conclusion
 According to gene expression results and other outcomes of metanalysis in this study, an effective step can be taken in predicting drought resistance and susceptibility in different wheat cultivars.

کلیدواژه‌ها [English]

  • drought
  • meta-analysis
  • R software
  • Triticum aestivum
منابع
خضری غفار؛ شبر زهرا سادات؛ ناجی امیرمحمد (2015) بررسی بیوانفورماتیکی ژن‌های خانواده‌ی عوامل رونویسی WRKY در گندم. دوفصلنامه فن آوری زیستی در کشاورزی 6 ، 39-44.‎
وفابخش جواد؛ نصیری محلاتی مهدی؛ کوچکی علیرضا و عزیزی مهدی (2009) اثر تنش خشکی بر کارآیی مصرف آب و عملکرد ارقام کلزا. پژوهشهای زراعی ایران 1، 292 - 285.
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