بررسی پایداری بیان برخی ژن‌های مرجع به‌روش Real-Time PCR تحت تنش زیستی در گیاه برنج

نوع مقاله : مقاله پژوهشی

نویسندگان

1 بخش تحقیقات علوم زراعی و باغی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی استان مازندران، سازمان تحقیقات، آموزش و ترویج کشاورزی،

2 استادیار گروه مهندسی ژنتیک و بیولوژی، پژوهشکده ژنتیک و زیست فناوری کشاورزی طبرستان، دانشگاه علوم کشاورزی و منابع طبیعی ساری

3 مربی پژوهش معاونت مازندران، موسسه تحقیقات برنج کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، آمل، ایران

چکیده

هدف: در پژوهش­های بیان ژن با استفاده از Real-time PCR که ابزار قدرتمندی برای تجزیه و تحلیل سازوکارهای مقاومت به تنش­­های زیستی در گیاهان است، انتخاب و اعتبارسنجی ژن­های مرجع به­منظور بهینه کردن بیان ژن­ها بسیار مهم می­باشد.
این تحقیق با هدف بررسی کارایی و انتخاب ژن­های مرجع مناسب برای بهینه­سازی اطلاعات حاصل از PCR زمان واقعی در گیاه برنج تحت تنش زیستی انجام شد.
مواد و روشها: بدین منظور، هشت ژن کنترل داخلی شاملUBQ5 ،eIF4A ، Actin11، Actin1، UBC، Cylophilin، 18SrRNA و GAPDH بررسی شد. تنش زیستی در بافت برگی گیاهچه­های 28 روزه رقم فجر و در حضور عامل بیماری قارچی Rhizoctonia solani RBL1، سیلیکات پتاسیم به­عنوان القاء­کننده مقاومت، جدایه­هایPseudomonas با خاصیت آنتاگونیستی و القاء­کنندگی و در سه دوره زمانی (6، 24 و 72 ساعت) اعمال شد.
نتایج:  بررسی پایداری بیان ژن­های مرجع با استفاده از نرم­افزار BestKeeper نشان داد؛ که ژن UBC از پایداری بیان بیشتری نسبت به سایر ژن­های مورد بررسی در شرایط تنش زیستی در نمونه بافت برگی گیاه برنج برخوردار بوده و بر اساس آماره توصیفی ژن­های UBC و Actin11 دارای بیشترین همبستگی (97/0) با شاخص BestKeeper بودند. همچنین، ژن­های UBC و Actin11 دارای بیشترین پایداری (کمترین ضریب تغییرات) بودند. بر اساس تجزیه و تحلیل داده­ها با استفاده از نرم­افزار NormFinder نیز ژن­های Actin11 و eIF4a (به­ترتیب 356/0 و 567/0) بیشترین میزان پایداری را نشان دادند.
نتیجهگیری کلی: بررسی بیان ژن‌های مرجع با استفاده از میانگین هندسی ژن­های UBC و Actin11 در مقایسه با ژن Actin1 نشان­دهنده ضرورت انتخاب مناسب ژن مرجع می­باشد. نتایج این تحقیق نشان می­دهد که ژن­های UBC و Actin11 ژن­های مرجع مناسبی برای بهینه­سازی داده­های بیان ژن به­وسیله تجزیه و تحلیل Real-Time PCR در گیاه برنج می­باشند.

کلیدواژه‌ها


عنوان مقاله [English]

Study of Some Reference Genes Expression Stability in Rice Using Real-Time PCR Method Under Biotic Stress

نویسندگان [English]

  • Hamidreza Ghorbani 1
  • Seyyed Hamidreza Hashemi-petroudi 2
  • Mehdi Rostami Rostami 3
1 Assistant Professor, Crop and Horticultural Science Research Department, Mazandaran Agricultural and Natural Resources Research and Education Center, AREEO, Sari, Iran
2 Sari University of Agricultural Sciences and Natural Resources, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT)
3 Rice Research Institute (Amol)
چکیده [English]

Objective
Gene expression studies by Real-Time PCR constitute a powerful tool to analyze the mechanisms underlying plant biotic-stress tolerance. One of the crucial steps of this technique is the selection and validation of reference genes to normalize target gene expression under different stress conditions. In this study, the expresion of candidate gene in oryza sativa was investigated under biotic stress at different developmental stages.
 
Materials and methods
Eight internal control genes consists of eIF-4A, UBQ5, UBC, Actin1, Actin11, GAPDH, Cyclophilin and 18SrRNA which are commonly used as housekeeping genes in plants, were selected and their expression stability were examined in present of Rhizoctonia solani RBL1 strain, potasium silicat as tolerance inducer and different growth stages in three time periods (6 h, 24 h and 72 h) using BestKeeper and NormFinder softwares.
 
Results and Conclusions
Based on the results gained through Best Keeper, the UBC has the higher expression than the other genes under biotic stress in rice leaf and also the UBC and Actin11 genes poses the highest correlations with the BestKeeper index (0.97). Additionally, it was shown that the UBC and Actin has the lowest coefficient variation. Also, the evaluation of the reference genes expression using geometric mean of the UBC and Actin11 compared to the Actin1 gene indicated the necessity of appropriate selection of the reference gene. Taken together, it was evidently demonstrated that the UBC and Actin11 genes are the proper reference gene to be employed for the normalization of expression data in the Oryza sativa L. using by Real-Time PCR.

کلیدواژه‌ها [English]

  • Gene expression
  • Inducer
  • Reference gene
  • Rice
  • R. solani
Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64, 5245-5250.
Burton RA, Shirley NJ, King BJ et al. (2004) The CesA gene family of barley. Quantitative analysis of transcripts reveals two groups of co-expressed genes. Plant Physiol 134, 224-236.
Bustin SA (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25, 169-93.
Caldana C, Scheible W, Mueller-Roeber B, Ruzicic S (2007) A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors. Plant Methods 3, 7.
Cao J, Wang L, Lan H (2016) Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy. Peer J 4, 16-97.
Chang EM, Shi SQ, Liu JF et al. (2012) Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR. Plos One 7(3): e33278.
Chomczynski P, Sacchi N (1987) Signal-Step method of RNA isolation by acid guanidiniumthiocyanate-henol-chloroform extraction. Anal Biochem 162, 156-159.
Dheda K, Huggett JF, Bustin SA et al. (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37, 112-114.
Fedoroff NV, Battisti DS, Beachy RN et al. (2010) Radically rethinking agriculture for the 21st century. Science 327, 833-834.
Gutierrez L, Mauriat M, Pelloux J et al. (2008) Towards a systematic validation of references in real-time RT-PCR. Plant Cell 20, 17-34.
Hashemi SHR, Nematzadeh GA, Ahmadian GR et al. (2016) Identification and validation of Aeluropus litoralis reference genes for quantitative real-time PCR normalization. J Biol Res-Thessalon 23, 18.
Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345, 646-651.
Kim BR, Nam HY, Kim SU et al. (2003) Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25, 1869-1872.
Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J Appl Genet 54, 391-406.
Li Y, Chen W, Wang Q et al. (2014) Assessment of reference genes for quantitative realtime PCR gene expression normalization in periwinkle during wheat blue dwarf phytoplasma infection. Australas Plant Pathol 43, 477-485.
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods 25, 402-408.
Ma QP, Hao S, Chen X, Li XH (2016) Validation of reliability for reference genes under various abiotic stresses in tea plant. Russ J Plant Physiol 63, 423-432.
Mallona I, Lischewski S, Weiss J et al. (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10, 4.
Martin KJ, Rygiewicz PT (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol 5, 28.
McCartney HA, Foster SJ, Fraaije BA, Ward E (2003) Molecular diagnostics for fungal plant pathogens. Pest Manag Sci 59, 129-42.
Naeimi Sh, Okhovvat SM, Javan-nikkhah M, Vagvolgyi C, Khosravi V, Kredics L (2010) Biological control of Rhizoctonia solani AG1-1A, the causal agent of rice sheath blight with Trichoderma strains. Phytopathol Mediterr 49, 287-300.
Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.      J Exp Bot 56, 2907-2914.
Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol 10, 11.
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-excel-based tool using pair-wise correlations. Biotechnol Lett 26, 509-515.
Qi J, Yu S, Zhang F et al. (2010) Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant Mol Biol Rep 28, 597-604.
Reddy DS, Bhatnagar-Mathur P, Reddy PS et al. (2016) Identification and validation of reference genes and their impact on normalized gene expression studies across cultivated and wild cicer species. Plos One 11: 2. DOI: 10.1371/journal.pone.0148451.
Scholtz JJ, Visser B (2013) Reference gene selection for qPCR gene expression analysis of rust-infected wheat. Physiol Mol Plant Pathol 81, 22-25.
Sinha P, Saxena RK, Singh VK et al. (2015) Selection and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under heat and salt stress conditions. Front Plant Sci 6, 1071.
Thellin O, Zorzi W, Lakaye B et al. (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75, 291-295.
Tian C, Jiang Q, Wang F et al. (2015) Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves. Plos One 10: 2.
Vandesompele J, De Preter K, Pattyn F et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, 7.
Vashisth T, Johnson LK, Malladi A (2011) An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry. Plant Cell Rep 30, 2167-2176.
Wu D, Dong J, Yao YJ et al. (2015) Identification and evaluation of endogenous control genes for use in quantitative RT-PCR during wheat (Triticum aestivum L.) grain filling. Genet Mol Res 14, 10530-10542.
Xu H, Bao JD, Dai JS et al. (2015) Genome-wide identification of new reference genes for qRT-PCR normalization under high temperature stress in rice endosperm. Plos One 10: 11.