بررسی ژن‏ های رفرنس موجود در برگ کنجد تحت تنش شوری به روش Real- Time PCR

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی‌ارشد اصلاح‌نباتات دانشگاه علوم کشاورزی و منابع طبیعی ساری

2 دانشیار دانشگاه علوم کشاورزی و منابع طبیعی ساری

3 دانشجوی دکتری بیوتکنولوژی پژوهشکده ژنتیک و زیست‌فناوری طبرستان، دانشگاه علوم کشاورزی و منابع طبیعی ساری

چکیده

تجزیه و تحلیل بیان ژن جزء لاینفک مطالعات ژنومیکس کاربردی در همه موجودات زنده بشمار می‏رود.  Real-time PCR تکنیک بسیار قوی برای بررسی بیان کمی ژن می‏باشد. با این حال، علاوه بر قابل اعتماد بودن، دارای یک‌ سری مشکلات خاص، از جمله انتخاب‏ ژن (های) کنترل داخلی مناسب برای نرمال‏سازی داده‏ها می‏باشد. این تحقیق در خصوص انتخاب ژن‏های رفرنس در گیاه کنجد در مراحل مختلف نمو و تحت تنش شوری، مورد بررسی قرار گرفت .بدین منظور چهار ژن کنترل داخلی شامل Alpha- Tubulin، Beta- Actin، eIF4- A و UBQ5 که معمولاً به عنوان ژن خانه‏دار در گیاهان استفاده می‏شود انتخاب و  پایداری بیان آنها در سطوح مختلف شوری (صفر و 75 میلی‏مولار) و مراحل مختلف نموی در شش دوره زمانی (0 ساعت، 6 ساعت، 1 روز، 4 روز، 8 روز و 16 روز) در بافت برگ مورد بررسی قرار گرفتند. بررسی بیان ژن‏های رفرنس با استفاده از نرم‏افزار geNORM نشان داد که ژن‏های eIF4- A و Beta-Actin از پایداری بیان بیشتری نسبت به سایر ژن‌های مورد بررسی در مراحل نمو و تنش شوری در نمونه بافت برگی برخوردار بودند. استفاده از این ژن‌ها می‌تواند در نرمال‌سازی بیان ژن به وسیله آنالیز Real-Time PCR مفید باشد. نتایج این تحقیق را می‏توان به عنوان ژن‏های رفرنس درآنالیز بیان ژن در Real-Time PCR در گیاه کنجد استفاده نمود.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Study of Reference Genes in Sesame Leaves under Salt Stress by Real-Time PCR Method

نویسندگان [English]

  • Samira Shakeri 1
  • Seyed kamal Kazemitabar 2
  • Seyed hamidreza Hashemi 3
1 M.Sc. Plant Breeding, Sari University of Agricultural Sciences and Natural Resources. Sari, Iran.
2 Associate Professor, Sari University of Agricultural Sciences and Natural Resources, Sari, Iran
3 Ph.D student in Biotechnology, Tabarestan Institute of Genetics and Biotechnology, Sari University of Agricultural Sciences and Natural Resources. Sari, Iran
چکیده [English]

Analysis of gene expression is considered as an essential part of functional genomics studies in all living organisms. Real-time PCR technique is very strong one to study the expression of a gene. However, despite its reliability, it has a set of specific problems, such as internal control gene selection which are suitable for normalization of the data. The study about selection reference of genes in sesame plant, at different developmental stages and under salinity stress of  were studied. For this purpose, four internal control genes consists of eIF4- A, UBQ5, Alpha-Tubulin and Beta-Actin which are commonly used as housekeeping genes in plants, are selected and the stability of its expression in different salinity levels (zero and 75 mM) and different growth stages in five time periods (0 h, 6 h, 1 day, 4 days, 8 days and 16 days) in leaf tissue were examined. Study of the expression of reference genes using geNORM software showed that, in developmental stages and salinity in the leaf tissues, eIF4-A and Beta-Actin genes have more stable expression than other investigated genes. Using these genes can be useful in normalization of gene expression by Real-Time PCR analysis. The results can be used as reference genes for gene expression analysis in the Real-Time PCR.

کلیدواژه‌ها [English]

  • Gene expression
  • Salt stress
  • Reference genes
  • Sesamum indicum L
Andersen C, Jensen J, Orntoft T (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. Canc. Res. 64(15): 5245-5250.

Barsalobres-Cavallari CF, Severino FE, Maluf MP, Maia IG (2009) Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol. Biol. 10: 1.

Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25: 169-193.

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 Meth. 3(7).

Chen L, Zhong H, Kuang J, Li J, Lu W, Chen J (2011) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta. 234(2): 377–390.

Chomczynski P, Sacchi N (1987) Signal-Step Method of RNA Isolation by Acid GuanidiniumThiocyanate-Phenol -Chloroform Extraction. Anal. Biochem. 162: 156-159.

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139: 5-17.

Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G,  Zumla A (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques. 37: 112-114.

Garg R, Sahoo A, Tyagi AK,  Jain M (2010) Validation of internal control genes for quantitative gene expression studies in chickpea (Cicer arietinum L.). Biochem. Biophys. Res. Comm. 396: 283–288.

 Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J. Exp. Bot. 60(2): 487-493.

 Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8(19).

 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. Comm. 345: 646-651.

 Lee PD, Sladek R, Greenwood CM, Hudson TJ (2002) Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome. Res. 12: 292-297.

 Liu LM, Liu HY, Tian BM (2012) Selection of reference genes from sesame infected by Macrophomina phaseolina. Acta Agron. Sin. 38: 471-478.

 Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrid. 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: 129-142.

Pattyn F, Speleman F, Depaepe A, Vandesompele J (2003) RTPrimerDB: the Real‑Time PCR primer and probe database. Nucleic Acids Res 1: 122-123.

 Pettengill AE, Parmentier-Line C, Coleman GD (2012) Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies. BMC Res Notes. 5: 366.

 Pfaffl M, Tichopad A, Prgomet C, Neuvians T (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: Best Keeper–Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26(6): 509-515.

 Suzuki T, Higgins PJ, Crawford DR (2000) Control selection for RNA quantitation. Biotechniques. 29: 332-337.

 Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A,  Heinen E (1999) Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75: 291-295.

 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N,  de Paepe A, Speleman F (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(12): 2167-2176.

 Wei L, Miao H, Zhao R, Han X, Zhang T, Zhang H (2013) Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR. Planta. 237(3): 873-889.