Evaluation of expression stability of reference genes ovEF1alpha and 18S rRNA under GA3 treatments and temperature and light stresses in Thyme genus

Document Type : Research Paper

Authors

1 M.Sc., Faculty of Agricultural Sciences, University of Guilan, Rasht, I.R. Iran

2 - Prof., Plant Breeding and Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, I.R. Iran

Abstract

Real-time PCR technique is available for determining changes of gene expression. However, normalizing gene expression data and obtaining reliable results depends on the selection of appropriate reference genes which are stably expressed under different experimental conditions. In this study, we investigated suitability of two commonly used reference genes [elongation factor 1 alpha (OvEF1alpha) and 18S ribosomal RNA (18S rRNA)] in seven Thyme populations and under exogenously applied gibberellic acid (GA3) hormone (control, 30, 60 and 90ppm), plant age (young plants and middle-aged plants and temperature/light stress. Results showed that expression levels of both genes in all populations were affected significantly. Plant age did not significantly effect on expression of the two genes. Also, ovEF1alpha unaffected by exogenously applied GA3. Under temperature/light stress, expression of OvEF1alpha increased in Babgorgy population but in Babzangy decreased. In contrast, expression of 18S rRNA in Babgorgy was significantly decreased but has stable expression in Thyme and Babzangy. Thus, these results showed that ovEF1alpha for comparison of gene expression between Thyme, Hanza, Sirch and Zarand and 18S rRNA for Babzangy, Sirch and Hanza or Zarand and Thyme populations are suitable as internal control gene. Also, for temperature/light stress both genes are suitable in Thymus vulgaris L. but only 18SrRNA is suitable in Thymus caramanicus Jalas.

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-      Bahador, S., 2014. Study on candidate genes expression related to biosynthesis pathway of phenolic monoterpenes in Thyme populations. University of Guilan, Rasht, Iran.
-      Bahador, S., Rabiei, B. and Hassani Kumleh, S.H., 2014. Comparison of different methods for isolating of total RNA from leaf of three Thyme species rich in secondary metabolites .Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 22: 11-24.
-      Bikdelu, M., 2012. Evaluation of morphological, genetical and phytochemical diversity of Thymus caramanicus Jalas. University of Tehran, Iran.
-      Crocoll, C., Asbach, J., Novak, J., Gershenzon, J. and Degenhardt, J., 2011. Terpene synthases of Oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis. Plant Molecular Biology, 73: 587-603.
-      Die, J.V., Roman, B., Nadal, S., and Gonzalez-Verdejo, C.I., 2010. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta, 232: 145-153.
-      Figueiredo, A., Loureiro, A., Batista, D., Filipa, M.,Várzea, V., Elijah, K. G. and Céu Silva, M., 2013. Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae. BMC Research Notes, 6: 388-410.
-      Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R. and Mathieu, C., 2001. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 25: 386–401.
-      Gopesh, C.S. and George, J.V., 2012. Evaluation of expression stability of candidate references genes among green and yellow pea cultivars (Pisum sativum L.) subjected to abiotic and biotic stress. American Journal of Plant Sciences, 3: 235-242.
-      Hu, R., Fan, C., Li, H., Zhang, Q., and Fu, Y.F., 2009. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Molecular Biology, 10:93.
-      Kowalewska, M., Danska-Bidzinska, A., Bakula-Zalewska, E. and Bidzinski, M., 2012. Identification of suitable reference genes for gene expression measurement in uterine sarcoma and carcinosarcoma tumors. Clinical Biochemistry, 45: 368–371.
-      Lee, J.M., Roche, J.R., Donaghy, D.J., Thrush, A. and Sathish, P., 2010. Validation of reference genes for quantitative RT PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Molecular Biology, 11:8
-      Lee, P.D., Sladek, R., Greenwood, C.M.T. and Hudson, T.J., 2001. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Research, 12: 292-297.
-      Li, Q.Q., Skinner, J. and Bennett, J.E., 2012. Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment. BMC Molecular Biology, 13: 22.
-      Peng, X.X., Zhao, L.R., Song, Wei., Chu, H.R., Song, S.Y., Li, M.,  Li, G.Z. and Liang, D.C., 2012. Selection of suitable reference genes for normalization of quantitative Real-Time PCR in cartilage tissue injury and repair in rabbits. International Journal of Molecular Sciences, 13: 14344-14355.
-      Pfaffl, M.W., 2004. Quantification strategies in Real-Time PCR. Physiology-Weihenstephan, Technical University of Munich, Center of Life and Food Science Weihenstephan, Freising, Germany Chaper, 3: 87 -112.
-      Podevin, N., Swennen, R., Krauss, A., Remy, S. and Henry, I., 2012. Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa. Molecular Breeding, 30: 1237–1252. 
Robert, D.B., Dan, W.H., Robert, A.C. and Brian, J.C., 2005. GAPDH as a housekeeping gene: