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Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

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Abstract

Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.

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Abbreviations

Actin :

actin

Ct:

cycle threshold

EF-1α :

eukaryotic elongation factor 1-alpha

eIF-4α :

eukaryotic-initiation factor 4α

GAPDH :

glyceraldehyde-3-phosphate dehydrogenase

M:

stability value

OsMT2b :

O. sativa METALLOTHIONEIN2b

SD:

standard deviation

UBC :

ubiquitin conjugating enzyme E2

UBQ 5:

ubiquitin 5

UBQ 10 :

ubiquitin 10

Vn/Vn + 1:

pairwise variation

β-TUB :

beta-tubulin

References

  1. Goyer, R.A., Toxic and essential metal interactions, Annu. Rev. Nutr., 1997, vol. 17, pp. 37–50.

    Article  PubMed  CAS  Google Scholar 

  2. Emumejaye, K., Heavy and trace elements in some brands of rice consumed in delta state, Nigeria, IOSR J. Appl. Phys., 2014, vol. 6, pp. 1–5.

    Article  Google Scholar 

  3. Clemens, C.S., Molecular mechanisms of plant metal tolerance and homeostasis, Planta, 2001, vol. 212, pp. 475–486.

    Article  PubMed  CAS  Google Scholar 

  4. Krämer, U., Metal hyperaccumulation in plants, Annu. Rev. Plant Biol., 2010, vol. 61, pp. 517–534.

    Article  PubMed  CAS  Google Scholar 

  5. Gachon, C., Mingam, A., and Charrier, B., Real-time PCR: what relevance to plant studies? J. Exp. Bot., 2004, vol. 55, pp. 1445–1454.

    Article  PubMed  CAS  Google Scholar 

  6. Remans, T., Keunen, E., Bex, G.J., Smeets, K., Vangronsveld, J., and Cuypers, A., Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy, Plant Cell, 2014, vol. 26, pp. 3829–3837.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Dheda, K., Huggett, J.F., Chang, J.S., Kim, L.U., Bustin, S.A., Johnson, M.A., Rook, G.A., and Zumla, A., The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization, Anal. Biochem., 2005, vol. 344, pp. 141–143.

    Article  PubMed  CAS  Google Scholar 

  8. Vandesompele, J., de Preter, K., Pattyn, F., Poppe, B., van Roy, N., de Paepe, A., and Speleman, F., Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes, Genome Biol., 2002, vol. 3. doi 10.1186/gb-2002-3-7-research0034

  9. Andersen, C.L., Jensen, J.L., and Orntoft, T.F., 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., 2004, vol. 64, pp. 5245–5250.

    Article  PubMed  CAS  Google Scholar 

  10. Pfaffl, M.W., Tichopad, A., Prgomet, C., and Neuvians, T.P., Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations, Biotechnol. Lett., 2004, vol. 26, pp. 509–515.

    Article  PubMed  CAS  Google Scholar 

  11. Xie, F., Peng, X., Chen, D., Xu, L., and Zhang, B., miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs, Plant Mol. Biol., 2012, vol. 80, pp. 75–84.

    Article  CAS  Google Scholar 

  12. Yoshida, S., Forno, D.A., Cock, J.H., and Gomez, K.A., Laboratory Manual for Physiological Studies of Rice, Manila, Philippines: Int. Rice Res. Inst., 1976.

    Google Scholar 

  13. Yuan, J., Chen, D., Ren, Y., Zhang, X., and Zhao, J., Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice, Plant Physiol., 2008, vol. 146, pp. 1637–1650.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Lanfranco, L., Bolchi, A., Ros, E.C., Ottonello, S., and Bonfante, P., Differential expression of a metallothionein gene during the presymbiotic versus the symbiotic phase of an arbuscular mycorrhizal fungus, Plant Physiol., 2002, vol. 130, pp. 58–67.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Pacher, P., Beckman, J.S., and Liaudet, L., Nitric oxide and peroxynitrite in health and disease, Physiol. Rev., 2007, vol. 87, pp. 315–424.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Ghoshal, N., Raychaudhuri, S.S., Talapatra, S., Moulick, A., and Chakraborty, A., Alterations in transcriptome and proteome on metallothioneins following oxidative stress induced by sub lethal doses of cadmium and gamma rays in Plantago ovate, Int. J. Radiat. Biol., 2013, vol. 89, pp. 571–582.

    Article  PubMed  CAS  Google Scholar 

  17. Lovdal, T. and Lillo, C., Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress, Anal. Biochem., 2009, vol. 387, pp. 238–242.

    Article  CAS  Google Scholar 

  18. Dekkers, B.J.W., Willems, L., Bassel, G.W., van Bolderen-Veldkamp, R.P., Ligterink, W., Hilhorst, H.W.M., and Bentsink, L., Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds, Plant Cell Physiol., 2012, vol. 53, pp. 28–37.

    Article  PubMed  CAS  Google Scholar 

  19. Dos Santos, L.F., Silva, R.J.S., do Amaral, D.O.J., de Paula, M.F.B., Falcao, L.L., Legavre, T., Alves, R.M., Marcellino, L.H., and Micheli, F., Selection of reference genes for expression study in pulp and seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum, PLoS One, 2016, vol. 11, no. 8: e0160646. http://www.ncbi.nlm.nih.gov/pubmed/27501324.

    Article  CAS  Google Scholar 

  20. Taylor, C.M., Jost, R., Erskine, W., and Nelson, M.N., Identifying stable reference genes for qRT-PCR normalization in gene expression studies of narrow-leafed lupin (Lupinus angustifolius L.), PLoS One, 2016, vol. 11, no. 2: e0148300. https://doi.org/10.1371/journal.pone.0148300

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Sun, C.W., Griffen, S., and Callis, J., A model for the evolution of polyubiquitin genes from the study of Arabidopsis thaliana ecotypes, Plant Mol. Biol., 1997, vol. 34, pp. 745–758.

    Article  PubMed  CAS  Google Scholar 

  22. Hernandez-Garcia, C.M., Martinelli, A.P., Bouchard, R.A., and Finer, J.J., A soybean (Glycine max) polyubiquitin promoter gives strong constitutive expression in transgenic soybean, Plant Cell Rep., 2009, vol. 28, pp. 837–849.

    Article  PubMed  CAS  Google Scholar 

  23. Dreher, K. and Callis, J., Ubiquitin, hormones and biotic stress in plants, Ann. Bot., 2007, vol. 99, pp. 787–822.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Correspondence to D. Ebadi Almas.

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Almas, D.E., Kamrodi, A.R. Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses. Russ J Plant Physiol 65, 890–897 (2018). https://doi.org/10.1134/S102144371806002X

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