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Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress

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  • Published:
Biologia Plantarum

Abstract

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.

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Abbreviations

ACT:

actin 7

βTUB:

β-tubulin

Cq:

quantification cycle

DNAJ:

DNAJ homolog subfamily A member 2

EF1α:

elongation factor 1-alpha

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

GST:

glutathione-S-transferase

M:

gene expression stability value

MDH:

malate dehydrogenase

qPCR:

quantitative PCR

U1:

uncharacterized protein 1

U2:

uncharacterized protein 2

UBC:

ubiquitin-conjugating enzyme E2

References

  • Andersen, C.L., Jensen, J.L., Ørntogt, 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. 64: 5245–5250, 2004.

    Article  CAS  PubMed  Google Scholar 

  • Asai, T., Tena, G., Plotnikova, J., Willmann, M.R., Chiu, W. L., Gomez-Gomez, L., Boller, T., Ausubel, F.M., Sheen, J.: MAP kinase signalling cascade in Arabidopsis innate immunity. — Nature 415: 977–983, 2002.

    Article  CAS  PubMed  Google Scholar 

  • Buchanan, C.D., Lim, S., Salaman, R.A., Kagiampakis, I., Morishige, D.T., Weers, B.D., Klein, R.R., Pratt, L.H., Cordonnier-Pratt, M.M., Klein, P.E., Mullet, J.E.: Sorghum bicolor’s transcriptome response to dehydration, high salinity and ABA. — Plant mol. Biol. 58: 699–720, 2005.

    Article  CAS  PubMed  Google Scholar 

  • Bustin, S.A., Benes, V., Garson, J.A., Hellemans J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T.: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. — Clin. Chem. 55: 611–622, 2009.

    Article  CAS  PubMed  Google Scholar 

  • Czechowski, T., Stitt, M., Altmann, T., Udvardi M.K., Scheible W.R.: 2005 Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. - Plant Physiol. 139: 5–17, 2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • De Oliveira, L.A., Breton, M.C., Bastolla, F.M., Camargo Sda, S., Margis, R., Frazzon, J., Pasquali, G.: Reference genes for the normalization of gene expression in Eucalyptus species. — Plant Cell Physiol. 53: 405–422, 2012.

    Article  PubMed  Google Scholar 

  • Eulgem, T., Rushton, P.J., Robatzek, S., Somssich, I.E.: The WRKY superfamily of plant transcription factors. — Trends Plant Sci. 5: 199–206, 2000.

    Article  CAS  PubMed  Google Scholar 

  • Fan, C., Ma, J., Guo, Q., Li, X., Wang, H., Lu, M.: Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis). — PLoS ONE. 8: e56573, 2013.

    Article  Google Scholar 

  • Geilfus, C.M., Zorb, C., Muhling, K.H.: Salt stress differentially affects growth-mediating β-expansins in resistant and sensitive maize (Zea mays L.). — Plant Physiol. Biochem. 48: 993–998, 2010.

    Article  CAS  PubMed  Google Scholar 

  • Han, X., Lu, M., Chen, Y., Zhan, Z., Cui, Q., Wang, Y.: Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development. — PLoS ONE 7: e43084, 2012.

    Article  Google Scholar 

  • Hong, S.Y., Seo, P.J., Yang, M.S., Xiang, F., Park, C.M.: Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. — BMC Plant Biol. 8: 112, 2008.

    Article  PubMed  PubMed Central  Google Scholar 

  • Hsu, F.C., Chou, M.Y., Chou, S.J., Li, Y.R., Peng H.P., Shih, M.C.: Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis. — Plant Cell 25: 2699–2713, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu, Y., Chen, H., Luo, C., Dong, L., Zhang, S., He, X., Huang, G.: Selection of reference genes for real-time quantitative PCR studies of kumquat in various tissues and under abiotic stress. — Sci. Hort. 174: 207–216, 2014.

    Article  CAS  Google Scholar 

  • Jain, M., Nuhawan, A., Tyagi, A.K., Khurana, J.P.: 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, 2006.

    Article  CAS  PubMed  Google Scholar 

  • Kumar, K., Muthamilarasan, M., Prasad, M.: Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions. — Plant Cell Tissue Organ Cult. 115: 13–22, 2013.

    Article  CAS  Google Scholar 

  • Kwon, Y.R., Lee, H.J., Kim, K., Hong, S.W., Lee, S.J., Lee, H.: Ectopic expression of Expansin3 or Expansinβ1 causes enhanced hormone and salt stress sensitivity in Arabidopsis. — Biotechnol. Lett. 30: 1281–1288, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Løvdal, T., Lillo, C.: Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. — Anal. Biochem. 387: 238–242, 2009.

    Article  PubMed  Google Scholar 

  • Libault, M., Thibivilliers, S., Bilgin, D., Radwan, O., Benitez, M., Clough, S.J., Stacey, G.: Identification of four soybean reference genes for gene expression normalization. — Plant Genome 1: 44–54, 2008.

    Article  CAS  Google Scholar 

  • Ling, H., Wu, Q., Guo, J., Xu, L., Que, Y.: Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative RTPCR. — PLoS ONE. 9: e97469, 2014.

    Article  Google Scholar 

  • Obertello, M., Wall, L., Laplaze, L., Nicole, M., Auguy, F., Gherbi, H., Bogusz, D., Franche, C.: Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca. — Mol. Plant-Microbe Interact. 20: 1231–1240, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Peter, B., Swarup, R., Jansen, L., Devos, G., Auguy, F., Collin, M., Santi, C., Hocher, V., Franche, C., Bogusz, D., Bennett, M., Laplaze, L.: Auxin influx activity is associated with frankia infection during actinorhizal nodule formation in Casuarina glauca. — Plant Physiol. 144: 1852–1862, 2007.

    Article  Google Scholar 

  • Paolacci, A.R., Tanzarella, O.A., Porceddu E., Ciaffi, M.: Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. — BMC mol. Biol. 10: 11, 2009.

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Saraiva, K.C., Fernandes de Melo, D., Morais, V., Vasconcelos, I., Costa, J.: Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions. — Plant cell. rep. 33: 1453–1465, 2014.

    Article  CAS  PubMed  Google Scholar 

  • Tani, C., Sasakawa, H.: Salt tolerance of Casuarina equisetifolia and FrankiaCeq1 strain isolated from the root nodules of C. equisetifolia. — Soil Sci. Plant Nutr. 49: 215–222, 2003.

    Article  Google Scholar 

  • Vanderauwera, S., Vandenbroucke, K., Inze, A., Van de Cotte, B., Muhlenbock P., De Rycke, R., Naouar, N., Van Gaever, T., Van Montagu, M.C., Van Breusegem, F.: AtWRKY15 perturbation abolishes the mitochondrial stress response that steers osmotic stress tolerance in Arabidopsis. — Proc. nat. Acad. Sci. USA 109: 20113–20118, 2012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F.: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. — Genome Biol. 3: 503–515, 2002.

    Article  Google Scholar 

  • VanGuilder, H.D., Vrana, K.E., Freeman, W.M.: Twenty-five years of quantitative PCR for gene expression analysis. — Biotechniques 44: 619, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Wang, H., Xu, Q., Kong, Y.H., Chen, Y., Duan, J.Y., Wu, W.H., Chen, Y.F.: Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation. — Plant Physiol. 164: 2020–2029, 2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xia, W., Z. Liu, Y. Yang, Y. Xiao, Mason A.S., Zhao, S., Ma, Z.: Selection of reference genes for quantitative real-time PCR in Cocos nucifera during abiotic stress. — Botany 92: 179–186, 2013.

    Article  Google Scholar 

  • Xu, M., Zhang, B., Su, X., Zhang, S., Huang, M.: Reference gene selection for quantitative real-time polymerase chain reaction in Populus. — Anal. Biochem. 408: 337–339, 2011.

    Article  CAS  PubMed  Google Scholar 

  • Xu, X., Chen, C., Fan, B., Chen, Z.: Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. — Plant Cell 18: 1310–1326, 2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang, L., Zhang, C., WU, P.Z., Chen, Y.P., Li, M., Jiang, H., Wu, G.: Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to salt stress. — PLoS ONE. 9: e97878, 2014.

    Article  Google Scholar 

  • Zhong, C., Zhang, Y., Chen, Y., Jiang, Q., Chen, Z., Liang, J., Pinyopusarerk, K., Franche, C., Bogusz, D.: Casuarina research and applications in China. — Symbiosis 50: 107–114, 2010.

    Article  Google Scholar 

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Authors and Affiliations

  1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, 510520, P.R. China

    C. Fan, Z. Qiu, B. Zeng, Y. Liu, X. Li & G. Guo

Authors
  1. C. Fan
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  2. Z. Qiu
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  3. B. Zeng
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  4. Y. Liu
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  5. X. Li
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  6. G. Guo
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Corresponding author

Correspondence to C. Fan.

Additional information

Acknowledgments: We are grateful to the Ministry of Science and Technology of China (2013AA102705) and the Fundamental Research Funds for central public welfare research institutes (RITFYWZX201304) for financial support. We are also grateful to Dr. Zhang Yong (the Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China) for providing experimental material.

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Fan, C., Qiu, Z., Zeng, B. et al. Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress. Biol Plant 61, 463–472 (2017). https://doi.org/10.1007/s10535-016-0670-y

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  • DOI: https://doi.org/10.1007/s10535-016-0670-y

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