Advertisement

Molecular Biology Reports

, Volume 46, Issue 6, pp 5767–5776 | Cite as

Selection of reliable reference genes for gene expression studies in Caenorhabditis elegans exposed to crystals (Cry1Ia36) protein of Bacillus thuringiensis

  • Dongwei Wang
  • Yong Liu
  • Deyong Zhang
  • Qingcong He
  • Bei Tang
  • Feixue ChengEmail author
Original Article

Abstract

Quantitative real time PCR (qRT-PCR) is a nucleic acid quantitative technique and is also considered as a validation tool. The Cry1Ia36 protein isolated from Bacillus thuringiensis (Bt) strain YC-10 has high nematicidal activity against nematodes. Caenorhabditis elegans is one of the major model organisms and a readily accessible source of biological material for gene expression studies. To evaluate the expression stability of 12 candidate reference genes of C. elegans for exposing to different concentrations of Cry1Ia36 protein and different treat time, five statistical approaches (the comparative delta-Ct method, BestKeeper, NormFinder, Genorm and RefFinder) were used to evaluate each individual candidate reference gene. The results indicated that cdc-42 and F35G12.2 were the best reference genes for performing reliable gene expression normalization in the impact of Cry1Ia36 protein. In addition, when C. elegans was exposed to Cry1Ia36 protein and other nematicides, avermectin and 5-aminolevulinic acid, cdc-42 was recommended as the most reliable reference genes. Y45F10D.4 was the least stable reference genes in our experimental settings. Therefore, cdc-42 was reliable reference gene for gene expression studies in C. elegans exposed to Cry1Ia36 protein and other nematicides.

Keywords

Reference genes Quantitative real time PCR Cry1Ia36 protein Caenorhabditis elegans Gene expression 

Notes

Acknowledgements

This work was supported by National Key Research and Development Program of China (2018YFD0201208), National Natural Science Foundation of China (No. 31871941) and the Hunan Province Key Research and Development Program (2016NK2196).

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

11033_2019_5010_MOESM1_ESM.doc (301 kb)
Supplementary material 1 (DOC 301 kb)

References

  1. 1.
    Rosa FE, Silveira SM, Silveira CG et al (2009) Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma. BMC Cancer 9:90.  https://doi.org/10.1186/1471-2407-9-90 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hoogewijs D, Houthoofd K, Matthijssens F et al (2008) Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol Biol 9(1):9.  https://doi.org/10.1186/1471-2199-9-9 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Taki FA, Zhang B (2013) Determination of reliable reference genes for multi-generational gene expression analysis on C. elegans exposed to abused drug nicotine. Psychopharmacology 230(1):77–88.  https://doi.org/10.1007/s00213-013-3139-0 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wu H, Taki FA, Zhang Y et al (2014) Evaluation and identification of reliable reference genes for toxicological study in Caenorhabditis elegans. Mol Biol Rep 41(5):3445–3455.  https://doi.org/10.1007/s11033-014-3206-6 CrossRefPubMedGoogle Scholar
  5. 5.
    Bravo A, Likitvivatanavong S, Gill SS et al (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41(7):423–431.  https://doi.org/10.1016/j.ibmb.2011.02.006 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Mohammed SH, El Saedy MA, Enan MR et al (2008) Biocontrol efficiency of Bacillus thuringiensis toxins against root-knot nematode, Meloidogyne incognita. J Cell Mol Biol 7(1):57–66Google Scholar
  7. 7.
    Read TD, Peterson SN, Tourasse N et al (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423(6935):81–86.  https://doi.org/10.1038/nature01586 CrossRefPubMedGoogle Scholar
  8. 8.
    van Frankenhuyzen K (2009) Insecticidal activity of Bacillus thuringiensis crystal proteins. J Invertebr Pathol 101(1):1–16.  https://doi.org/10.1016/j.jip.2009.02.009 CrossRefPubMedGoogle Scholar
  9. 9.
    Schnepf E, Nv Crickmore, Van Rie J et al (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3):775–806.  https://doi.org/10.0000/PMID9729609 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cheng F, Wang J, Song Z et al (2015) Complete genome sequence of Bacillus thuringiensis YC-10, a novel active strain against plant-parasitic nematodes. J Biotechnol 210:17–18.  https://doi.org/10.1016/j.jbiotec.2015.06.395 CrossRefPubMedGoogle Scholar
  11. 11.
    Liu L, Knauth S, Eickhorst T (2018) Adsorption and desorption of Cry1Ab proteins on differently textured paddy soils. Pedosphere 28(1):94–102.  https://doi.org/10.1016/S1002-0160(18)60006-2 CrossRefGoogle Scholar
  12. 12.
    Sajid M, Geng C, Li M, Wang Y et al (2018) Whole genomic analysis of Bacillus thuringiensis revealing partial genes as a source of novel Cry toxins. Appl Environ Microbiol 84(14):e00277-18.  https://doi.org/10.1128/AEM.00277-18 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sanahuja G, Banakar R, Twyman RM et al (2011) Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol J 9(3):283–300.  https://doi.org/10.1111/j.1467-7652.2011.00595.x CrossRefPubMedGoogle Scholar
  14. 14.
    Shi C, Zhou G, Liu Z et al (2018) A novel Bacillus thuringiensis Cry57 protein domain swap influence on insecticidal activity. J Northeast Agric Univ 25(3):44–52Google Scholar
  15. 15.
    Song Z (2017) Identification of several key crop pathogenic nematodes in hunan province and nematicidal role of Bt Cry1Ia36 protein. Hunan Agricultural University, ChangshaGoogle Scholar
  16. 16.
    Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94PubMedPubMedCentralGoogle Scholar
  17. 17.
    Bone LW, Bottjer KP, Gill SS (1985) Trichostrongylus colubriformis: egg lethality due to Bacillus thuringiensis crystal toxin. Exp Parasitol 60(3):314.  https://doi.org/10.1016/0014-4894(85)90037-2 CrossRefPubMedGoogle Scholar
  18. 18.
    Griffitts JS, Huffman DL, Whitacre JL et al (2003) Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions. J Biol Chem 278(46):45594–45602.  https://doi.org/10.1074/jbc.M308142200 CrossRefPubMedGoogle Scholar
  19. 19.
    Marroquin LD, Elyassnia D, Griffitts JS et al (2000) Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genetics 155(4):1693–1699.  https://doi.org/10.1017/S0016672300004560 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wei JZ, Hale K, Carta L et al (2003) Bacillus thuringiensis crystal proteins that target nematodes. Proc Natl Acad Sci USA 100(5):2760–2765.  https://doi.org/10.1073/pnas.0538072100 CrossRefPubMedGoogle Scholar
  21. 21.
    Kagias K, Podolska A, Pocock R (2014) reliable reference miRNAs for quantitative gene expression analysis of stress responses in Caenorhabditis elegans. BMC Genom 15(1):222.  https://doi.org/10.1186/1471-2164-15-222 CrossRefGoogle Scholar
  22. 22.
    Peres TV, Arantes LP, Miah MR et al (2018) Role of Caenorhabditis elegans AKT-1/2 and SGK-1 in manganese toxicity. Neurotox Res 34(3):584–596.  https://doi.org/10.1007/s12640-018-9915-1 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Prasansuklab A, Meemon K, Sobhon P et al (2017) Ethanolic extract of Streblus asper leaves protects against glutamate-induced toxicity in HT22 hippocampal neuronal cells and extends lifespan of Caenorhabditis elegans. BMC Complement Altern Med 17(1):551.  https://doi.org/10.1186/s12906-017-2050-3 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Polak N, Read DS, Jurkschat K et al (2014) Metalloproteins and phytochelatin synthase may confer protection against zinc oxide nanoparticle induced toxicity in Caenorhabditis elegans. Comp Biochem Physiol C 160:75–85.  https://doi.org/10.1016/j.cbpc.2013.12.001 CrossRefGoogle Scholar
  25. 25.
    Gao X, Teng Y, Luo J et al (2014) The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions. RNA Biol 11(9):1148–1160.  https://doi.org/10.4161/rna.36100 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lapierre LR, De Magalhaes Filho CD, McQuary PR et al (2013) The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans. Nat Commun 4:2267.  https://doi.org/10.1038/ncomms3267 CrossRefPubMedGoogle Scholar
  27. 27.
    Zhang Y, Chen D, Smith MA et al (2012) Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity. PLoS ONE 7(3):e31849.  https://doi.org/10.1371/journal.pone.0031849 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Silver N, Best S, Jiang J et al (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:33.  https://doi.org/10.1186/1471-2199-7-33 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Pfaffl MW, Tichopad A, Prgomet C et al (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515.  https://doi.org/10.1023/B:BILE.0000019559.84305.47 CrossRefPubMedGoogle Scholar
  30. 30.
    Andersen CL, Jensen JL, Orntoft 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(15):5245–5250.  https://doi.org/10.1158/0008-5472.CAN-04-0496 CrossRefPubMedGoogle Scholar
  31. 31.
    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):34.  https://doi.org/10.1186/gb-2002-3-7-research0034 CrossRefGoogle Scholar
  32. 32.
    Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622.  https://doi.org/10.1373/clinchem.2008.112797 CrossRefPubMedGoogle Scholar
  33. 33.
    Conery AL, Larkins-Ford J, Ausubel FM et al (2014) High-throughput screening for novel anti-infectives using a C. elegans pathogenesis model. Curr Protoc Chem Biol 6(1):25–37.  https://doi.org/10.1002/9780470559277.ch130160 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Zhang C, Xie H, Xu CL et al (2012) Differential expression of Rs-eng-1b in two populations of Radopholus similis (Tylenchida: pratylecnchidae) and its relationship to pathogenicity. Eur J Plant Pathol 133:899–910.  https://doi.org/10.1007/s10658-012-0015-4 CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Dongwei Wang
    • 1
    • 2
  • Yong Liu
    • 1
    • 2
  • Deyong Zhang
    • 1
    • 2
  • Qingcong He
    • 1
    • 3
  • Bei Tang
    • 1
    • 3
  • Feixue Cheng
    • 1
    • 2
    Email author
  1. 1.Institute of Plant ProtectionHunan Academy of Agricultural ScienceChangshaChina
  2. 2.Key Laboratory of Integrated Management of the Pests and Diseases on Horticultural Crops in Hunan ProvinceChangshaChina
  3. 3.Long Ping Branch, Graduate School of Hunan UniversityHunan UniversityChangshaChina

Personalised recommendations