Validation of suitable reference genes for quantitative gene expression analysis in Tripterygium wilfordii
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Validation of suitable reference genes is critical in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Suitable and reliable reference genes for the normalization of gene expression data are characterized by high gene expression stability across tissues and different experimental conditions. This study evaluated the gene expression stability of ten reference genes commonly used in Arabidopsis thaliana for their suitability in qRT-PCR analysis in Tripterygium wilfordii Hook.f. The orthologous sequences of these ten candidate genes were identified from T. wilfordii transcriptomic data (Project No. SRX472292). Five algorithms including GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to assess the gene expression stability of these putative reference genes in different plant tissues and different stress conditions. The results identified ACTINT7 and TBP as the most suitable reference genes across all samples. The gene expressions of TwHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase, KU246037.1) and of TwDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase, KJ174341.1) were investigated to validate the suitability of the reference genes. The validation analysis confirmed the suitability of ACTINT7 and TBP as the best reference genes for elucidating secondary metabolite biosynthesis pathway in T. wilfordii. In summary, this study identified the most suitable and reliable reference genes for future qRT-PCR- based studies in T. wilfordii.
KeywordsTripterygium wilfordii qRT-PCR Reference gene Gene expression
This work was supported by the National Natural Science Foundation of China (Grant No. 31272110) and the Natural Science Foundation of Shaanxi Province (Grant No. 2016JM3036).
CZ and JZ conceived and designed the study. JZ, YH and BZ performed the experiments. CZ and JZ wrote the paper. CZ, JF, ZM and XZ reviewed and edited the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
- 7.Liao ZH, Gong YF, Kai GY, Zu KJ, Chen M, Tan QM, Wei YM, Guo L, Tan F, Sun XF, Tang KX (2005) An intron-free methyl jasmonate inducible geranylgeranyl diphosphate- synthase gene from Taxus media and its functional identification in yeast. Mol Biol 39:11–17. https://doi.org/10.1007/s11008-005-0002-3 CrossRefGoogle Scholar
- 9.Antonoff MB, Chugh R, Borja-Cacho D, Dudeja V, Clawson KA, Skube SJ, Sorenson BS, Saltzman DA, Vickers SM, Saluja AK (2009) Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo. Surgery 146:282–290. https://doi.org/10.1159/000236859 CrossRefGoogle Scholar
- 11.Zhu WB, He SM, Li Y, Qiu PX, Shu MF, Ou YQ, Zhou YH, Leng TD, Xie J, Zheng XK, Xu D, Su XW, Yan GM (2010) Anti-angiogenic activity of triptolide in anaplastic thyroid carcinoma is mediated by targeting vascular endothelial and tumor cells. Vascul Pharmacol 52:46–54. https://doi.org/10.1016/j.vph.2009.10.006 CrossRefGoogle Scholar
- 19.Huang YX, Tan HX, Yu J, Chen Y, Guo ZY, Wang GQ, Zhang QL, Chen JF, Zhang L, Diao Y (2017) Stable internal reference genes for normalizing real-time quantitative PCR in Baphicacanthus cusia under hormonal stimuli and UV irradiation, and in different plant organs. Front Plant Sci 8:668. https://doi.org/10.3389/fpls.2017.00668 CrossRefGoogle Scholar
- 22.Miao GP, Li W, Zhang B, Zhang ZF, Ma ZQ, Feng JT, Zhang X, Zhu CS (2015) Identification of genes involved in the biosynthesis of Tripterygium wilfordii Hook.f. secondary metabolites by suppression subtractive hybridization. Plant Mol Biol Rep 33:756–769. https://doi.org/10.1007/s11105-014-0792-3 CrossRefGoogle Scholar
- 23.Miao GP, Han J, Zhang JF, Zhu CS, Zhang X (2017) A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f. Plant Mol Biol 95:51–62. https://doi.org/10.1007/s11103-017-0634-4 CrossRefGoogle Scholar
- 25.Su P, Guan HY, Zhang YF, Wang X, Gao LH, Zhao YJ, Hu TY, Zhou JW, Ma BW, Tu LC, Tong YR, Huang LQ, Gao W (2017) Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-Cytochrome P450 Reductases in Tripterygium wilfordii. Front Plant Sci 8:1756. https://doi.org/10.3389/fpls.2017.01756 CrossRefGoogle Scholar
- 29.Guan HY, Zhao YJ, Su P, Tong YR, Liu YJ, Hu TY, Zhang YF, Zhang XA, Li J, Wu XY, Huang LQ, Gao W (2017) Molecular cloning and functional identification of sterol C24-methyltransferase gene from Tripterygium wilfordii. Acta Pharmaceut Sin B 7(5):603–609. https://doi.org/10.1016/j.apsb.2017.07.001 CrossRefGoogle Scholar
- 30.Su P, Guan HY, Zhao YJ, Tong YR, Xu MM, Zhang YF, Hu TY, Yang J, Cheng QQ, Gao LH, Liu YJ, Zhou JW, Reuben JP, Huang LQ, Gao W (2018) Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii. Plant J 93(1):50–65. https://doi.org/10.1111/tpj.13756 CrossRefGoogle Scholar
- 32.Nikolaj LH, Allison MH, Britta H, Carl EO, Björn MH, Johan AR, Björn H (2017) The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily. Plant J 89:429–441. https://doi.org/10.1111/tpj.13410 CrossRefGoogle Scholar
- 33.Zhu CS, Miao GP, Guo J, Huo YB, Zhang X, Xie JH, Feng JT (2014) Establishment of Tripterygium wilfordii Hook.f. hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine. J Microbiol Biotechnol 24:823–834. https://doi.org/10.4014/jmb.1402.02045 CrossRefGoogle Scholar
- 36.Miao GP, Zhu CS, Yang YQ, Feng MX, Ma ZQ, Feng JT, Zhang X (2014) Elicitation and in situ adsorption enhanced secondary metabolites production of Tripterygium wilfordii Hook. f. adventitious root fragment liquid cultures in shake flask and a modified bubble column bioreactor. Bioprocess Biosyst Eng 37:641–650. https://doi.org/10.1007/s00449-013-1033-0 CrossRefGoogle Scholar
- 40.Vandesompele J, Preter KD, Pattyn F, Poppe B, Roy NV, Paepe AD, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):0034.1–0034.11. https://doi.org/10.1186/gb-2002-3-7-research0034 CrossRefGoogle Scholar
- 41.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. Can Res 64:5245–5250. https://doi.org/10.1158/0008-5472.can-04-0496 CrossRefGoogle Scholar
- 43.Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: Bestkeeper - excel-based tool using pair-wise correlations. Biotech Lett 26:509–515. https://doi.org/10.1023/B:BILE.0000019559.84305.47 CrossRefGoogle Scholar
- 45.Herz S, Wungsintaweekul J, Schulir CA, Hecht S, Luttgen H, Sagner S, Fellermeier M, Eisenreich W, Zenk MH, Bacher A, Rohdich F (2000) Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-d-erythritol 2,4-cyclodiphosphate. Proc Natl Acad Sci USA 97:2486–2490. https://doi.org/10.1073/pnas.040554697 CrossRefGoogle Scholar
- 50.Enjuto M, Balcells L, Campos N, Caelles C, Arró M, Boronat A (1994) Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme. Proc Natl Acad Sci USA 91:927–931. https://doi.org/10.1073/pnas.91.3.927 CrossRefGoogle Scholar
- 55.Won-Jae L, Ryoung-Hoon J, Si-Jung J, Ji-Sung P, Seung-Chan L, Raghavendra BS et al (2015) Selection of reference genes for quantitative gene expression in porcine mesenchymal stem cells derived from various sources along with differentiation into multilineages. Stem Cells Int 2015:1–14. https://doi.org/10.1155/2015/235192 Google Scholar