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Reference Gene Selection for Gene Expression Studies Using Quantitative Real-Time PCR Normalization in Atropa belladonna

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Abstract

Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels. Accurate and reproducible results are dependent on the correct choice of reference genes for data normalization. Atropa belladonna is a commercial plant species from which pharmaceutical tropane alkaloids are extracted. In this study, eight candidate reference genes, namely 18S ribosomal RNA (18S), actin (ACT), cyclophilin (CYC), elongation factor 1α (EF-1α), β-fructosidase (FRU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and beta-tubulin (TUB), were selected and their expression stabilities studied to determine their suitability for normalizing gene expression in A. belladonna. The expression stabilities of these genes were analyzed in the root, stem, and leaf under cold, heat, NaCl, UV-B, methyl jasmonate, salicylic acid, and abscisic acid treatments using geNorm, NormFinder, and BestKeeper. The statistical algorithms indicated that PGK was a reliable gene for normalizing gene expression under most of the experimental conditions. The pairwise value analysis showed that two genes were sufficient for proper expression normalization, except when analyzing gene expression in heat-treated roots. However, the choice of the second reference gene depended on specific conditions. Finally, the relative expression level of the PMT gene of A. belladonna was detected to validate the selection of PGK a reliable reference gene. In summary, our results should guide the selection of appropriate reference genes for gene expression studies in A. belladonna under different organs and abiotic stress conditions.

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References

  • 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 datasets. Cancer Res 64:5245–5250

    Article  CAS  PubMed  Google Scholar 

  • Biastoff S, Brandt W, Dräger B (2009) Putrescine N-methyltransferase—the start for alkaloids. Phytochemistry 70(15–16):1708–1718

    Article  CAS  PubMed  Google Scholar 

  • Brunner A, Yakovlev I, Strauss S (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14

    Article  PubMed Central  PubMed  Google Scholar 

  • Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huguett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  PubMed  Google Scholar 

  • Chang EM, Shi SH, Liu GF, Cheng TL, Xue L, Yang XY, Yang WJ, Lan Q, Jiang ZP (2012) Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR. Plos One 7:7

    Article  Google Scholar 

  • Chen K, Fessehaie A, Arora R (2011) Selection of reference genes for normalizing gene expression during seed priming and germination using qPCR in Zea mays and Spinacia oleracea. Plant Mol Biol Rep 30:478–487

    Article  Google Scholar 

  • Crismani W, Baumann U, Sutton T, Shirley N, Webster T, Spangenberg G, Langridge P, Able JA (2006) Microarray expression analysis of meiosis and microsporogenesis in hexaploid bread wheat. BMC Genomics 7:267

    Article  PubMed Central  PubMed  Google Scholar 

  • Czechowski T, Bari R, Stitt M, Scheible W, Udvardi M (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. The Plant J 38:366–379

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • de Almeida MR, Ruedell CM, Ricachenevsky FK, Sperotto RA, Pasquali G, Fett-Neto AG (2010) Reference gene selection for quantitative reverse transcription–polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill. BMC Mol Biol 11:73

    Article  PubMed Central  PubMed  Google Scholar 

  • Demidenko NV, Logacheva MD, Penin AA (2011) Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data. PLoS ONE 6(5):e19434. doi:10.1371/journal.pone.0019434

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GAW, Zumla A (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344:141–143

    Article  CAS  PubMed  Google Scholar 

  • Die JV, Roman B, Nadal S, Gonzalez-Verdejo CI (2010) Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232:145–153

    Article  CAS  PubMed  Google Scholar 

  • Fernandez P, Di Rienzo JA, Moschen S, Dosio GAA, Aguirrezabal LAN, Hopp HE, Paniego N, Heinz RA (2011) Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis. Plant Cell Rep 30:63–74

    Article  CAS  PubMed  Google Scholar 

  • Fukami H, Asakura T, Hirano H, Shimomura K, Yamakawa T (2002) Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogenously added salicylic acid. Plant Cell Physiol 43:1054–1058

    Article  CAS  PubMed  Google Scholar 

  • Gachon C, Mingam A, Charrier B (2004) Real-time PCR: what relevance to plant studies. J Exp Bot 55:1445–1454

    Article  CAS  PubMed  Google Scholar 

  • Ghareeb H, Bozso Z, Ott PG, Wydra K (2011) Silicon and Ralstonia solanacearum modulate expression stability of housekeeping genes in tomato. Physiol Mol Plant 75:176–179

    Article  CAS  Google Scholar 

  • Goossens K, Soom A, Poucke M, Vandaele L, Vandesompele J, Van Zeveren A, Peelman LJ (2007) Identification and expression analysis of genes associated with bovine blastocyst formation. BMC Dev Biol 7:64

    Article  PubMed Central  PubMed  Google Scholar 

  • Goulao LF, Fortunato AS, Ramalho JC (2012) Select ion of reference genes for normalizing quantitative real-time PCR gene expression data with multiple variables in Coffea spp. Plant Mol Biol Rep 30:741–759

    Article  CAS  Google Scholar 

  • Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60:487–493

    Article  CAS  PubMed  Google Scholar 

  • Gutierrez N, Gimenez MJ, Palomino C, Avila CM (2011) Assessment of candidate reference genes for expression studies in Vicia faba L. by real-time quantitative PCR. Mol Breed 28:13–24

    Article  Google Scholar 

  • Hibbeler S, Scharsack JP, Becker S (2008) Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Mol Biol 9:18

    Article  PubMed Central  PubMed  Google Scholar 

  • Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalization; strategies and considerations. Genes Immu 6:279–284

    Article  CAS  Google Scholar 

  • Ingerslev HC, Pettersen EF, Jakobsen RA, Petersen CB, Wergeland HI (2006) Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Mol Immunol 43:1194–1201

    Article  CAS  PubMed  Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Bioph Res Co 345:646–651

    Article  CAS  Google Scholar 

  • Jarosova J, Kundu J (2010) Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR. BMC Plant Biol 1:146

    Article  Google Scholar 

  • Kang SM, Jung YH, Kang YM, Yun DJ, Bahk JD, Yang JK, Choi MS (2004) Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora. Plant Sci 166:745–751

    Article  CAS  Google Scholar 

  • Kim BR, Nam HY, Kim SY, Kim SI, Chang YJ (2003) Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25:1869–1872

    Article  CAS  PubMed  Google Scholar 

  • Klie M, Debener T (2011) Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida). BMC Res Notes 4:518

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lee PD, Sladek R, Greenwood CM, Hudson TJ (2002) Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res 12:292–297

    Article  PubMed Central  PubMed  Google Scholar 

  • Lee JM, Roche JR, Donaghy DJ, Thrush A, Sathish P (2010) Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Mol Biol 11:8

    Article  PubMed Central  PubMed  Google Scholar 

  • Li QF, Sun SM, Yuan DY, Yu HX, Gu MH, Liu QQ (2010) Validation of candidate reference genes for the accurate normalization of real-time quantitative RT-PCR data in rice during seed development. Plant Mol Biol Rep 28:49–57

    Article  Google Scholar 

  • Ma S, Niu H, Liu C, Zhang J, Hou C, Wang D (2013) Expression stabilities of candidate reference genes for RT-qPCR under different stress conditions in soybean. PLoS ONE 8(10):e75271. doi:10.1371/journal.pone.0075271

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mackay IM (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004:190–212

    Article  Google Scholar 

  • Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10:4

    Article  PubMed Central  PubMed  Google Scholar 

  • Manaa A, Ben Ahmed H, Valot B, Bouchet JP, Aschi-Smiti S, Causse M, Faurobert M (2011) Salt and genotype impact on plant physiology and root proteome variations in tomato. J Exp Bot 62:2797–2813

    Article  CAS  PubMed  Google Scholar 

  • Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    Article  PubMed Central  PubMed  Google Scholar 

  • Migocka M, Papierniak A (2010) Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators. Mol Breeding 28:343–357

    Article  Google Scholar 

  • Nam MH, Huh SM, Kim KM, Park WJ, Seo JB, Cho K, Kim DY, Kim BG, Yoon IS (2012) Comparative proteomic analysis of early salt stress-responsive proteins in roots of SnRK2 transgenic rice. Proteome Sci 10:25

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914

    Article  CAS  PubMed  Google Scholar 

  • Oksman-Caldentey KM (2007) Tropane and nicotine alkaloid biosynthesis—novel approaches towards biotechnological production of plant-derived pharmaceuticals. Curr Pharm Biotechno 8:203–210

    Article  CAS  Google Scholar 

  • 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. Biotechnol Lett 26:509–515

    Article  CAS  PubMed  Google Scholar 

  • Pombo-Suarez M, Calaza M, Gomez-Reino JJ, Gonzalez A (2008) Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage. BMC Mol Biol 9:17

    Article  PubMed Central  PubMed  Google Scholar 

  • Rothe G, Garske U, Drager B (2001) Calystegines in root cultures of Atropa belladonna respond to sucrose, not to elicitation. Plant Sci 160:1043–1053

    Article  CAS  PubMed  Google Scholar 

  • Rubie C, Kempt K, Hans J, Su TF, Tilton B, Georg T, Brittner B, Ludwig B, Schilling M (2005) Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues. Mol Cell Probe 19:101–109

    Article  CAS  Google Scholar 

  • Suzuki K, Yamada Y, Hashimoto T (1999a) Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiol 40:289–297

    Article  CAS  PubMed  Google Scholar 

  • Suzuki K, Yun DJ, Chen XY, Yamada Y, Hashimoto T (1999b) An Atropa belladonna hyoscyamine 6 β-hydroxylase gene is differentially expressed in the root pericycle and anthers. Plant Mol Biol 40:141–152

    Article  CAS  PubMed  Google Scholar 

  • Thornton S, Anand N, Purcell D, Lee J (2003) Not just for housekeeping: protein initiation and elongation factors in cell growth and tumorigenesis. J Mol Med 81:536–5481

    Article  CAS  PubMed  Google Scholar 

  • Tong ZG, Gao ZH, Wang F, Zhou J, Zhang Z (2009) Selection of reliable reference genes for gene expression studies in peach using realtime PCR. BMC Mol Biol 10:71

    Article  PubMed Central  PubMed  Google Scholar 

  • Valasek MA, Repa JJ (2005) The power of real-time PCR. Advan Physiol Edu 29:151–159

    Article  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:34

    Article  Google Scholar 

  • Walker NJ (2002) A technique whose time has come. Science 296:557–559

    Article  CAS  PubMed  Google Scholar 

  • Wang XR, Chen M, Yang CX, Liu XQ, Zhang L, Lan XZ, Tang KX, Liao ZH (2011) Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes. Physiol Plant 143(4):309–315

    Article  CAS  PubMed  Google Scholar 

  • Yang CX, Chen M, Zeng LJ, Zhang L, Liu XQ, Lan XZ, Tang KX, Liao ZH (2011) Improvement of tropane alkaloids production in hairy root cultures of Atropa belladonna by overexpressing pmt and h6h genes. Plant Omics 4:29–33

    CAS  Google Scholar 

  • Zhao S, Fernald RD (2005) Comprehensive algorithm for quantitative real-time polymerase chain reaction. J Comput Biol 12:1047–1064

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhao WJ, Li Y, Gao PF, Sun ZH, Sun TS, Zhang HP (2011) Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang. J Ind Microbiol Biotechnol 38:1279–1286

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported financially by the NSFC project (31370333), the Program for New Century Excellent Talents in University (NCET-12-0930), the National 863 Hi-Tech Plans (2011AA100605; 2011AA100607), Chongqing Sciences and Technology Project (CSTC2012GGYYJS80013), and the Fundamental Research Funds for the Central Universities (XDJK2013A024).

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Authors and Affiliations

  1. Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), Chongqing Engineering and Technology Research Center for Sweetpotato, School of Life Sciences, Southwest University, Chongqing, 400715, China

    Jindi Li, Fei Qiu, Baifu Qin, Nengbiao Wu & Zhihua Liao

  2. Beihai No. 7 Middle School, Guangxi, 536000, China

    Jindi Li

  3. College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, China

    Min Chen

  4. School of Chemistry and Chemical Engineering, Chongqing University of Science and Technology, Chongqing, 401331, China

    Wanhong Liu

  5. Agricultural and Animal Husbandry College, Tibet University, Nyingchi, Tibet, 860000, China

    Xiaozhong Lan

  6. China Rural Technology Development Center, Ministry of Science and Technology, Beijing, 100045, China

    Qiang Wang

  7. Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Kexuan Tang

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  1. Jindi Li
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  2. Min Chen
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  3. Fei Qiu
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  9. Zhihua Liao
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Correspondence to Zhihua Liao or Kexuan Tang.

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Li, J., Chen, M., Qiu, F. et al. Reference Gene Selection for Gene Expression Studies Using Quantitative Real-Time PCR Normalization in Atropa belladonna . Plant Mol Biol Rep 32, 1002–1014 (2014). https://doi.org/10.1007/s11105-014-0701-9

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