Chinese Science Bulletin

, Volume 52, Issue 22, pp 3110–3117 | Cite as

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

  • Tu LiLi 
  • Zhang XianLong 
  • Liu DiQiu 
  • Jin ShuangXia 
  • Cao JingLin 
  • Zhu LongFu 
  • Deng FengLin 
  • Tan JiaFu 
  • Zhang CunBin 
Articles Plant Development Biology

Abstract

The mechanisms of cotton fiber development and somatic embryogenesis have been explored systematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples, with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression results, normalization of real-time PCR data against one or several internal control genes is required, which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes, including 18S rRNA, Histone3, UBQ7, Actin, Cyclophilin, Gbpolyubiquitin-1 and Gbpolyubiquitin-2, in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene (arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quantification should be an efficient and convenient method for the fiber developmental series. The expression of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3, UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.

Keywords

cotton fiber development housekeeping genes internal control real-time PCR somatic embryogenesis 

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References

  1. 1.
    Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002, 3: RESEARCH0034Google Scholar
  2. 2.
    Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards: Use and limits. J Biotechnol, 1999, 75: 291–295CrossRefGoogle Scholar
  3. 3.
    Lee P D, Sladek R, Greenwood C M, et al. Control genes and variability: Absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res, 2002, 12: 292–297CrossRefGoogle Scholar
  4. 4.
    Czechowski T, Stitt M, Altmann T, et al. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol, 2005, 139: 5–17CrossRefGoogle Scholar
  5. 5.
    Pfaffl M W, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett, 2004, 26: 509–515CrossRefGoogle Scholar
  6. 6.
    Goidin D, Mamessier A, Staquet M J, et al. Ribosomal 18S rRNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem, 2001, 295: 17–21CrossRefGoogle Scholar
  7. 7.
    Andersen C L, Jensen J L, 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, 64: 5245–5250CrossRefGoogle Scholar
  8. 8.
    Brunner A M, Yakovlev I A, Strauss S H. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol, 2004, 4: 14CrossRefGoogle Scholar
  9. 9.
    Dheda K, Huggett J F, Bustin S A, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques, 2004, 37: 112–114Google Scholar
  10. 10.
    Radonic A, Thulke S, Mackay I M, et al. Guideline to reference gene selection for quantitative realtime PCR. Biochem Biophys Res Commun, 2004, 313: 856–862CrossRefGoogle Scholar
  11. 11.
    Jain M, Nijhawan A, Tyagi A K, et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun, 2006, 345: 646–651CrossRefGoogle Scholar
  12. 12.
    Arpat A B, Waugh M, Sullivan J P, et al. Functional genomics of cell elongation in developing cotton fibers. Plant Mol Biol, 2004, 54: 911–929CrossRefGoogle Scholar
  13. 13.
    Li X B, Fan X P, Wang X L, et al. The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell, 2005, 17: 859–875CrossRefGoogle Scholar
  14. 14.
    Lee J J, Hassan O S, Gao W, et al. Developmental and gene expression analyses of a cotton naked seed mutant. Planta, 2006, 223: 418–432CrossRefGoogle Scholar
  15. 15.
    Shi Y H, Zhu S W, Mao X Z, et al. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell 2006, 18: 651–664CrossRefGoogle Scholar
  16. 16.
    Samuel Yang S, Cheung F, Lee J J, et al. Accumulation of genome-specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton. Plant J, 2006, 47: 761–775CrossRefGoogle Scholar
  17. 17.
    Zeng F, Zhang X, Zhu L, et al. Isolation and characterization of Genes associated to cotton somatic embryogenesis by suppression subtractive hybridization and macroarray. Plant Mol Biol, 2006, 60: 167–183CrossRefGoogle Scholar
  18. 18.
    Wu J H, Zhang X L, Nie Y C, et al. Factors affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genotypes of Chinese cotton (Gossypium hirsutum L.). In Vitro Cell Del Biol Plant, 2004, 40: 371–375CrossRefGoogle Scholar
  19. 19.
    Zhu L F, Tu L L, Zeng F C, et al. An improved simple protocol for isolation of high quality RNA from Gossypium spp. suitable for cDNA library construction. Acta Agronomica Sinica, 2005, 31: 1657–1659Google Scholar
  20. 20.
    Sun Y, Zhang X, Huang C, et al. Factors influencing in vitro regeneration from protoplasts of wild cotton (G. klotzschianum A) and RAPD analysis of regenerated plantlets. Plant Growth Regul, 2005, 46: 79–86CrossRefGoogle Scholar
  21. 21.
    Liu D, Zhang X, Tu L, et al. Isolation by suppression-subtractive hybridization of genes preferentially expressed during early and late fiber development stages in cotton. Mol Biol (Mosk), 2006, 40: 825–834Google Scholar
  22. 22.
    Kim B R, Nam H Y, Kim S U, et al. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett, 2003, 25: 1869–1872CrossRefGoogle Scholar
  23. 23.
    Nicot N, Hausman J F, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot, 2005, 56: 2907–2914CrossRefGoogle Scholar
  24. 24.
    Goncalves S, Cairney J, Maroco J, et al. Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta, 2005, 222: 556–563CrossRefGoogle Scholar
  25. 25.
    Reid K E, Olsson N, Schlosser J, et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol, 2006, 14: 27CrossRefGoogle Scholar
  26. 26.
    Laney J D, Hochstrasser M. Substrate targeting in the ubiquitin system. Cell, 1999, 97: 427–430CrossRefGoogle Scholar
  27. 27.
    Callis J, Raasch J A, Vierstra R D. Ubiquitin extension proteins of Arabidopsis thaliana. J Bio Chem, 1999, 265: 12486–12493Google Scholar
  28. 28.
    Ciechanover A. The ubiquitin-proteasome pathway: On protein death and cell life. EMBO J, 1998, 17: 7151–7160CrossRefGoogle Scholar
  29. 29.
    Hochstrasser M. Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol, 2000, 2: E153–E157CrossRefGoogle Scholar
  30. 30.
    Basra A, Malik C P. Development of the cotton fiber. Int Rev Cytol, 1984, 89: 65–113CrossRefGoogle Scholar
  31. 31.
    Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the \(2^{ - \Delta \Delta C_T } \) method. Methods, 2001, 25: 402–408CrossRefGoogle Scholar

Copyright information

© Science in China Press 2007

Authors and Affiliations

  • Tu LiLi 
    • 1
  • Zhang XianLong 
    • 1
  • Liu DiQiu 
    • 1
  • Jin ShuangXia 
    • 1
  • Cao JingLin 
    • 1
  • Zhu LongFu 
    • 1
  • Deng FengLin 
    • 1
  • Tan JiaFu 
    • 1
  • Zhang CunBin 
    • 1
  1. 1.National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina

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