Plant Cell Reports

, Volume 24, Issue 4, pp 237–245 | Cite as

Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons

  • Litao Yang
  • Jianxiu Chen
  • Cheng Huang
  • Yuhui Liu
  • Shirong Jia
  • Liangwen Pan
  • Dabing Zhang
Genetics and Genomics


Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicat that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons.


Gossypium hirsutum Stearoyl-Acyl Carrier Protein desaturase gene Endogenous reference gene Genetically modified organism Conventional and real-time PCR 



This work was supported by the Fund of National Key Basic Research Developments Program of the Ministry of Science and Technology P. R. China (2001CB109002), National Transgenic Plant Special Fund (JY03-B-20), National Natural Science Foundation of China (30370893) and Shanghai Municipal Committee of Science and Technology (03DZ19307, 03DZ05032). We also acknowledge Prof. Sandui Guo for supplying the insect-resistant cotton GK19 seeds


  1. Ahmed FE (2002) Detection of genetically modified organisms in foods. Trends Biotechnol 5:215–223Google Scholar
  2. Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218Google Scholar
  3. Beazleyn KA, Hillyard JR, Pang S, Roberts JK (2004) Cotton event PV-GHBK04 (531) and compositions and methods for detection thereof. US Patent. Pub. No.: US2004/0045054 A1Google Scholar
  4. Bonfini L, Heinze P, Kay S, Van den Eade G (2002) Review of GMO detection and quantification techniques. EUR 20348 ENGoogle Scholar
  5. Ding J, Jia J, Yang L, Wen H, Zhang C, Liu W, Zhang D (2004) Validation of a rice-specific gene, sucrose-phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. J Agric Food Chem 52:3372–3377CrossRefPubMedGoogle Scholar
  6. Duijn GV, Biert RV, Marcelis HB, Peppelman H, Hessing M (1999) Detection methods for genetically modified crops. Food Control 10:375–378Google Scholar
  7. Guo S, Ni W, Xu Q (1996) Expressive carrier with coded insect-killing protein fusion gene, and transfer gene plant. Chinese Patent PN: 1134981Google Scholar
  8. Harwood JL (1988) Fatty acid metabolism. Ann Rev Plant Physiol Plant Mol Biol 39:101–138Google Scholar
  9. Hernández M, Río A, Esteve T, Prat S, Pla M (2001) A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and real-time quantitative PCR detection of transgenes from mixed food samples. J Agric Food Chem 49:3622–3627Google Scholar
  10. Huang J, Rozelle S, Pray C, Wang Q (2002) Plant biotechnology in China. Science 295:674–676Google Scholar
  11. James C (2003) Global status of commercialized transgenic crops ISAAA Briefs, no.30Google Scholar
  12. James D, Schmidt AM, Wall E, Green M, Masri S (2003) Reliable detection and identification of genetically modified maize, soybean and canola by multiplex PCR analysis. J Agric Food Chem 51:5839– 5834Google Scholar
  13. Jeanna RH, Roberts JK, Ye M (2003) Cotton event PV-GHBK04 (757) and compositions and methods for detection thereof. US Patent. Pub. No.: US2003/0024005 A1Google Scholar
  14. Kok EJ, Kuiper HA (2003) Comparative safety assessment for biotech crops. Trends Biotechnol 10:439–444Google Scholar
  15. Liu Q, Singh S, Sharp P, Green A, Marshall DR (1996) Nucleotide sequence of a cdna from Gossypium hirsutum encoding a stearoyl-acyl carrier protein desaturase. Plant Physiol 110:1436Google Scholar
  16. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  17. Meyer R (1995) Nachweis gentechnologisch veränderter Pflanzen mittels der Polymerase Kettenreaktion (PCR) am Beispiel der Flavr Savr-Tomate. Z Lebensm Unters Forsch 201:583–586Google Scholar
  18. Meyer R, Chardonnens F, Hubner P, Luthy J (1996) Polymerase Chain Reaction (PCR) in the quality and safety assurance of food: detection of soya in processed meat products. Z Lebensm Unters Forsch 203:339–344Google Scholar
  19. Pietsch K, Waiblinger HU, Brodmann P, Wurz A (1997) Screening verfahren zur Identifizierung “gentechnisch veränderter” pflanzlicher Lebensmittel. Dtsch Lebensm Rundsch 93:35–38Google Scholar
  20. Rangwala TS, Ye M (2002) Cotton event PV-GHGT07 (1445) and compositions and methods for detection thereof. World patent, Pub. No.: WO 02/34946 A2Google Scholar
  21. Saiki RK, Scharf SJ, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 230:1350–1354Google Scholar
  22. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  23. Studer E, Dahinden I, Luthy J, Hubner P (1997) Nachweis des gentechnisch veranderten “Maximizer”-mais mittels der polymerase-kettenreaktion (PCR). Mitt Geb Lebensmittelunter Hyg 88:515–524Google Scholar
  24. Terry CF, Harris N (2001) Event-specific detection of roundup ready soya using two different real time PCR detection chemistries. Eur Food Res Technol 213:425–431CrossRefGoogle Scholar
  25. VaÏtilingom M, Pijnenburg H, Gendre F, Brignon P (1999) Real-time quantitative PCR detection of genetically modified maximizer maize and roundup ready soybean in some representative foods. J Agric Food Chem 47:5261–5266CrossRefPubMedGoogle Scholar
  26. Vollenhofer S, Burg K, Schmidt J, Kroath H (1999) Genetically modified organism in food-screening and specific detection by polymerase chain reaction. J Agric Food Chem 47:5038–5043Google Scholar
  27. Weng H, Yang L, Liu Z, Ding J, Pan A, Zhang D (2005) A novel reference gene, high-mobility-group protein I/Y, can be used in qualitative and real-time quantitative PCR detection of transgenic rapeseeds. J AOAC Int (in press)Google Scholar
  28. Wurz A, Bluth P, Zeltz P, Pfeifer C, Willmund R (1999) Quantitative analysis of genetically modified organism (GMO) in processed food by PCR-based methods. Food Control 10:385–389Google Scholar
  29. Yang L, Pan A, Jia J, Ding J, Chen J, Huang C, Zhang C, Zhang D (2005) Validation of a tomato specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes. J Agric Food Chem (in press)Google Scholar
  30. Zhang Y, Zhang D, Li W, Chen J, Peng Y, Cao W (2003) A novel real-time quantitative PCR method using attached universal template probe. Nucleic Acids Res 31:20e123Google Scholar
  31. Zimmermann A, Hemmer W, Liniger M, Luthy J, Pauli U (1998) A sensitive detection method for genetically modified MaisGard corn using a nested PCR-system. Lebensm Wiss Technol 31:664–667Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Litao Yang
    • 1
    • 2
  • Jianxiu Chen
    • 1
  • Cheng Huang
    • 1
  • Yuhui Liu
    • 4
  • Shirong Jia
    • 4
  • Liangwen Pan
    • 5
  • Dabing Zhang
    • 2
    • 3
  1. 1.Department of Biological Science and TechnologyNanjing UniversityNanjingP. R. China
  2. 2.Key Laboratory of Agricultural Genetics and Breeding, Agro-biotech Research CenterShanghai Academy of Agricultural SciencesShanghaiP. R. China
  3. 3.School of life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiP. R. China
  4. 4.Biotechnology Research InstituteChinese Academy of Agricultural SciencesBeijingP. R. China
  5. 5.Shanghai Entry-Exit inspection and Quarantine BureauShanghaiP. R. China

Personalised recommendations