Skip to main content
Log in

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

  • Genetics and Genomics
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Ahmed FE (2002) Detection of genetically modified organisms in foods. Trends Biotechnol 5:215–223

    Google Scholar 

  • Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218

    CAS  Google Scholar 

  • 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 A1

  • Bonfini L, Heinze P, Kay S, Van den Eade G (2002) Review of GMO detection and quantification techniques. EUR 20348 EN

  • 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 52:3372–3377

    Article  CAS  PubMed  Google Scholar 

  • Duijn GV, Biert RV, Marcelis HB, Peppelman H, Hessing M (1999) Detection methods for genetically modified crops. Food Control 10:375–378

    Google Scholar 

  • Guo S, Ni W, Xu Q (1996) Expressive carrier with coded insect-killing protein fusion gene, and transfer gene plant. Chinese Patent PN: 1134981

    Google Scholar 

  • Harwood JL (1988) Fatty acid metabolism. Ann Rev Plant Physiol Plant Mol Biol 39:101–138

    Google Scholar 

  • 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 49:3622–3627

    Google Scholar 

  • Huang J, Rozelle S, Pray C, Wang Q (2002) Plant biotechnology in China. Science 295:674–676

    Google Scholar 

  • James C (2003) Global status of commercialized transgenic crops ISAAA Briefs, no.30

  • 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– 5834

    Google Scholar 

  • 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 A1

  • Kok EJ, Kuiper HA (2003) Comparative safety assessment for biotech crops. Trends Biotechnol 10:439–444

    Google Scholar 

  • 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 110:1436

    Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Meyer R (1995) Nachweis gentechnologisch veränderter Pflanzen mittels der Polymerase Kettenreaktion (PCR) am Beispiel der Flavr Savr-Tomate. Z Lebensm Unters Forsch 201:583–586

    Google Scholar 

  • 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 203:339–344

    Google Scholar 

  • Pietsch K, Waiblinger HU, Brodmann P, Wurz A (1997) Screening verfahren zur Identifizierung “gentechnisch veränderter” pflanzlicher Lebensmittel. Dtsch Lebensm Rundsch 93:35–38

    Google Scholar 

  • Rangwala TS, Ye M (2002) Cotton event PV-GHGT07 (1445) and compositions and methods for detection thereof. World patent, Pub. No.: WO 02/34946 A2

  • 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 230:1350–1354

    Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Studer E, Dahinden I, Luthy J, Hubner P (1997) Nachweis des gentechnisch veranderten “Maximizer”-mais mittels der polymerase-kettenreaktion (PCR). Mitt Geb Lebensmittelunter Hyg 88:515–524

    Google Scholar 

  • Terry CF, Harris N (2001) Event-specific detection of roundup ready soya using two different real time PCR detection chemistries. Eur Food Res Technol 213:425–431

    Article  CAS  Google Scholar 

  • 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 47:5261–5266

    Article  PubMed  Google Scholar 

  • 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 47:5038–5043

    Google Scholar 

  • 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)

  • 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 10:385–389

    Google Scholar 

  • 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)

  • 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 31:20e123

    Google Scholar 

  • 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 31:664–667

    Google Scholar 

Download references

Acknowledgments

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

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dabing Zhang.

Additional information

Communicated by I.S. Chung

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, L., Chen, J., Huang, C. et al. Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons. Plant Cell Rep 24, 237–245 (2005). https://doi.org/10.1007/s00299-005-0929-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-005-0929-9

Keywords

Navigation