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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

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.

Keywords

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

Notes

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

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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

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