Plant Cell Reports

, Volume 23, Issue 10–11, pp 759–763 | Cite as

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

  • Litao Yang
  • Jiayu Ding
  • Chengmei Zhang
  • Junwei Jia
  • Haibo Weng
  • Wenxuan Liu
  • Dabing Zhang
Genetics and Genomics

Abstract

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous β-glucuronidase (GUS) and hygromycin phosphortransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.

Keywords

Transgenic rice TaqMan real-time PCR GUS HPT Copy number 

Notes

Acknowledgements

This investigation 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).

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Litao Yang
    • 1
    • 2
  • Jiayu Ding
    • 1
    • 3
  • Chengmei Zhang
    • 4
  • Junwei Jia
    • 4
  • Haibo Weng
    • 4
  • Wenxuan Liu
    • 3
  • Dabing Zhang
    • 1
    • 4
  1. 1.School of life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.Department of Biological Science and TechnologyNanjing UniversityNanjingPeople’s Republic of China
  3. 3.School of Life ScienceShanghai UniversityShanghaiPeople’s Republic of China
  4. 4.Key Laboratory of Agricultural Genetics and Breeding, Agro-biotech Research CenterShanghai Academy of Agricultural SciencesShanghaiPeople’s Republic of China

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