Plant Molecular Biology Reporter

, Volume 22, Issue 3, pp 289–300 | Cite as

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay withHMG I/Y as an endogenous reference gene

  • Haibo Weng
  • Aihu Pan
  • Litao Yang
  • Chengmei Zhang
  • Zhili Liu
  • Dabing Zhang
Protocols

Abstract

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T0) were calculated by comparing threshold cycle (CT) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsCT value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.

Key words

Brassica napus HMG I/Y real-time PCR transgene copy number 

Abbreviations

CT

threshold cycle

CTAB

cetyltrimethylammonium bromide

real-time PCR

real-time polymerase chain reaction

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Callaway AS, Abranches R, Scroggs J, Allen GC, and Thompson WF (2002) High-throughput transgene copy number estimation by competitive PCR. Plant Mol Biol Rep 20: 265–277.CrossRefGoogle Scholar
  2. Celi FS, Cohen MM, Antonarakis SE, Wertheimer E, Roth J, and Shuldiner AR (1994) Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences. Genomics 21: 304–310.PubMedCrossRefGoogle Scholar
  3. Chengmei Z, Bingjun Q, Yun X, Yahong H, Aihu P, Wanqi L, and Dabing Z (2003) Transferring an antisenseacyl-acyl carrier protein (ACP) DNA fragment intoBrassica napus by high efficientAgrobacterium tumefaciens genetic transformation system. Acta Agriculturae Shanghai 19: 5–8.Google Scholar
  4. De Preter K, Speleman F, Combaret V, Lunec J, Laureys G, Eussen BH, Francotte N, Board J, Pearson AD, De Paepe A, Van Roy N, and Vandesompele J (2002) Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. Mod Pathol 15: 159–166.PubMedCrossRefGoogle Scholar
  5. Flavell RB (1994) Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc Natl Acad Sci USA 91: 3490–3496.PubMedCrossRefGoogle Scholar
  6. Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30: 503–512.PubMedCrossRefGoogle Scholar
  7. Huang Y, Liang W, Pan A, Zhou Z, Huang C, Chen J, and Zhang D (2003) Production of FaeG, the major subunit of K88 fimbriae, in transgenic tobacco plants and its immunogenicity in mice. Infect Immun 71: 5436–5439.PubMedCrossRefGoogle Scholar
  8. Ingham DJ, Beer S, Money S, and Hansen G (2001) Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 31: 136–140.Google Scholar
  9. Iyer LM, Kumpatla SP, Chandrasekharan MB, and Hall TC (2000) Transgene silencing in monocots. Plant Mol Biol 43: 323–346.PubMedCrossRefGoogle Scholar
  10. Jiayu D, Junwei J, Litao Y, Weng H, Chengmei Z, Wenxuan L, and Dabing Z (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.CrossRefGoogle Scholar
  11. Khachatourians GG, McHughen A, Scorza R, Nip W, and Hui Y (2002) Transgenic plants and crops. Marcel Dekker, New York.Google Scholar
  12. Kooter JM, Matzke TA, and Meyer P (1999) Listening to the silent genes: transgene silencing, gene regulation, and pathogen control. Trends Plant Sci 4: 340–347.PubMedCrossRefGoogle Scholar
  13. Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25: 402–408.PubMedCrossRefGoogle Scholar
  14. Luo ZY, Zhou G, and Chen XH (2001) Isolation of high-quality genomic DNA from plants. Hunan Yi Ke Da Xue Xue Bao 26: 178–180.PubMedGoogle Scholar
  15. Masek T, Smykal P, Janotova II, Honys D, Capkova VV, and Pechan PM (2002) Isolation of aBrassica napus L. cDNA encoding a putative high-mobility-group HMG I/Y protein. Plant Sci 159: 197–204.CrossRefGoogle Scholar
  16. Mason G, Provero P, Vaira AM, and Accotto GP (2002) Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2: 20.PubMedCrossRefGoogle Scholar
  17. Murray MG and Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4325.PubMedCrossRefGoogle Scholar
  18. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  19. Song P, Cai CQ, Skokut M, Kosegi BD, and Petolino JF (2002) Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS-derived transgenic maize. Plant Cell Rep 20: 948–954.CrossRefGoogle Scholar
  20. Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Mourrain P, Palauqui JC, and Vernhettes S (1998) Transgene-induced gene silencing in plant. Plant J 16: 651–659.PubMedCrossRefGoogle Scholar
  21. Weng H, Yang L, Liu Z, Ding J, Pan A, and Zhang D (2004) A novel reference gene,high-mobility-group protein I/Y, can be used in qualitative and real-time quantitative PCR detection of transgenic rapeseed cultivars. J AOAC Int. Forthcoming.Google Scholar
  22. Zhang Y, Zhang D, Li W, Chen J, Peng Y, and Cao W (2003) A novel real-time quantitative PCR method using attached universal template probe. Nucleic Acids Res 31: e123.PubMedCrossRefGoogle Scholar

Copyright information

© International Society for Plant Molecular Biology 2004

Authors and Affiliations

  • Haibo Weng
    • 1
    • 2
  • Aihu Pan
    • 3
  • Litao Yang
    • 1
  • Chengmei Zhang
    • 3
  • Zhili Liu
    • 1
  • Dabing Zhang
    • 2
    • 3
  1. 1.Department of Biological Science and TechnologyNanjing UniversityNanjingPR China
  2. 2.School of Life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiPR China
  3. 3.Shanghai Key Laboratory of Agricultural Genetics and BreedingAgro-biotech Research Center, Shanghai Academy of Agricultural SciencesShanghaiPR China

Personalised recommendations