Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay withHMG I/Y as an endogenous reference gene
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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 (C T) 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 itsC T 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 wordsBrassica napus HMG I/Y real-time PCR transgene copy number
- real-time PCR
real-time polymerase chain reaction
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- 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
- 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
- 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
- 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
- Khachatourians GG, McHughen A, Scorza R, Nip W, and Hui Y (2002) Transgenic plants and crops. Marcel Dekker, New York.Google Scholar
- 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
- 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