Assessment of transgene copy number and zygosity of transgenic maize overexpressing Cry1Ie gene with SYBR® Green qRT-PCR
2015
Zhang, Yuwen | Liu, Yan | Zhang, Jie | Wang, Guoying | Wang, Jianhua | Liu, Yunjun
Transgenic maize expressing the Cry1Ie gene was shown to efficiently kill susceptible and Cry1Ac-resistant insects, indicating that Cry1Ie might be a good candidate for the development of a new Bt-maize variety in China. As transgenic maize events with potential commercial usage are required to have one copy of a transgene, it is important to develop high-throughput methods to determine the transgene copy number initially and zygosity during subsequent breeding. In this study, a SYBR® Green quantitative real-time PCR (qRT-PCR) method was established and used to estimate the transgene copy number in Agrobacterium-derived transgenic maize lines expressing Cry1Ie gene. The amplification efficiencies of Cry1Ie, bar, and Adh1 with specific primers were calculated as 0.987, 1.194, and 0.992, respectively. The transgene copy number was calculated using the formula Ratio = 2 Cᵗ_ʳᵉᶠᵉʳᵉⁿᶜᵉ ⁻ Cᵗ_ᵗʳᵃⁿˢᵍᵉⁿᵉ, where a calibrator line with a single copy of the transgene was not required. Six homozygous transgenic maize lines were initially analyzed using this calculation method, and the results were as accurate as those obtained using 2∆∆Cᵗ, a common method for calculation of relative expression levels. More T₀generation transgene lines were compared using both the SYBR Green qRT-PCR and TaqMan qRT-PCR methods, and the results showed a high correlation (R² = 0.8942) between SYBR Green qRT-PCR and TaqMan qRT-PCR values, confirming the reliability of the SYBR Green qRT-PCR method. Using this method, we successfully determined the transgene copy number and zygosity in transgenic maize lines expressing Cry1Ie.
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