Assessment of transgene copy number and zygosity of transgenic maize overexpressing Cry1Ie gene with SYBRA (R) Green qRT-PCR

doi:10.1007/s11627-014-9658-5

	Assessment of transgene copy number and zygosity of transgenic maize overexpressing Cry1Ie gene with SYBRA (R) Green qRT-PCR
	Zhang, Yuwen 1,2; Liu, Yan 2; Zhang, Jie 2; Wang, Guoying 1; Wang, Jianhua 2; Liu, Yunjun 1
刊名	IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT
	2015
卷号	51 期号:2 页码:125-134
关键词	Copy number Transgenic maize Quantitative real-time PCR SYBR (R) Green
ISSN号	1054-5476
DOI	10.1007/s11627-014-9658-5
通讯作者	Zhang, Yuwen
英文摘要	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 SYBRA (R) 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 -aEuro parts per thousand 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(a dagger a dagger Ct), a common method for calculation of relative expression levels. More T-0 generation transgene lines were compared using both the SYBR Green qRT-PCR and TaqMan qRT-PCR methods, and the results showed a high correlation (R (2) = 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.
学科主题	Plant Sciences ; Cell Biology ; Developmental Biology
语种	英语
出版者	SPRINGER
WOS记录号	WOS:000352920100001
内容类型	期刊论文
源URL	[http://111.203.20.206/handle/2HMLN22E/4732]
专题	作物科学研究所_分子生物学系
作者单位	1.Chinese Acad Agr Sci, Inst Crop Sci, Beijing 100081, Peoples R China 2.China Agr Univ, Coll Agr & Biotechnol, Beijing 100094, Peoples R China
推荐引用方式 GB/T 7714	Zhang, Yuwen,Liu, Yan,Zhang, Jie,et al. Assessment of transgene copy number and zygosity of transgenic maize overexpressing Cry1Ie gene with SYBRA (R) Green qRT-PCR[J]. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT,2015,51(2):125-134.
APA	Zhang, Yuwen,Liu, Yan,Zhang, Jie,Wang, Guoying,Wang, Jianhua,&Liu, Yunjun.(2015).Assessment of transgene copy number and zygosity of transgenic maize overexpressing Cry1Ie gene with SYBRA (R) Green qRT-PCR.IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT,51(2),125-134.
MLA	Zhang, Yuwen,et al."Assessment of transgene copy number and zygosity of transgenic maize overexpressing Cry1Ie gene with SYBRA (R) Green qRT-PCR".IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT 51.2(2015):125-134.