Analysis of Integrated Structure of the Exogenous Gene Insertion and the Establishment of Event-specific Detection Method in Transgenic Insect Resistant Cotton
1 Tobacco Research Institute of CAAS, Qingdao, Shandong, 266101, P.R.China
2 Biotechnology Research Institute, CAAS, Beijing, 100081, P.R.China
GMO Biosafety Research, 2012, Vol. 3, No. 1 doi: 10.5376/gmo.2012.03.0001
Received: 04 May, 2012 Accepted: 18 Jun., 2012 Published: 21 Jun., 2012
© 2012 BioPublisher Publishing Platform
This article was first published in Molecular Plant Breeding (2012, 10(3): 317-323) in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Hou et al., 2012, Analysis of Integrated Structure of the Exogenous Gene Insertion and the Establishment of Event-specific Detection Method in Transgenic Insect Resistant Cotton, GMO Biosafety Research, Vol.3, No.1 1-7 (doi: 10.5376/gmo.2012.03.0001)
In this study, the complete sequence of exogenous DNA insertion was obtained from transgenic cotton line 06N-119 by using 3'-terminal high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) two times, 5'-terminal hiTAIL-PCR four times and long distance PCR (LD-PCR) once. The insertion of the exogenous gene is 11578 bp in length and consists of the Nptâ…¡ gene expression cassette and the Bt gene expression cassette in series. There is a 75 bp fragment lost at insertion site but no gene recombination occurred. The event-specific qualitative PCR method was established based on the 3'-terminal flanking sequence, which was proved to be highly specific and sensitive. The detection limit was as low as 0.05% every 100 ng of template, that is equivalent 11 target sequence copies. The results of this study would be importance on the detection of exogenous gene in transgenic cotton and biosafety assessment.
Transgenic resistant cotton; Integrated structure; PCR detection; Event-specificity