4 - GMO-Vol.03-No.01页

GMO Biosafety Research 2012, Vol.3, No.1, 1-7

http://gmo.sophiapublisher.com

Research Report Open Access

Analysis of Integrated Structure of the Exogenous Gene Insertion and the

Establishment of Event-specific Detection Method in Transgenic Insect

Resistant Cotton

Na Hou

1, 2

, Huiqun He

2

, Mei Dong

2

, Rongqi Xu

2

, Yusong Wan

2

, Jin Wujun

2

, Liu Haobao

1

1. Tobacco Research Institute of CAAS, Qingdao, 266101, Shandong, P.R. China

2. Biotechnology Research Institute, CAAS, 100081, Beijing, P.R. China

Corresponding author email:

jinwujun1218@yahoo.com.cn; L88702236@163.com;

Authors

GMO Biosafety Research, 2012, Vol.3, No.1 doi: 10.5376/gmo.2012.03.0001

Received: 04 May, 2012

Accepted: 18 Jun., 2012

Published: 20 Jun., 2012

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)

Abstract

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

Npt

Ⅱ

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.

Keywords

Transgenic resistant cotton; Integrated structure; PCR detection; Event-specificity

Since the transgenic cotton initiated to study in 1989,

China has made great achievements in the field of

genetically modified cotton research. The domestic

transgenic

Bt

cotton being successful developed in

1994 made China become the second country

succeeding in the United States who had independent

intellectual property rights of the insect-resistant genes

(Cui and Guo, 1996; Mao and Guo, 1998; Huang et al.,

1998). With the successful development of the

bivalent transgenic cotton harboring Bt and CpTI in

1996 (Guo and Cui, 1998; Cui and Guo, 1998; Guo et

al., 1999), which not only delays the bollworm’s

resistance to Bt protein but also enhances the toxic to

insect, China entered the international advanced level

in the field of cotton research. It is said that the

Bt

cotton made by China occupied for more than 93

percent of China's

Bt

cotton market in 2010.

1

In recent years, with the continuous expansion of the

transgenic

Bt

cotton planting area, safety assessment

and supervision of genetically modified crops have

been maturing in China. Therefore, it has become

increasingly urgent to study the integrated structure of

the exogenous gene and to establish a scientific testing

method in the events of genetically modified crop

varieties (lines). Guo and Zhang (2010) conducted a

preliminary study to obtain the flanking sequence of

left and right borders of T-DNA insertion sites by

TAIL-PCR, which the exogenous DNA fragments

were transformed by cotton pollen tube pathway

approach into; Cui (2004) preliminary analyzed

integration flanking sequence of insecticidal gene in the

Bt cotton GK12, providing a reference for the integration

mechanism of the insecticidal gene; Wang (2011)

obtained flanking sequences in EZaMian 1Hao by the

Genome Walking combined with the long-chain method,

and further screened and identified strain-specific

detection primers. However, the complete structure of

the inserted fragment did not report yet.

At present, the most popular methods for acquiring the

flanking sequence of the foreign gene are the target

genome walking PCR (Parker et al., 1991), and

thermal asymmetric interlaced PCR ( TAIL-PCR) that