Analysis of Insertion Copy Number and Integration Site of T-DNA in the Genome of Transgenic High Oelic Rapeseed (Brassica napus L.)  

Song Chen , Jiefu Zhang , Huiming Pu , Aijuan Shen , Xiaoying Zhou , Weihua Long , Maolong Hu , Cunkou Qi
Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, P.R. China
Author    Correspondence author
Plant Gene and Trait, 2011, Vol. 2, No. 3   doi: 10.5376/pgt.2011.02.0003
Received: 09 Nov., 2011    Accepted: 10 Dec., 2011    Published: 17 Jan., 2012
© 2011 BioPublisher Publishing Platform
This article was first published in Molecular Plant Breeding 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:

Chen et al., 2011, Analysis of Insertion Copy Number and Integration Site of T-DNA in the Genome of Transgenic High Oelic Rapeseed (Brassica napus L.), Plant Gene and Trait, Vol.2, No.3 15-22 (doi: 10.5376/pgt.2011.02.0003)

Abstract

In order to acquire the information about the insertion copy number and integration site of T-DNA in the genome of transgenic high oleic acid rapeseed (Brassica napus L.), we isolated the genomic DNA from the transgenic line W-4, the T2 generation plants, then the transgenic genomic DNA was digested with BamHI prior to conducting Southern bolt with the probe of a segment of NPT II labeled with Dig. The results showed that only one copy of T-DNA was detected to be integrated into the genome of transgenic line W-4. To isolate the flanking sequence of both right border and left border of T-DNA insertion in the genome, the thermal asymmetric interlaced PCR (TAIL-PCR) was employed by using three or four nested specific primers designed based on the sequence of the vector pCNFIRnos and a short arbitrary degenerate primer (AD1 or AD2) , respectively. The PCR products corresponding to flanking sequences of the right and left border were specifically amplified. The flanking sequence of right border is 470 bp in length which includes a 290 bp genomic sequence and a 180 bp vector sequence based on analysis of VecScreen, while the left border flanking sequence is 641 bp in length including a 365 bp genomic sequence and a 276 bp vector sequence. Further sequence alignment analysis revealed that the 180 bp sequence is identical to the RB border of pCNFIRnos vector, which exists a 62 bp deletion, whereas the later of 276 bp is better identical to the left border of pCNFIRnos vector besides a change from G to A happened. Therefore, it was suggested that the integration of the T-DNA in the genome of transgenic line W-4 should be a kind of vector backbone-free integration. Furthermore, there is no any information about homologous sequence of acquired flanking sequences found in the genome based on the Blastn analysis, which implied that the T-DNA should be integrated into non-coding region of the genome. In conclusion, we considered that the information about the T-DNA insertion copy number, integration site and flanking sequences in the genome of the transgenic rapeseed W-4 might be very useful for the biosafety assessment of genetically modified rapeseed and for the indentification of the transgenic high oleic acid rapeseed.

Keywords
Rapeseed (Brassica napus L.); Transgenic high oleic acid rapeseed; Gene insertion copy; Integration site; T-DNA
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