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

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 Bam HI 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.