Construction of the Plasmid Reference Molecule for Detection of Transgenic Soybean MON89788

[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.

Analysis of the Flanking Sequence and EventSpecific Detection of Transgenic Line W-4 of Brassica napus

The genetically modified high-oleic rapeseed （Brassica napus L.） line W-4 was obtained by transforming a binary vector which harbored an inverted repeat expression cassette of fad2 gene into the rapeseed cultivar Westar.The transformation was mediated by Agrobacterium.The flanking sequences to both the left and right borders of T-DNA insertion site were amplified by thermal asymmetric interlaced PCR （TAIL-PCR） from the genomic DNA of the transgenic rapeseed line W-4.The flanking sequences to the right border was 290 bp in length and the nucleotide composition was 31.27％ for G＋C content while 68.73％ for A＋T content.The flanking sequence to the left border was 365 bp in length and the G＋C content was 32.6％ and the A＋T content was 67.4％,indicating that the T-DNA was integrated in the A/T-rich region.Further more,sequence alignment analysis showed a deletion of 62 bp including the right border of pCNFIRnos and the integration of the whole left border except a change of G to A.That was to say,the integration of the T-DNA in the transgenic line W-4 not involved in the vector sequences.Based on both flanking sequences as well as the left and right borders of the T-DNA sequences,two pairs of specific primers TLF/TLR and TRF/TRR were designed.Using the primers the event-specific PCR detection method for transgenic rapeseed line W-4 was established.By the PCR,two fragments of 485 and 405 bp were amplified from the W-4 genomic DNA as expected,while no products were amplified from the genomic DNA of other transgenic rapeseed lines and non-transgenic rapeseed line.And by the PCR it is possible to detect the W-4 genomic DNA from a mixed sample of genomic DNA.The limit of the detection for the qualitative PCR assay was 0.1％.The method developed in this work is highly specific,sensitive and suitable for event-specific detection of the transgenic rapeseed line W-4.