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Abstract In order to execute the labeling regulations and solve the inadequate availability of positive plant genome DNA, the existence of insect resistant rice (Oryza sativa L.) Bt Shanyou 63 has to be monitored, In this study, the complete sequence of foreign DNA insertion of transgenic cry1Ab/cry1Ac fusing gene rice line Bt Shanyou 63 transformed by particle bombardment method was determined using thermal asymmetric interlaced PCR (TAIL-PCR), Genome Walking and long distance PCR (LD-PCR). The Real-time PCR assay was established using the standard plasmid pMDBt63 containing 3' flanking sequence of Bt Shanyou 63 and rice endogenous gos9 as the reference material. The length of the complete sequence of foreign DNA was 9 818 bp, located at position 5348630 of chromosome 10 (AP008214) in rice genome and composed of eight recombinant fragments including two expression boxes of cry1Ab/cry1Ac fused gene at tail to head direction. The square regression coefficients (R2) of the two standard curves for gos9 and 3' flanking sequence were 0.9997 and 0.9992 and the efficiencies (E) were 98.05% and 99.46%, respectively. The detection limit (LOD) was 10 copies and the quantitative limit (LOQ) was 100 copies in 100 ng of DNA template for one reaction. In addition, two mixed samples with known Bt Shanyou 63 contents (5% and 1%) were quantified using the established Real-time PCR and the mean values were 4.98% and 1.07%, respectively. The above results showed that the established method employing pMDBt63 as the reference material can be used reliably for Bt Shanyou 63 quatification.
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Received: 21 October 2010
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