Event Specific Detection Of Transgenic Rapeseed

According to the regulations of GMO biosafety management in China, transgenic crops are not allowed import and cultivation without obtaining the biosafety certificate. The transgenic varieties that have been approved for commercialization, are required to provide methods of detection and monitoring. In 2004, six varieties of transgenic rapeseed (MS1×RF1, MS1×RF2, MS8×RF3, OXY235, Topas19/2 and T45) developed by Bayer Cropscience and one variety RT73 (GT73) from Monsanto company were permitted to import into China as processing materials. With the introduction of the labelling policy of genetically modified products, it is extremely urgent and important to establish the qualitative and quantitative detection methods for genetically modified products. Using the six transgenic rapeseed varieties (MS1×RF1, MS1×RF2, MS8×RF3, OXY235, Topas19/2 and T45) as experimental materials, this study isolated and characterized the junction fragments between insert DNA and plant genomic DNA in 8 transgenic rapeseed lines by means of the GenomeWalker strategy. And according to the isolated junction fragment, the event-specific qualitative and quantitative detection methods were established for the transgenic lines MS1, RF1, RF2, MS8, RF3, OXY235, Topas19/2 and T45. The specificity, sensitivity, repeatability, accuracy and reliability of each detection system were evaluated.1. The size of MS1 right border (5'-) junction fragment was 1165 bp, based on which, the event-specific primer pair MS1RG/MS1RV was designed, generation an amplicon of 141 bp. The detection sensitivity of MS1 qualitative PCR assay were 2.5-copy (0.013%), the correlation coefficient value (R2)for the MS1 standard curve was 0.995, the limit of detection (LOD) and limit of quantification ( LOQ ) of the real-time PCR assay were 2.5 and 25 copies , respectively.2. The fragment size of RF1 left and right border (3'- and 5'-) flanking sequence was 1592 bp and 3192bp, respectively. The event-specifc qualitative primer sets RF1RG/RF1RV and RF1LG-F/RF1LV-R produced amplicons of 99 bp and 284 bp, respectively, the detection sensitivity of conventional PCR assays was 5 copies (0.013%) for both primer sets. The quantitative primer sets RF1RG/RF1RV and RF1LG/RF1LV amplified 99 bp and 117bp, respectively. The R2 value for the two RF1 standard curves were 0.996 and 0.988. The LOD values of real-time assays were 5 copies and the LOQ values were 25 copies.3. The fragment size of RF2 right border (5'-) flanking sequence was 2978 bp. Based on the RB junction fragment, the RF2 event-specific PCR assay was developed with the primer pair RF2RG/RF2RV producing 138 bp of band. The detection sensitivity of RF2 qualitative assay was 5 copies (0.013%). The R2 value for RF2 standard curve was 0.998. The LOD and LOQ of quantitative assay were 5 and 25 copies, respectively.4. 907 bp of RB (3'-) junction fragment was obtained from MS8 rapeseed, the event-specific primer pair Ms8RG/Ms8RV was designed, and used to generate an amplicon of 123 bp. The detection sensitivity of MS8 conventional PCR assay was 2.5 copies (0.013%). The R2 value for the MS8 standard curve was 0.991. The LOD and LOQ values of real-time PCR were 20 and 25 copies, respectively.5. The fragment size of RF3 right border (3'-) flanking sequence was 694 bp and the fragment size generated by the event-specific primers Rf3RG/Rf3RV was 92 bp. The detection sensitivity of RF3 conventional PCR assay was 50 copies (0.13 %). The R2 value for the RF3 standard curve was 0.995, and the LOD and LOQ values being 20 and 25 copies, respectively.6. Both the left and right border (5'-and 3'-) junction fragments were both obtained for OXY235 rapeseed.The fragment spanning plant DNA and left border was 923 bp in length and that spanning plant DNA and right border was 1109 bp. OXY235 event-specific PCR assay was established, with one primer pair OXYLG/OXYLV producing 105bp product, with another primer pair OXYRG/OXYRV producing 124 bp amplicon. The detection sensitivity was 100 copies (0.13%) for primer set OXYLG/OXYLV and 10 copies (0.013%) for OXYRG/OXYRV. The R2 value for the established standard curves was 1.000 and 0.998 with the LOD values for both real-time PCR assay were about 10 copies and the LOQ values being 50 copies.7. 823 bp Topas19/2 LB (3'-) junction was isolated. The event-specific primers TOPLG/TOPLV produced 110 bp amplicon specific for the LB junction. The detection sensitivity of conventional PCR was 10 copies (0.013%), the R2 value for the established standards curve were 0.995, LOD and LOQ of real-time PCR were 5 and 50 copies, respectively.8. The fragment size of T45 left and right border (3'-and 5'-) flanking sequence was 2154bp and 1616bp, respectively. The primers T45LG/T45LV amplified a 107 bp fragment specific for LB (3'-) junction and T45RG/T45RV amplified a 117bp fragment specific for RB (5'-) junction. The fluorescent signal rapidly decreased when the primer pair T45RG/T45RV was used to amplify low copy number of templates. Therefore, the primer pair T45LG/T45LV wasselected to develope T45 event-specific assay, the R2 value for the standard curve was 0.996.The established event-specific qualitative detection method can identify one transgenic rapeseed line from others, and the quantitative detection methods can accurately quantify a specific transgenic rapeseed content in samples, providing technical support and scientific basis in safety management and the imminent implementation of labelling policy for genetically modified rapeseed in China.