| In transgenic detection field, reference plasmid has been becoming a preferable reference material(RM) with the advantage of easier availability, higher purity, lower cost and better stability, which is more suitable for the detection of multi-target genes. In order to overcome the problem of inadequate availability of positive plant materials, the present study developed a set of four reference plasmids that could be used for PCR detection of products derived from transgenic rice and cotton. Four transgenic rice lines and 16 transgenic cotton varieties were used to evaluate the potential suitability of the constructed reference plasmids for transgenic detection in this study. The main results are as follows:1. The reference plasmid pMD-KF6 suitable for the event-specific detection for transgenic rice (Oryza sativa L.) Kefeng6 line was constructed and the real-time quantitative PCR method was established. The plasmid was 3100 bp in length constructed by inserting gos9 sequence and 5?-junction of Kefeng6 into the cloning site and HindIII restriction site in pMD-18T vector, respectively. The event-specific real-time PCR based on the 5?-junction of Kefeng6 products was developed. The square regression coefficients(R2) of the two standard curves for 5?-junction and gos9 were 0.999 and 0.998, and the efficiencies (E) were 97.2% and 96.8%, respectively. The limit of quantification was 10 copies. Thereafter, two different transgenic amounts of mixed samples (5%, 1%) were quantified to assess and verify the performance characteristics of the established real-time PCR. The accuracy expressed as biases were 4.0% and 17.0%, the precision expressed as relative standard deviation (RSD) were 3.96% and 3.42%. Concluded from above results, the developed quantitative PCR assays could be used for the event-specific quantitative detection for Kefeng6 products, and the reference plasmid pMD-KF6 could be a substitute for Kefeng6 genomic DNA as the RM.2. The reference plasmid pMDBt63 suitable for the event-specific detection for transgenic rice Bt Shanyou63 line was constructed and the real-time quantitative PCR method was established. The plasmid was 3137 bp in length constructed by inserting gos9 sequence and 3?-junction of Bt Shanyou63 into the cloning site and the EcoRâ… restriction site in pMD-18T vector, respectively. The event-specific real-time PCR based on the 3?-junction of Bt Shanyou63 products was developed. The square regression coefficients(R2)of the two standard curves for 3?-junction and gos9 were 0.9997 and 0.9992, and the efficiencies (E) were 98.05% and 99.46%. The limit of detection and the limit of quantitative were 10 copies and 100 copies, respectively. Thereafter, two different transgenic contents of mixed samples (5%, 1%) were quantified to assess and varify the performance characteristics of the real-time PCR. The accuracy expressed as biases were 0.02% and 0.07%, the precision expressed as relative standard deviation (RSD) were 1.23% and 5.16%. Concluded from above results, the developed quantitative PCR assays could be used for the event-specific quantitative detection for Bt Shanyou63 products, and the reference plasmid pMDBt63 could be a substitute for Bt Shanyou63 genomic DNA as the RM.3. We attempted to construct the reference plasmid pMD68DH suitable for the event-specific detections for Kemingdao, Kefeng8, Kefeng6 and Bt Shanyou63 was constructed and established the real-time quantitative PCR method considering the requirement of detection for multiple targets. The plasmid with was 5152 bp in length constructed by inserting 5? and 3?-junction of rice genome at the insertion site of these four transgenic rice lines and rice endogenous gos9, PLD, SPS fragments into the cloning site, EcoRâ… , BamHâ… and HindIII site in pMD-18T vector, respectively. In order to test the suitability of pMD68DH, Kefeng 8 and Kemingdao lines were used to develop the event-specific real-time PCR methods using gos9 endogenous gene with a limit of quantification of 10 template copies. Two mixed samples with known Kefeng8 and Kemingdao contents (5% and 1%) were quantified using the developed real-time PCR. The accuracy for KF8 expressed as bias were 21.6% and 7.0%, the precision for KF8 expressed as RSD were 2.3% and 7.48%. The accuracy for Kemingdao expressed as bias were 2.8% and 1.0%, the precision for Kemingdao expressed as RSD were 3.29% and 7.92%. Concluded from above results, pMD68DH was suitable for the transgenic rice products quantification as a substitute for the RM.4. Considering the requirement of the multi-target detection, the multi-target plasmid pMDMCS suitable for insect-resistant cotton (Gossypium hirsutum L.) products was constructed and the real-time quantitative PCR methods were developed targeting the major elements and foreign genes in China. The plasmid was 4309 bp in length constructed by inserting seven targets, i.e., CaMV35S promoter, NOS terminator, 7S terminator, pNOS promoter, nptII reporter , CpTI gene, cry1A gene and endogenous Sad1 gene into the cloning vector pMD-20T. The real-time PCR assays were developed based on the requirement of the detection technical for above targets such as cry1A gene, Sad1 gene and etc. The testing parameters of the standard curves met the requirements for quantification. The dosages of seven target genes for sixteen transgenic cotton varieties were determined using the developed assays. CaMV35S promoter, NOS terminator, 7S terminator, nptII reporter and cry1A gene were stable in many insect-resistant cotton varieties, but pNOS promoter and CpTI gene dosages were less than 0.3 copies.
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