The Real-time Fluorescence Quantitative PCR Detection Of Gm Maize MON88017 And Its Products Abstract

MON88017 was a kind of genetically modified maize discovered by the Monsanto Company through Agrobacterium mediated transformation method, which had transferred the plasmid vector PV-ZMIR39 containing two genes cry3Bb1 and cp4 epsps expression cassette into maize Hi-II cells. Molecular biological analysis on MON88017 confirmed that there was only a single copy of cp4 epsps and cry3Bb1 gene cassette integrated into a single point of the genome of MON 88017; various expression elements were intact; there was no bacterial plasmid backbone sequences inserted into MON88017 genome.In this study the genome walking method and nested PCR method were employed to determine the lead-in sites flanking sequence of MON88017 exogenous DNA, and flanking sequences of transformants under specific (event specific) PCR detection primers are used for qualitative PCR, further to determine the optimum conditions of primers so as to establish the transgenic maize lines transformants PCR method. Based on the Qualitative PCR, appropriate primers and probes were designed, and with the help of quantitative fluorescent real-time quantitative PCR, compared with the standard curve to determine the content of genetically modified products, a comprehensive anti-insect and herbicide-resistant corn MON88017 system and its quantitative detection of genetically modified products were established. Detailed results were as follows:(1) The left border flanking sequence 504bp of transgenic maize MON88017 foreign gene insertion site was obtained through the genome walking method and nested PCR method. Sequence analysis showed that from the 5' end to 168bp lays the maize genome sequence, and from 169bp to 3' end was the vector sequence that was inserted into the corn, among which 169bp~479bp were inserted sequences, while from 480bp to 3' end were promoter sequences P-ract1 for the rice.(2) According to the left border sequences of GM maize MON88017, three pairs of MON 88017 transformants specific primers were designed with Primer premier 5.0 software, and the three pairs of specific primers were screened and optimized with reaction conditions. The optimal specific primers MON88017-1F/R (446bp) were determined, and the optimal annealing temperature was 56℃. Through validation, the primers MON88017-1F/R had a high specificity and accuracy. Tests on different concentrations of DNA with specific primers MON88017-1F/R showed that when MON88017 DNA was diluted to 0.1%, specific fragment of 446bp could still be amplified, indicating that the transgenic maize minimum detection limit (i.e., sensitivity) was high to 0.1%. The qualitative PCR method could meet the specific detection requirements of transgenic maize lines transformants.(3) According to the left border sequences of GM maize MON88017, transformants specific quantitative primer MON88017-F/R (87bp) and Taqman probe MON88017-P were designed with Primer Express 2.0 software, through the screening and optimization of reaction conditions, the optimal annealing temperature was determined as 60℃. Real-time fluorescence quantitative PCR detection was conducted and Taqman probe quantitative PCR results showed that 0.01% of GM content could be detected. Therefore, it's more sensitive than qualitative PCR detection method. The results indicated that the quantitative PCR primers and probes were quite specific and the established Real-time fluorescence quantitative PCR detection system was accurate, reliable and sensitive.