Establishment Of Genetically Modified Crops And Products Of Exogenous Resistance Gene Detection Technology System

Since the first genetically modified crop tobacco came into being in the word in 1983, the genetically modified crops as a cluster of highest economic return crops have been widely spread in an incomparable speed throughout the globe, however, the ecological risk, food safety anxiety, intellectual property rights and product tagging etc. have been on debate. Thus, the detection for genetically modified crops is urgently needed.In this paper, 73 lines in 16 kinds of resistance gene plasmid schemes contained in the genetically modified crops were collected and classified. Based on the information, the researches on advanced DNA molecular detection approaches were carried out: by using reverse PCR cloning-sequencing modified gene decoding to obtain differentiating flanking gene and modified gene sequence and modified gene sequences, designing specific primer to amplify specific region by conventional PCR(qualitative detection) and by real time PCR (qualitative and quantitative detections) and high density genetic chip multi-crop line detection, those newly developed detection protocols were for the first time in the world utilized to detect exogenous resistance genes in genetically modified crops and products, which have momentous meanings for detecting exogenous resistant genes in imported and exported GMO and products, tagging GMO and products, removing farm crop product trade barriers and drawbacks imposed by developed nations and judging international disputes on agricultural crops and products in accordance with principle of WTO.Crop line differentiating flanking sequences of 6 lines of genetically modified (GM) maize (MON810, BT11, BT176, GA21, T25 and CBH-351) and 1 line of GM soybean GTS 40-3-2, 2 lines of GM canola (RT73 and MS8) and 2 lines of GM cotton (MON531 and MON1445) were known using inverted PCR cloning-sequencing approach to determine unknown sequences. Sequences of modified genes GOX and CP4-EPSPS in GM canola and that of modified gene Cryla(c) in GM cotton were decoded and the conservative sequences of exogenous resistant genes : PLRVrep, PVYcp and CryIIIA in GM potato: New leaf?PLUS and New leaf?Y were confirmed using the modified exogenous gene decoding technique and isogenous sequence similarity BLAST analysis. Bases on DNA sequences studied above, conventional PCR primers were designed and selected in an optimized way and the conventional PCR detection protocol for exogenous resistant genes in 19 GM crops was established. Based on crop line differentiating flanking sequences and crop line specific resistant genes in GMO, specific primers and probes suitable for real time PCR detection were designed and selected.Different protocols for real time PCR with probe Taqman and fluorescein SYBR瓽reen and three quantitative detection methods for GMO were systematically compared, from these studies, the real time PCR detection protocol for 11 resistance genes and the quantitative protocol based on standard molecular plasmids in GMO were established.In the study of genetic chip of resistance gene detection 29 primers and 29 probes which are uniform in PCR detection profile and condition were selected and optimized, which could allow 10 pairs of primers in one multiple PCR reaction to perform amplifying reaction simultaneously, so the interaction interferences between primer pairs in the process of multiple PCR reaction condition optimization , between every pair of primers and products of multiple PCR reaction and between oligonucleotide length and structure of primers, probes and fluorescein labeled products were overcome. The studies shown above clearly indicated that oligonucleotide chip was better than cDNA chip in consideration of exogenous gene detection in GMO. Further more, taking the advantage of the special traits of common exogenous resistance genes, species specific genes and crop line differentiating Flanking sequences, the GMO and product sieving detection chip, species structural gene detection chip, crop line differentiating chip and 32 gene integrated detection chip were...