Development And Application Of Standardized Detection Technology Of Main Commercialized Genetically Modified Organisms

As the fully development of genetically modified technology, a lot of genetically modified organisms (GMO) have already been commercialized, and traded into the global market. Many of them, such as soybean, maize and rapeseed have been traded into the China market. In order to intensify the safety administration of GMO, State Counsel of China issued the "Biosafety Administration Regulations on Agricultural GMO" on May 23th, 2001. And in Jan. 5th, 2002, Ministry of Agriculture issued the "Biosafety Evaluation and Administration Regulations on Agricultural GMO", "Safety Administration Regulations on Imported Agricultural GMO" and "Labeling Administration Regulations on Agricultural GMO" as the follow-up regulations, which demand the safety assessment for agricultural GMO before they enter the market, as well as the labeling administration for agricultural GMO that enter the circulation.1 The content of the safety assessment of GMOThe safety of GMO has already attracted a worldwide attention. The international organisms such as: Organization for Economic Co-operation and Development (OECD), International Food Biotechnology Council (IFBC), Food and Agriculture Organization of the United Nations/ World Health Organization (FAO/WHO) and The International Life Sciences Institute (ILSI), all commence establishing the biosafety assessment system for GMO.The contents of the safety assessment of GMO include: (l)Carefully check the background information of the GMO; (2)Establish the method for tests of GMO; (3)The safety concern for antibiotic resistance gene; (4)Toxicological problems; (5)Nutritional concerns; (6)The environmental risk of the GM crops.One of the key item in the safety assessment of GMO is the detection of GMO, which is the basis for safety assessment. The tests of GMO consists of qualitative and quantitative tests. The usual detection methods include: General PCR, Real-Time Quantitative PCR, gene chip and protein detection.2 The establishment of the method for screening test of GMOAmong the GM crops approved for commercial planting at present, over 95% containCaMV 35S promoter, Nos terminator or NPTII gene, thus the samples could be preliminarily screened as GMO by detecting these special genes. The study has set up the method of screening tests of CaMV 35S promoter, Nos terminator and NPTII gene.3 The establishment of the method for test of GM rapeseedThe Phosphoenolpyruvate-Carboxylase (PE3-PEPCase) gene was chosen as endogenous reference gene of rape.Roundup-ready raprseed is one of a few commercial GMO without CaMV35S promoter, Nos terminator and NPTII gene. The study has established the detection method for exogenous FMV 35S promoter, modified CP4-EPSPS gene and modified GOX gene in GM Roundup-ready raprseed, that by means of: (1) Compared the gene sequences of FMV 35S promoter and FMV 34S promoter; (2)Compared the nucleotide sequences as well as the amino acid sequences expressed by CP4-EPSPS gene transferred into RT73 rape and by agrobacterium CP4-EPSPS gene transferred into 40-3-2 soybean; (3)Compared the nucleotide sequences as well as the amino acid sequences expressed by modified GOX gene and GOX gene.The rape genome sequences of both flanking region of the insert site are specific because the exogenous gene was inserted into the crops genome randomly during the genetically modified manipulation. Using Thermal Asymmetric Interlaced PCR (TAIL-PCR), the rape genome sequence near the FMV 35S promoter on the insert site of RT73 transgenic glyphosate-tolerant raprseed was identified, and the method for event-specific detection of RT73 was set up by designing an primer based on the genome sequence of rape, and another primer based on the FMV 35S promoter sequence. The PCR products contain not only the sequence of rape genome, but also the sequence of exogenous promoter gene. Because of the specificity of target sequence, the false-positive results that caused by the factors such as infection of plant Cauliflower Mosaic Virus could be excluded as well.In addition, the methods for detection of th...