Detection Technology Transfer Of Genetically Modified Soybean And Its Processed Products

In recent years, a lot of agricultural enterprises have developed new plant varieties by adopting modern biotechnology, including genetic transformation. Labeling of food from transgenic crops has become one of the key issues dominating public concern due to their safety concerns, and laws or regulations have been issued to label the GMOs and their derived products in more than thirty countries. At present, the genetically modified (GM) Roundup-Ready soybean has entered Chinese market without labeling and claiming, which makes it necessary to confirm the existence of transgenic ingredient in food and the appropriate analytical methodology to detect these GM crops is becoming urgent and important.GM products contain an additional trait encoded by an introduced gene(s), which generally produce an additional protein(s) that confers the trait of interest. Raw material (e.g. grains) and processed products (e.g. foods) derived from GM crops might thus be identified by testing for the presence of introduced DNA, or by detecting expressed novel proteins encoded by the genetic material.Accordingly, in the dissertation here is composed of two related but different level parts, and GM soybean and soybean derived products have been used as the materials. One is the study of detection methods on GM soybean ?(Roundup-Ready soybean) and its derived processed foods at DNA-based level and the other is the study of the detection methods on GM soybean at Protein-based level.On the level of DNA-based, the method of extracting the DNA from soybean and its derived processed products such as bean cake, bean curd, soy milk and sauce was optimized firstly, and the high quality DNA that OD value is between 1.7-1.9 have been obtained. Subsequently, we established a method using the polymerase chain reaction (PCR) technique and Multiplex PCR reactions which could simultaneously amplify two fragments in one tube for detectingtransgenic component EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, obtained from A. tumefaciens strain CP4) and endogenous gene Lectin (used to identify the GM soybeans). According the specific sequence of EPSPS and Lectin gene, two pairs of primers were designed using Primer Premier 5. 0 software. The PCR analysis using the two pairs of primers showed that as little as 0. 01ng of genomic DNA could be detected in the soybean, and the two pairs of primers could successfully used for Multiplex PCR reactions to detect GM soybean and its derived processed products. Finally, Corresponding primers and probe were designed using Primer Premier 5. 0 and Beacon Designer 2.0 software to set up relative PCR cycling conditions for real-time fluorescence quantitative PCR ( Taqman PCR ), and established the standard curve to quantify the GM component of soybean. The Taqman PCR analysis showed that the sensitivities of the detection could reach as little as 0. Olng.The detection methods on protein-based level involve Western-blot analysis and ELISA (enzyme linked immunosorbent assays, ELISA).In order to preparation the monoclonal and polyclonal antibodies that could be used in the immunoassay, an E. coli expression vector p28aYTY containing EPSPS was constructed to produce abundance protein that would be used as antigen. The SDS gel electrophoresis analysis shows that the expression of EPSPS recombined protein is over 55% and the purified EPSPS recombined protein reached as 90%. Subsequently, polyclonal antibodies were obtained from the serum of immunised New Zealand rabbits. Western-blot analysis shows the polyclonal antibodies could be applied for the detecting of the expressed proteins encoded by the EPSPS of transgenic soybean.Seventeen hybridoma cell lines that could stably secret monoclonal antibody (McAb) against EPSPS recombined protein were established after fusion of mouse X63-Ag8. 6. 5. 3 meloma cells with spleen cells isolated from BAL b/c mice that had been immunized with the purified EPSPS recombined protein. All of the hybridoma cell lines can produce tumor ascites in Bal b/c mice whichpr...