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Detection of Exogenous Recombinan DNA of Transgenic Rice and Its Rice Product

Author: SongShangXin
Tutor: ZhouGuangHong
School: Nanjing Agricultural College
Course: Animal Nutrition and Feed Science
Keywords: Transgenic rice DNA extraction multiplex PCR food processing DNA degradation
CLC: S511
Type: Master's thesis
Year: 2011
Downloads: 13
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Abstract


The purpose of study was to establish PCR methods for the detection of exogenous recombinant DNA in transgenic rice Xiang 125S/Bar 68-1, and investigate the degradation of exogenous recombinant DNA in the course of rice product processing. It will provide the technological and theoretical basis for the detection, safety evaluation and tracing research of transgenic rice in food chains. The experiment included there parts:The first experiment was designed to establish a simple, fast and effective DNA extraction method of transgenic rice, which will provide the technological basis for the establishment of PCR detection method of transgenic rice. In this study, traditional CTAB method, improved alkali-treated method and boiling-treated method were used to extract DNA from transgenic brown rice, non-transgenic brown rice and transgenic milled rice samples. For each method, six biological replicates were prepared for each sample. According to the results, the integrity, purity and PCR result of template DNA extracted by improved alkali-treated method were similar with the DNA extracted by traditional CTAB method. Meanwhile, compared with traditional CTAB method, improved alkali-treated method had higher DNA extraction rate, less consuming time and lower cost. Therefore, improved alkali treated method can replace traditional CTAB method as DNA extraction method for transgenic rice.The second experiment was designed to establish a multiplex PCR dedection method for transgenic rice, which will provide the theoretical and technological basis for fast detection of transgenic rice and its rice product in food chains. In this study, four pairs of primers for the amplifications of SPS gene, CAMV35S promoter, Bar gene and NOS terminator were designed. The multiplex PCR dedection method was finally established by optimizing the concentration of primers and annealing temperature. The reaction system of multiplex PCR consists of 12.5μL of PCR Mix, the addition volum of primers for CAMV35S, Bar, SPS and NOS were 0.5,0.2,1.0 and 0.5μL, respectively,100 ng DNA template, add sterile H2O to 25μL. The reaction condition is as follows:the sample solution was kept at 94℃for 5 min, subjected to 40 cycles of amplification (94℃for 1 min,57℃for 1 min, and 72℃for lmin). and kept at 72℃for 7min. According to the results, this mutiplex PCR method has a good specificity, and can be used for the detection of herbicide-tolerant rice and its rice product as sweet rice wine.The third experiment was designed to examine the degradation of recombinant DNA of transgenic rice in the course of rice product processing, which will provide the technological and theoretical basis for the detection and safety evaluation of transgenic rice in food chains. In this study, sweet rice wine and rice cracker were made by using transgenic rice as main material, and genomic DNA was extracted from food samples by alkaline lysis or CTAB method. The degradation of SPS gene, CAMV35S promoter, Bar gene and NOS terminator were detected by using different fragments-size PCR method. It was found that the purity, quantity and quality of genomic DNA by alkaline lysis method was higher than by CTAB method. For sweet rice wine, the endogenous gene SPS was degraded on the first day of fermentation, and CAMV35S promoter and NOS terminator were degraded by the third day, whereas the exogenous Bar gene remained unaffected. The largest detectable fragments of SPS, Bar, CAMV35S and NOS were 916,568,446 and 180 bp, respectively. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. The maximal detectable fragments of SPS and CAMV35S after frying were 168 and 446 bp, but Bar and NOS were not detectable with primers in this study. In conclusion, sufficient DNA for PCR can be quickly extracted from transgenic rice food by alkaline lysis method. The stability of recombinant DNA in transgenic was different under different processing conditions.

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