Safety Assessment Of Bar-transgenic Rice With Herbicide Resistance Using A Mouse(Mus Musculus) Model

Applying the methods of ethology, environmental ecology, toxicology, physiology, Genetics, developmental biology, proteomics and mathematical ecology, comprehensive assessment of food safety of Bar-transgenic rice with herbicide resistance was made using Mus musculus as animal model in this study on physiology and biochemistry, toxicology and pathology, reproductive development, genetic diversity and so on.1. Kunming mice(Mus musculus) of200SPF-grade (20g±2g), half of which were male and the other half female, were randomly divided into five groups with four replications per group and ten mice per replication to assess the safety of Bar-transgenic rice. They were fed with diets containing low and high doses of genetically modified (GM) Bar68-1rice, D68(non-GM) rice, and routine feed for180days. After90days, parental generation (P) was bred to produce the first filial generation (F1), and then F1, which was fed for90days, produced the second filial generation (F2). Each generation was fed for180days. On the180th day, ten mice from each group were randomly sampled, and organ coefficient, and blood biochemistry values were quantified. The quantification of these chemicals was done for three generations of mice. The results indicated that no significant difference in organ coefficient, and blood biochemistry values between the Bar68-1GM rice group and the non-GM D68rice groups (P>0.05), and the Bar-transgenic rice did not have allergenic effects on the growth, development of organs and blood biochemistry values.2. To assess the safety of Bar-transgenic rice, its allergenicity were tested on120Kunming mice (Mus musculus). The test mice were randomly divided into five groups with four replications per group and six mice per replication, and were given diets containing varying doses of genetically modified (GM) Bar68-1rice, D68(non-GM) rice, and routine feed. Database SDAP, Farrp and NCBI were used in protein sequence alignment of PAT, and the three-dimensional conformation of PAT was formed and analysed in SWISS-MODEL. On the180th day,6mice from each group were sampled at random and the IgE and DAO levels in their serum and the sIA in their small intestinal mucus were quantified. The quantification of these chemicals was done for three generations of mice. The results indicated that no significant difference in the serum sIgA, DAO, and IgE between the Bar68-1GM rice group and the non-GM D68rice groups (P>0.05), and the Bar-transgenic rice did not have allergenic effects on the mice. Besides, there was no positive match through the protein-bioinformatics comparison, PAT had no homology with any of the known allergens. The three-dimensional conformation inspection indicated that PAT had a similar structure with other members in NATSF. There was little possibility to cause anaphylactic reaction. we could get a conclusion that Bar-transgenic rice had no obvious allegenicity.3.100Kunming mice (Mus musculus) were randomly divided into five groups of twenty mice each and were given diets containing varying doses of genetically modified (GM) Bar68-1rice, D68(non-GM) rice, and routine feed. On the180th day,6mice from each group were sampled at random to detecte the existence of Bar-gene segments and PAT in mice crureus, liver, kidney, spleen, small intestine to discuss the residue of the genetically modified component in the body metabolism. Besides, the preliminary investigation about the degradation of foreign protein in mice digestive system was discussed. Four pairs of oligonucleotide primers were designed for the12S rDNA and16S rDNA conserved region of the mice intestinal mitochondrial DNA (mtDNA), the PCR amplicons were examed for mutation study. It showed a negative result of the existence of Bar-gene segments and PAT enzyme in all parts of mice body that mentioned above. The foreign protein digestive experiment indicated that PAT were degradable in mice gastrointestinal tract, it had no tolerance for the digestion. No mutational site was found in the12S rDNA and16S rDNA conserved region of the intestinal mtDNA. 4. Microbial molecular ecology approaches were used to the effects of Bar-transgenic rice on Intestinal Microflora of the Mice(Mus musculus). Kunming mice(Mus musculus) of120SPF-grade (20g±2g), half of which were male and the other half female, were randomly divided into five groups with four replications per group and six mice per replication to assess the safety of Bar-transgenic rice. Five diets meetinging or exceeding the minimum nutrient requirement were fed for180days. After90days, parental generation (P) was bred to produce the first filial generation (F1). Each generation was fed for180days. On the180th day, six mice from each group were randomly sampled, and their intestinal contents were collected for DNA isolation. The V3region of the16S rDNA was amplified by polymerase chain reaction (PCR) and analyzed via denaturing gradient gel electrophoresis (DGGE). The resulting PCR-DGGE band number(bacterial species) was counted, and the banding patterns were analyzed by calculating the Sorenson's pairwise similarity coefficients (Cs), an index used to measure bacterial species found among all samples. The sequence analysis of bands was performed to identify the intestinal predominant microflora of the mice. The intergroup Cs values of the samples across all groups did not differ (P>0.05) from each other. The effect of Bar-transgenic rice on the intestinal microflora of the mice was considered insignificant.5. To investigate the cytotoxicity of Bar68-1genetically modified(GM) rice in Mus musculus lymphocytes with different assays in vitro. Mus musculus lymphocytes were exposed to whole protein of Bar68-1GM rice at doses of25,50,100and200μg/mL and incubated for2h,6h and24h, respectively. The cytotoxicity induced by whole protein of Bar68-1M rice were measured by CCK-8assay and neutral red uptake (NRU) assay. After different incubation periods, the survival rate of lymphocytes in positive control group was significantly less than that of lymphocytes in blank control group(P<0.05). Moreover, the exposure time-effect relationship was observed in positive control group with CCK-8assay and neutral red uptake (NRU) assay. There was no significant difference in survival rate between Bar68-1GM rice group and non-GM rice group(P>0.05). Also, the Bar68-1GM rice group did not show higher survival rate than that of D68non-GM rice group(P>0.05). The results of this study indicated that Bar68-1GM rice had similar cytotoxicity with D68non-GM rice, and Bar68-1GM rice had no cytotoxicity effect on Mus musculus lymphocytes in vitro.6. To study the influence of long-period PAT force on genetic polymorphism of mice (Mus musculus), the genetic polymorphism of the second filial generation(F2) mice fed with of Bar-transgenic rice were analysed. F2mice (Mus musculus) of36SPF-grade (20g±2g), half of which were male and the other half female, were randomly divided into one experimental group (Group Z2, fed with rice Bar68-1)and one control group (Group C2, fed with rice D68) with three replications per group and six mice per replication. With six pairs of AFLP selective primers,108AFLP loci were recorded, of which25.9%were polymorphic. There was a tiny difference between liver and small intestine samples of Group Z2and Group C2based on dendrogram, but the difference wasn't obvious. The results indicated that the influence of long-period PAT force on genetic polymorphism of mice (Mus musculus) was insignificant.