【作者】 刘金；

【导师】 颜亨梅；

【作者基本信息】 湖南师范大学 ， 动物学， 2012， 博士

【摘要】 本研究以昆明小鼠为动物模型(animal model),应用行为学、环境生态学、毒理学、生理学、遗传学、发育生物学、蛋白质组学和数理生态学等研究方法,从生理生化、毒理病理、生殖发育和遗传多样性等方面对转Bar基因抗除草剂稻谷的安全性进行了综合评价。1.选取6周龄、体重18-22g的SPF级昆明小鼠200只,按编号随机分成5组,每组4个重复,每个重复10只小鼠,雌雄各半。用不同含量(40%和60%)的转Bar基因抗除草剂稻谷及其亲本非转基因稻谷日粮分别喂养小鼠,亲代小鼠饲养90天后开始繁殖子一代(F1),子一代饲养90天后开始繁殖子二代(F2),每代小鼠饲养180天。在180后分别从亲代、子一代和子二代每组按编号随机抓取10只小鼠对其血常规、血生化指标和主要脏器指数进行了检测。结果显示,P、F,和F2三代小鼠中,各处理组间器官指数和血生理生化指标均不存在显著差异(P>0.05),转Bar基因抗除草剂稻谷对小鼠的生长、器官发育以及血生理生化未产生显著影响。2.将120只SPF级昆明小鼠分为5组,每组4个重复,每个重复6只小鼠,雌雄各半,分别饲喂含不同剂量转基因稻谷Bar68-1和常规非转基因稻谷D68180天,持续3代。采用SDAP、FARRP和NCBI三大数据库对磷丝菌素乙酰转移酶(PAT)进行致敏原序列对比,并用蛋白质结构模拟SWISS-MODEL预测PAT的三维构象并对其进行分析。每组按编号随机抽样6只小鼠,用ELISA分别检测其肠道粘液免疫球蛋白A(sIgA)、血清二胺氧化酶(DAO)和血清免疫球蛋白E(IgE)。蛋白质生物信息学比对结果表明,PAT酶与数据库中已知致敏原无同源性,三维构象亦表明其与其他N-乙酰转移酶大家族(NATSF)成员相似,无致敏性；转基因组与非转基因组小鼠比较,sIgA、 DAO和IgE三项检测指标值均无显著性差异(P>0.05)。结果显示,转Bar基因的稻谷对小鼠无明显致敏性。3.选择体重在18～22g SPF级昆明小鼠100只,按编号随机分成5组,分别饲喂含不同剂量(40%和60%)的转基因稻谷Bar68-1和常规非转基因稻谷D68180天,持续2代。180天后每组按编号随机抓取6只小鼠,分别检测其腿肌、肝脏、肾脏、脾脏、小肠中是否含有Bar基因片段和其表达蛋白——磷丝菌素乙酰转移酶(PAT),进而探讨转基因成分在小鼠体内的代谢残留状况；同时,还进行了小鼠消化道内对外源蛋白消化降解情况的初步检测,并对小肠线粒体(mtDNA)基因组12S rDNA和16S rDNA保守区域测序。结果表明：采用定性PCR法检测的实验组小鼠各脏器组织中没有发现Bar基因片段和其表达的PAT蛋白酶；而小鼠消化道外源蛋白检测结果表明PAT酶在胃肠道内无耐受性,外源蛋白成分能够被机体完全消化；小鼠小肠线粒体(mtDNA)基因组12S rDNA和16S rDNA保守区域的测序结果也无异常,没有发现突变点。4.选取6周龄、体重18-22g的SPF级昆明小鼠120只,按编号随机分成5组,每组4个重复,每个重复6只小鼠,雌雄各半。用不同含量(40%和60%)的转Bar基因抗除草剂稻谷和非转基因稻谷日粮分别喂养小鼠,亲代小鼠饲养90天后开始繁殖子一代(F1),每代小鼠饲养180天。在180后分别从亲代和子一代每组按编号随机抓取6只小鼠,提取肠道内容物基因组DNA,利用细菌通用引物对细菌16S rDNA的V3区序列进行PCR扩增,并将扩增产物用变性梯度凝胶电泳(DGGE)后进行比较、分析,并对小鼠肠道微生物主要优势菌群进行了鉴别。结果显示,饲喂转Bar基因抗除草剂稻谷和非转基因稻谷日粮的小鼠肠道细菌种类和数量相似度大,无显著差异(P>0.05),转Bar基因抗除草剂稻谷对小鼠肠道微生物区系的种类、数量和分布没有产生显著影响。5.通过体外实验评价转基因大米Bar68-1的细胞毒性,分别以25、50、100和200μg/mL转基因大米Bar68-1全蛋白作用于昆明小鼠淋巴细胞,并各孵育2h、6h、24h,然后用CCK-8及中性红摄取试验检测细胞毒性大小。在经过不同的孵育时间段后,阳性对照组淋巴细胞的细胞存活率与空白对照组相比,存在显著差异(P<0.05)。其中,CCK-8试验、中性红试验测得细胞存活率存在着明显的损伤作用-时间效应关系。转基因大米Bar68-1全蛋白暴露组淋巴细胞存活率与非转基因大米D68全蛋白暴露组淋巴细胞相比无明显差异(P>0.05),且与空白对照组细胞差异不显著(P＞0.05)。结果显示,转基因大米Bar68-1与非转基因大米D68两者在细胞毒性上性状相似,实质等同,对淋巴细胞无明显毒性作用。6.为探测PAT酶长期胁迫下小鼠遗传多态性的变化,应用AFLP技术对饲喂转Bar基因抗除草剂稻谷子二代(F2)小鼠遗传多样性进行了研究分析。36只体重18-22g的F2代小鼠分为实验组(Z2)和对照组(C2),分别饲喂Bar基因稻谷Bar68-1和非转基因稻谷D68。每组3个重复,每个重复6只小鼠,雌雄各半。6对引物组合扩增出108个AFLP条带,其中多态性条带占25.9%。在聚类图上Z2组肝脏和小肠样品和C2组肝脏和小肠样品有微小差异,但差异不明显。结果表明,PAT对小鼠肝脏和小肠的DNA多态性没有明显影响。更多还原

【Abstract】 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.更多还原