【作者】 周兴华；

【导师】 董英；

【作者基本信息】 江苏大学 ， 食品科学， 2012， 博士

【摘要】 水稻(Oryza sativa L.)是我国最重要的粮食作物之一,世界上近一半以上的人口都以大米为主食,因此大米的品质也越来越受到人们的关注。利用转基因技术对传统水稻品种进行改良,可以提高大米的营养价值或拓展其功能。但是,由于对转基因水稻的安全性一直存在争议,因此,有必要对转基因水稻的食用安全性进行深入系统的研究,为转基因水稻的发展提供科学依据。本论文以转GL融合蛋白基因和转双反义分支酶基因大米为研究对象,通过系统的毒理学试验研究与分析两种转基因大米的食用安全性,为转基因大米食用安全性评价技术体系的构建提供参考。主要研究内容与结果如下：1.对转双反义分支酶基因大米进行了急性毒性试验研究。将转基因大米以2.15、4.64、10.00和21.50 g/kg·bw的剂量灌胃小鼠,连续观察14天。结果表明,转基因大米的LD50>21.50 g/kg·bw;试验期间,动物状态良好,无死亡和异常现象；对各组小鼠体重、血常规、脏器系数和主要脏器病理学观察也未见显著性差异。因此,转双反义分支酶基因大米属无毒级别。2.对转双反义分支酶基因大米进行了遗传毒性试验研究。对转基因大米进行小鼠骨髓细胞微核试验、精子畸形试验、Ames试验和彷徨试验研究。结果表明,以上试验均呈阴性,未见转双反义分支酶基因大米产生遗传毒性。3.对转双反义分支酶基因大米进行了致畸毒性试验研究。用含有转基因大米的饲料喂养大鼠,观察和分析转基因大米对孕鼠生长及胎鼠发育的影响。结果表明,转基因大米对孕鼠体重增长、孕鼠生殖能力、胎鼠发育(外观、内脏及骨骼)均未产生不良影响,即转双反义分支酶基因大米未对试验大鼠产生致畸作用。4.对转双反义分支酶基因大米进行了90天喂养试验研究。用含有转基因大米的饲料喂养大鼠90天,对大鼠体重、进食量、进水量、血常规、血生化、尿常规、性激素、脏器系数及组织病理学进行检测。结果表明,转基因大米组和亲本大米组相比未见显著性差异；转基因大米组和对照组相比,小部分生化指标存在差异,但不具有生物学意义,即转双反义分支酶基因大米未对试验大鼠产生毒性作用。5.对两种转基因大米进行了三代繁殖试验研究。对转基因大米进行了大鼠三代繁殖试验研究,观察FO代、F1代和F2代孕鼠繁殖指标(交配成功率、受孕率、妊娠时间和活产率)；F1代、F2代和F3代仔鼠繁殖指标(出生成活率、窝平均仔鼠数、仔鼠雌雄比率、出生窝总重、仔鼠出生体重、身长、尾长、肛殖距)：大鼠生长发育及妊娠哺乳期的体重、进食量及食物利用率；雌雄成年大鼠血常规、血生化指标；断乳仔鼠及成年大鼠脏器系数及病理切片。结果表明,转基因大米组的小部分血液学指标和脏器系数指标存在显著性差异,这些差异主要集中在转基因大米组和对照组之间,且这些差异具有随机分散性,所以不具有生物学意义。因此转基因大米未影响母鼠及仔鼠的繁殖能力,未对大鼠产生毒性作用。6.对两种转基因大米进行了生殖毒性试验研究。给大鼠饲喂转基因大米,观察与测定雌性大鼠的动情周期、卵巢组织抗氧化能力；观察与测定雄性大鼠的精子数量和畸变率,睾丸细胞周期变化；测定雌雄大鼠的性激素水平、肛殖距、生殖器官脏器系数及病理切片。结果表明,转基因大米组与亲本大米组相比,各项指标均无显著性差异；与对照组相比,小部分指标(睾丸细胞周期、生殖器官脏器系数)存在差异,但是不具有生物学意义。因此转基因大米未对大鼠生殖系统的结构和功能产生不利影响。7.对转GL融合蛋白基因大米进行了免疫毒性试验研究。对转基因大米进行了balb/c小鼠免疫毒性及免疫原性试验研究,观察并测量小鼠血常规、淋巴细胞亚群、迟发型超敏反应(DTH)、刀豆球蛋白(ConA)诱导淋巴细胞转化、溶血素水平、腹腔巨噬细胞吞噬鸡红细胞、免疫器官脏器系数及外源蛋白检测情况。结果表明,转基因大米组、亲本大米组及对照组之间均无显著性差异,未在动物体内及其粪便中检测到转基因大米的外源蛋白。因此未见转GL融合蛋白基因大米对小鼠产生免疫毒性,转基因大米中的外源蛋白能被机体完全消化分解,不具有免疫原性。更多还原

【Abstract】 Rice (Oryza sativa L.) is one of the most important crops in our country, and it is used to feed half of the world population, as a result there are concerns on the quality of rice consumed by people. Genetic engineering technology can be used to improve the nutritional and functional value of traditional rice, but there are some controversies on the usage of this technology with regards to the safety of transgenic rice. Therefore studying the food safety of transgenic rice and providing scientific basis for the development of transgenic rice is of great significance.Transgenic rice lines expressing GL fusion protein gene and double antisense branching enzymes gene were used in this study. Through the systematic toxicological research, the safety of the two transgenic rice were assessed in order to provide reference for the evaluation of food safety with regard to transgenic rice. The main research contents and results are as following:1. The acute toxicity study of the transgenic rice expressing double antisense branching enzymes gene. The mice were observed for 14 days following acute oral exposure at a dose of 2.15,4.64,10.00 and 21.50g/kg of body weight. The results indicated that no adverse toxic effect appeared in mice growth. There were no significant differences in the body weight, haematology, organ relative weights and histopathology in the different groups. So this transgenic rice can be classified as a nontoxic rice.2. The mutagenicity study of the transgenic rice expressing double antisense branching enzymes gene. On the mutagenicty study the following test were conducted: mice bone marrow cell micronucleus test, mice sperm abnormality test, Ames test and fluctuation test. Test results gave no indication that this transgenic rice possessed any mutagenic potential.3. The teratogenicity toxicology study of the transgenic rice expressing double antisense branching enzymes gene. The rats were fed with genetically modified rice feed, while pregnant rats and their fetuses development were studied. The results indicated that no adverse effect were observed on the body weight gain and fecundity of pregnant rats and fetuses growth (appearance, viscera and bone), the transgenic rice expressing double antisense branching enzymes gene have no teratogenesis on test rats.4. The ninety days feeding study of the transgenic rice expressing double antisense branching enzymes gene. The rats were fed with genetically modified rice feed and was analysed on the basis of the rats body weight, food consumption, water intake, hematology, serum chemistry, urinalysis, serum sex hormoneorgan, organ weight, ratio and pathology. The results showed that there were no significant differences in the transgenic rice group compared with the non-transgenic rice group, some statistical differences were observed in the transgenic rice group compared with the control diet, but it were not considered biologically significant or related to exposure to the transgenic rice.5. The three generation study of two transgenic rice. The three generation experiment of the two transgenic rice were studied on rats, some parameters were observed such as reproductive parameters for F0, F1 and F2 pregnant rats (including copulation index, fertility index, gestation length and live-birth rate), F1, F2 and F3 offspring (including viability index at birth, number of pups birth, sex ratio of pups, body weight, body length, tail length and anogenital distance at birth), body weight and food consumption during the pre-mating, gestation and lactation periods, hematology and serum chemistry for adult rats, organ ratio and pathology for litters after weanlings and adult rats. The results indicated that small serum chemistry and organ ratio parametersshowed statistical differences, this was found mostly in the transgenic rice group compared with the control rice. These differences had random dispersion and no gender unity, so it was not considered biologically significant. Therefore this form of transgenic rice have no adverse effect on the reproductive abilities of the rats.6. The reproductive study of two transgenic rice. The rats were fed with genetically modified rice feed, this was analysed based on estrous cycle and antioxidant capacity of the ovarian tissue of the female rats, number of sperm and morphologically abnormal sperm, testicular cells cycle for male rats, serum sex hormoneorgan, anogenital distance, reproductive organs ratio and the pathology of the male and female rats. The results revealed that no statistical difference was observed in the transgenic rice group compared with the non-transgenic rice group. The small parameters (testicular cells cycle and reproductive organs ratio) have statistical differences in the transgenic rice group compared with the control rice, but it were not considered biologically significant. Therefore this transgenic rice have no adverse effect on the structure and the function of the reproductive system of the rats.7. The immunotoxicity study of the transgenic rice expressing GL fusion protein gene. The immunotoxicologic and immunogenic experiment of the transgenic rice expressing GL fusion protein gene were studied on balb/c mice, this was analysed on the bases the rats hematology, lymphocyte subsets, delayed type hypersensitivity (DTH), T lymphocyte transformation induced by ConA, hemolysin level, phagocytosis ratio of peritoneal macrophages, immune organs ratio and exogenous protein detection. The findings of this study showed no statistical difference was observed in the transgenic rice group compared with the non-transgenic rice group and as well as the control diet. Moverover, the exogenous protein of the transgenic rice were not detected in the animal organs and their feces consequently, no immunotoxicity was observed on mice fed with transgenic rice this indicates that exogenous protein of transgenic rice can be digested completely and have no immunogenicity.更多还原