【作者】 高辉；

【导师】 韩春杨； 张运海；

【作者基本信息】 安徽农业大学 ， 临床兽医学， 2013， 硕士

【摘要】 随着动物转基因技术在育种新材料创制、生物反应器、医药、食品以及生物学基础研究等方面的应用日益广泛，转基因动物的生物安全问题也越来越为人们所关注。然而，由于对转基因技术的科学普及等工作开展不足、科学家与媒体以及大众的沟通存在问题等原因，尤其是相关评价体系、内容、方法的建立等还不健全，使得人们对这一崭新领域知之相对甚少。在转基因家畜中，水平基因转移或转基因的无意释放对生物多样性有何影响，以及基因修饰对健康动物的潜在威胁、产品安全性等都是人们非常关心的问题。本研究以转磷酸烯醇式丙酮酸羧激酶（PEPCK）-1基因的体细胞克隆母猪为研究对象，通过检测其粪便、接触的土壤以及血液中DNA是否发生了水平基因转移评估其环境安全性；通过检测血液生理生化指标来评估自身健康状况，以及转基因猪母乳（PEPCK-1母乳）饲喂普通仔猪健康状况，来评估转基因动物的产品对其他动物的影响。为了评估转基因猪的环境安全性，采取了三种方法：1）利用PCR技术检测水平基因转移，即考察是否有外源基因转移到了普通猪血液、肠道菌或土壤微生物中。结果显示，血液、肠道菌和土壤微生物的基因组中均未检测到外源基因。2）利用PCR检测转基因阳性猪进行本交过的公猪和饲喂转基因猪母乳的仔猪是否发生水平基因转移。结果表明，进行本交的公猪和饲喂转基因猪母乳的仔猪同样没有发生水平基因转移。3）利用生理生化的方法测定粪便和土壤中的营养元素，进而评价转基因阳性猪代谢变化和土壤营养结构的变化。结果发现，土壤和粪便中营养元素的变化很可能是由土壤空间距离上差异和猪个体之间差异的引起的，而与外源基因无相关性。以上结果表明所研究的转基因阳性猪对环境安全可能无影响。为了考察转基因阳性猪的健康状况，开展了生长性能测定、血常规、血清生理生化分析、免疫指标测定、生殖激素水平监测。结果如下：1）在测定的生长性能指标中，转基因阳性猪在2、3月龄时体重、日增重明显高于普通猪，与对照组比较差异显著。虽然4、5月龄差异不显著，但其平均体重、日增重仍然优于对照组。2）在测定的7项血常规指标中，实验组和对照组比较，在6月龄时白细胞（WBC）测定值差异显著。其他未见显著差异。3）在测定的14项血清生理生化指标中，6月龄甘油三酯（TG）含量实验组与对照组差异显著，在3、7月龄总蛋白（TP）含量实验组显著低于对照组，并且在整个试验阶段TG含量均偏低。其他生化指标转基因阳性猪与对照组之间没有显著性差异。4）免疫指标中，IgG含量在6月龄实验组显著高于对照组。5）生殖激素水平检测结果表明，A1转基因母猪基本符合母畜在发情期内生殖激素变化的规律，A2转基因母猪可能受外界因素的影响出现了生殖机能紊乱，但其生殖激素水平处于正常范围。由以上结果可初步推测，除去繁殖性能方面，外源基因（PEPCK-1）对转基因猪的身体健康未见明显的影响。最后，为了研究转基因猪母乳对普通仔猪健康的影响，通过给仔猪饲喂转基因猪母乳的试验组与饲喂普通乳的对照组来比较其差异，实验开始后每周称量一次猪的体重，在实验初和实验末采取血液样品，测定血常规、血清生理生化指标。结果显示：试验组与对照组的体重、增长速度无显著差异，血常规、生化指标也无显著差异。由此可初步推断转基因猪母乳对仔猪的健康未见不良影响。综合以上三个方面，本试验初步得出，PEPCK-1基因对环境安全以及猪的自身安全未发现不良影响。更多还原

【Abstract】 With the animal transgenic technology being used widely in creating of newbreeding materials, bioreactors, medicine, food and basic research of biology,bio-safety issues of transgenic animals is also getting more concern. However, due tothe inadequate work of science popularization of the transgenic technology, andlacking of communications to the media and public for scientists, etc, Especially theestablishment of related evaluation system, the contents, methods are not perfect,sothat people have little acquaintance with this field. For transgenic animals, people paymore and more attention on bio-safety issues including the impact of horizontal genetransfer or unintentional release of trans-genes on biodiversity, the potential risk ofgene modification on animal health, and food safety of genetically engineeredanimals.In the study, transgenic pigs of expressing PEPCK-1were the research object; theenvironmental safety was assessed by detecting the manure, the soil and the bloodDNA. The health of genetically engineered pigs was assessed by detecting thephysiological and biochemical indicators. The impacts of the milk of PEPCK-1transgenic sow on the health of piglets were also assessed.The environmental safety of genetically engineered pigs were assessed through thefollowing protocols:1）The horizontal gene transfer was detected with PCR to detectthe transgenes in microbial DNA extracts of blood or manure and soil, The resultswere negative.2）Using PCR to detect the boars in mating transgenic sows and pigletsof feeding milk of genetically modified sows whether occur to horizontal genetransfer,The results were negative.3）Analyzing nutrient element of transgenic pigsmanure or soil by physio-biochemical method. It was found that the variation ofnutrient element of soil or manure was probably caused by soil spacial difference oranimal-to-animal variation but not the transgenes. These results suggested thattransgenic pigs may be safe for the environment.In order to examine the health status of the transgenic pigs, carrying out theanalysis of growth performance, hematology, physio-biochemical analysis, immuneindexes and reproductive hormone level. The results were as follows:1)The resultsshowed that the weight and average daily gain were significantly higher than ordinarypigs in2,3months of age. Although the growth production was not significantdifference in4or5months of age, but the average body weight or average daily gainwas still higher than the control group.2) Most values of hematology were normal either for genetically engineered pigs or control ordinary pigs.only white blood cells(WBC) was significant difference in6months of age.3) For physio-biochemicalindicators, Triglyceride (TG) was significant difference between the experimentalgroup and control group in6months of age, and significantly lower than the controlgroup in3or7months of age, and relatively low in the whole test. Otherphysio-biochemical indicators had no significant difference between transgenic pigsand the control group.4) The IgG level of the experimental group at6months of agewas significantly higher than the control group.5)The level of reproductive hormoneshowed that reproductive hormone level change significantly, and the transgenic sowsof A1was in line with the law of dams reproductive hormonal changes during theestrus period. Transgenic sows of A2may be affected by external factors appeared toreproductive disorders, but the content of reproductive hormones in the normal range. Theseresults initially speculated that the exogenous gene (PEPCK-1) may not have impacton the health of pigs except the reproductive hormone.Finally, the piglets were fed with the milk of genetically modified sows andcommon milk to study the impact of the milk of genetically modified sows on piglets.The piglets were weighed once a week after the start of the experiment, and collectedthe blood samples to detect the serum physiological and biochemical at the beginningand end of the experiment. The results showed: there was no significant difference onthe weight, growth between the experimental group and the control group. We canpreliminarily conclude that the milk of genetically modified sows have no adverseeffect to the health of piglets.Based on the three aspects above, this test preliminarily demonstrates PEPCK-1gene has no adverse impact on environmental safety and the health of pigs.更多还原