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题名: 转GH基因鲤鱼的生物安全性评价
作者: 崔宗斌
答辩日期: 1998
导师: 朱作言
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 长激素基因 ; 转基因鱼 ; 繁殖力 ; 发育选择 ; 性选择 ; 生存能力 ; 基因流动 ; 食品安全
摘要: 鱼类基因转移研究取得了突破性进展。与此同时,转GH基因鱼释放的生物安全问题引起了公众的广泛重视。本文在详细介绍了鱼类基因转移研究取得的成就的基础上,对目前存在的问题,尤其是转基因鱼的安全问题进行了深入分析和探讨,并针对转GH基因鲤鱼的生态、遗传和食品安全问题中的几个重要方面进行了研究,就转GH基因鲤鱼的生物安全性进行了综合评价。1.转MThGH基因鱼的PCR鉴定 研究了Mg~(++)浓度、dNTP浓度、引物浓度、Taq酶用量和退火温度对PCR法扩增MThGH基因中特异DNA片段的影响。在此基础上,建立起PCR检测MThGH基因的优化方法,可检测出单个拷贝的MThGH基因以及0.2μg的鲤鱼尾鳍总DNA中l * l0~4个拷贝的MThGH基因。利用这一优化方法,对转MThGH基因红鲤(Cyprinus carpio L. red var.)的五个世代进行了PCR检测,结果指出转MThGH基因鱼的阳性率由P_0代的58.3%逐渐提高到F_4代的94.3%,表明通过逐代筛选阳性亲本繁殖后代,外源基因在受体鱼中的遗传逐代趋于稳定。2.转GH基因鲤鱼的基本生物学特征 用转MThGH基因红鲤的F_4代以及F_2代与普通镜鲤(Cyprinus carpio L.mirrow var.)杂交得到的杂交鲤为材料,比较研究了转移的GH基因对受体鱼的生长、形态、内脏器官重量、繁殖、肌肉生化组成等生物学性状的影响。结果指出,转基因鱼在生长和肌肉蛋白质含量方面明显高于对照鱼,而其形态、生存能力、繁殖特性、发育特征和内脏器官的重量比等性状与对照鱼无显著差异。3.转GH基因鲤鱼的生化组成分析 研究了20至80日龄期间转MThGH基因红鲤鱼的F_3代和对照鲤鱼的生化成分。结果发现,F_3代红鲤的生长较对照鱼快,并以幼鱼阶段最显著;20、40、60和80日龄时转基因鱼干物质中的蛋白质和17种氨基酸的总含量显著高于对照鱼,而转基因鱼和对照鱼在干物质含量、含水量、总脂肪和灰分含量方面无显著差异。20至80日龄,随着鱼体的生长,转基因鱼和对照鱼的干物质和脂肪含量增加,而其干物质中的蛋白质、17种氨基酸的总量、水分和灰分含量降低。幼鱼阶段,转基因鱼的不饱和脂肪酸含量显著高于对照鱼,饱和脂肪酸含量无显著差异,但在80日龄时,转基因鱼和对照鱼的饱和与不饱和脂肪酸含量均无显著差异。20至80日龄,随着鱼体的生长,转基因鱼和对照鱼的脂肪酸数目、饱和与不饱和脂肪酸的含量增加,但同一生长阶段转基因鱼和对照鱼在脂肪酸组成方面无显著区别。4.外源GH基因在鲤鱼和鲫鱼群体之间的流动 利用PCR技术检测了F_2代红鲤群体与非转基因红鲤、普通鲤鱼(Cyprinus carpio L.)、镜鲤和鲫鱼(Carassius auratus L.)群体在同一水体内自然繁殖的子代中,MThGH基因存在的可能性和频率。结果显示,转基因红鲤携带的MThGH基因可以通过婚配扩散到同种鱼的不同品系中,但不能扩散到鲫鱼群体中;大小相同的转基因红鲤与非转基因红鲤在性选择能力方面无显著区别。5.转GH基因鲤鱼对几种主养鱼类生长的影响 混养状态下,F_4代红鲤和对照鱼对普通鲤鱼、草鱼(Ctenopharyngodon idellus Cuvier et Valeficiennes)、团头鲂(Megalobrama amblycephala Yih)、白鲢(mypophthalmichthys molitrix Cuvier et Valeneiennes)和花鲢(Aristichthys nobilis Richardson)生长的影响相同,二者的存活率无显著区别。混养草鱼时,红鲤生长最快,混养花白鲢和团头鲂时次之,混养普通鲤鱼时最慢,表明混养鱼类之间的食性差别大,有利于鱼的生长和水体中饵料资源的充分利用。合理混养转基因鱼可以提高水体的鱼产量。6.肉食动物摄食转GH基因鲤鱼后的血液理化指标观测 利用F_3代红鲤和对照红鲤的生鱼喂养波斯猫。结果显示,摄食转GH基因鲤鱼后,波斯猫的生长速率和食物转化效率显著提高,实验后期其体重增加,而其静脉血的常规和生化指标、外部形态和习性无病理变化,表明在强制性地饲喂转MThGH基因鱼的情况下,波斯猫的体重增加但生长与发育正常。7.转GH基因鲤鱼的安全性评价 在本研究结果的基础上,参照我国《农业生物基因工程安全管理实施办法》,就受体鲤鱼、基因操作和转GH基因鱼三个方面的安全性进行初步评价。结论指出,本研究所用受体鲤的安全等级为I级,基因操作为类型2,转“全鱼”GH基因鲤的安全等级为I级,即对人类健康和生态环境不存在危险。
英文摘要: Transgenic common carp (Cyprinus carpio L. red var.) have been produced by microinjecting a construct pMThGH, a human growth hormone (hGH) gene capped with a mouse metallothionein-1 (MT-1) gene promoter in pBR322, into fertilized eggs. The following generations F_1 to F_4 of the transgenics were reproduced by artificial fertilization. In comparison with the control, the transgenics exhibited higher growth rate, especially at the early developmental stages. Optimized conditions of PCR was established for identifying a single copy of MThGH and 1 * 10~4 copies of MThGH in 0.2μg of non-transgenic fish caudal fin DNA, which equivalent to less than one copy of MThGH per genome.The positive rates of MThGH-transgenics were 58.3% in P_0 generation and 94.3% in F_4 generation, which suggested that the transmission of MThGH tended to be stable in the successor generations of MThGH-transgenics. There were no significant differences between the transgenics and the control in the taxonomic characteristics, weight ratio of internal organs, viability, sex ratio, age of sexual maturity, fecundity and developmental selection. The transgenics had higher contents of protein and 17 amino acids than the control at a same developmental stage, while there were no significant differences between the transgenics and the control in the contents of dry matter, moisture, total lipid, ash and the composition of fatty acids. Comparing with the control, the transgenics showed higher contents of unsaturated fatty acid at early developmental stages but this significant difference disappeard along with the increase of body weight of the fishes. In polyculture system, the transgene carried by the transgenics could only spread amoung individuals within species but not between species by natural reproduction. The ability of sexual selection between MThGH-transgenics and non-transgenics in the same size showed no significant difference. Separately stocking the transgenics or the control with common carp(Cyprinus carpio L.), grass carp(Ctenopharyngodon idellus Cuvier et Valenciennes), Wuchang fish(Megalobrama amblycephala Yih), bighead(Aristichthys nobilis Richardson) and silver carp (Hypophthalmichthys molitrix Cuvier et Valenciennes), the growth rate of the transgenics was higher than that of the control, while the transgenics and the control had the same effects on the growth of common carp, grass carp, Wuchang fish, bighead and silver carp. The total output stocked the transgenics with other fishes in a pond was higher than that shocked the control with other fishes. Two groups of cats were used in the experiment. The experimental group (n=4) were fed with the raw transgenic fish and the control group (n=4) were fed with the raw control fish for 180 days. The pathological diagnosis and blood analysis for two groups of cats showed no significant difference at the same developmental stages, even though growth, appetite and food conversion efficiency of experimental cats were higher than that of the control, suggesting that MThGH-transgenic fish is safe as a daily food for cats. Based on the results of this study and the standardized procedures of safety assessment in the "Safety Administration Implementation Regulation on Agricultural Biological Genetic Engineering" issued by the Ministry of Agriculture, People' s Republic of China in 1996, we determined the safety classes of recipient common carp and "all-fish" GH-transgenic common carp as Safety Class I, since the gene transfer has no effect on the safety of recipient common carp.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12516
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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转GH基因鲤鱼的生物安全性评价.崔宗斌[d].中国科学院水生生物研究所,1998.20-25
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