【作者】 刘顺梅；

【导师】 张学成；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 鱼类生长因子能够促进鱼类生长,在水产养殖中具有重要的应用价值,受到了人们的广泛关注。鱼类生长因子的开发应用主要通过培育转生长因子基因鱼和在基因工程菌中表达重组生长因子应用于养殖鱼类。由于转基因鱼潜在的食品安全性和环境安全性问题,重组生长因子在水产养殖业中的应用引起了人们极大的兴趣。本文研究了本实验室构建的转牙鲆生长因子(GH)基因集胞藻(Synechocystis PCC6803)对鱼类的促生长作用,评价了转牙鲆生长因子基因集胞藻对小鼠、牙鲆、大菱鲆以及牙鲆和舌鳎原代培养肝细胞的安全性,为将其应用于生产实践提供了理论依据和实践经验。对转基因集胞藻的促生长作用研究包括三部分:一是对牙鲆的促生长作用研究;二是对大菱鲆促生长作用研究;三是对牙鲆和舌鳎原代培养肝细胞的促生长作用研究。通过7周牙鲆喂养和40天的大菱鲆喂养实验发现,转牙鲆生长因子基因集胞藻在促进鱼类生长、提高饵料转化率方面具有显著的作用。0.5%和2.0 %添加量分别使牙鲆体重比对照提高32.09 %和49.04 %,饵料转化率比对照组提高20.75%和35.85%;1.0%转基因藻使大菱鲆体重增长比对照提高18.54% (P < 0.05),饵料转化率比对照提高9.84% (P < 0.05)。将转基因藻添加到牙鲆和舌鳎原代肝细胞培养液中,通过检测细胞对中性红的吸收和对MTS的代谢,证实转基因集胞藻能够提高两种细胞的活力,100μg/ml和200μg/ml两个添加量显著提高了牙鲆和舌鳎肝细胞的相对存活率(P < 0.05)。通过小鼠急性毒性、传统致畸、骨髓微核及精子畸形检查,牙鲆和大菱鲆的成活率、肌肉成分、血液生理生化指标、红细胞微核及核异常率、脏体系数、组织病理学检测,以及牙鲆和舌鳎原代肝细胞LDH释放和基因组DNA片段检查,评价了转基因集胞藻的安全性。小鼠毒理学试验结果表明转基因集胞藻的LD50 > 10g/kg,转基因集胞藻对小鼠生殖能力、胎鼠的体重和体长没有影响,对胎鼠骨骼和器官没有致畸作用,对小鼠骨髓细胞微核及精子畸形也不具有诱导作用。对牙鲆和大菱鲆心、肝、脾的脏体比以及胃、肠、盲肠、肝、脾、肾进行组织病理学检查,证明了转基因藻不会引起鱼类组织器官损伤。对大菱鲆血液指标和红细胞微核的检测结果显示转基因藻对血液细胞数量、血红蛋白含量、转氨酶活性、以及蛋白质、葡萄糖、甘油三脂、胆固醇,肌酐等代谢物含量不会产生影响,转基因集胞藻组鱼的红细胞微核率及核异常率与对照没有差别。鱼类肌肉成分分析显示,摄食转基因藻对牙鲆肌肉水分、蛋白、脂肪及氨基酸含量没有影响(P > 0.05),但却引起大菱鲆肌肉水分减少(P < 0.05)、蛋白质含量提高(12.73%),对脂肪和氨基酸的组成及比例没有影响,各种氨基酸均衡增加。原代肝细胞研究结果表明,转基因藻处理的细胞其形态结构和LDH释放率与对照没有差别,基因组中没有出现小片段DNA。本文还通过摄食转基因藻的牙鲆鱼肉喂养小鼠实验研究了转基因藻能否通过食物链对食鱼生物产生影响。实验结果证明以摄食转基因藻的牙鲆喂养的小鼠,其生长、血细胞数量、血红蛋白含量、血液转氨酶活性、组织器官结构以及生殖能力与对照小鼠没有显著差异。本论文的研究结果表明转牙鲆生长因子基因集胞藻是一种安全有效的鱼类饲料添加剂,可能会给鱼类养殖业带来巨大的经济效益。更多还原

【Abstract】 Fish growth hormone (GH), a promoter of fish growth, should be of great value in fish culture, and it has already gained great attention. The exploiture of fish GH for its application in aquaculture are mainly through the breeding of GH transgenic fish and the expression of GH gene in gene engineering host. Because of the food safety and environmental safety issues accompanied with transgenic fish, the use of recombinant growth hormone (rGH) in aquaculture has generated considerable interest. In this paper, the growth-promoting effect of transgenic Synechocystis sp. PCC6803 containing flounder GH gene constructed by our lab is studied, and the safety of this transgenic alga on mouse, flounder, turbot, hepatocytes of flounder and tonguefishin in primary culture are evaluated. The present study provides theory and experience for the use of the transgenic alga in fish culture production.Studies on growth-promoting effect of transgenic Synechocystis include three parts: the first is growth-promoting effect on flounder, the second is growth-promoting effect on turbot and the third is growth-promoting effect on hepatocytes of flounder and tonguefishin. 7-week flounder feeding experiment and 40-day turbot feeding trial show that transgenic Synechocystis sp. PCC6803 containing flounder GH gene can significantly enhance fish growth and improve feed conversion ratio. 0.5% and 2.0% dose of transgenic alga increase body weight of flounder by 32.09 % and 49.04 % than that of control group, respectively. Enhancement in feed conversion ratio of flounder in 0.5% and 2.0% transgenic alga groups are 20.75% and 35.85%, respectively. In turbot, 1.0% transgenic alga causes 18.54% and 9.84% increase in body weight and feed conversion ratio (P < 0.05), respectively. Neutral red uptake and MTS reduction assays verified that hepatocytes of flounder and tonguefishin in primary culture exposed to 100μg/ml and 200μg/ml of transgenic Synechocystis significantly elevated relative survival rate (P < 0.05).To evaluate the safety of transgenic Synechocystis containing flounder GH gene, acute toxicity, traditional abnormality induction, bone marrow erythrocyte micronucleus and sperm abnormality test in mice; survival rate, muscle composition, blood physiological and biochemical parameters, peripheral blood erythrocyte micronucleus and other nuclear abnormality, organ/weight ratio and histopathology examination in fish; LDH release and DNA fragmentation assay in hepatocytes of flounder and tonguefishin are conducted. Mice toxicity assays show that the LD50 of transgenic Synechocystis is more than 10g/kg; reproductive function of female mice and body weight and length of fetal mice are not affected by transgenic alga; neither abnormality of skeleton, organ and sperm nor micronucleus of bone marrow erythrocyte is induced by the transgenic alga. Test on organ/weight ratio of heart, liver, spleen, and the histopathology examination on stomach, intestine, pyloric caecum, liver, spleen and kidney in flounder and turbot verify that transgenic alga can not damage fish organ structure. Analyses on haematological parameters reveal that the transgenic alga has no effect on fish blood cell count, haemoglobin level, serum aminotransferase activities, and metabolite content, such as protein, glucose, triglyceride, cholesterol, creatinine and so on. Frequencies of micronucleus and other nuclear abnormality in erythrocytes of turbot show no difference among transgenic alga groups and control. Muscle composition analyses show that feeding transgenic alga can not influence proximate composition of muscle in flounder, but significant (P<0.05) decrease in moisture content and significant (P<0.05) increase in protein content (12.73%) is induced by transgenic Synechocystis. However, lipid and amino acid profile is not changed by transgenic alga. Examination on the morphology, LDH release and DNA fragmentation of hepatocytes in flounder and tonguefishin demonstrate that the cell exposed to transgenic alga showed similar structure and LDH release to the control. No DNA fragment is detected in cell treated with transgenic Synechocystis.Muscle of flounder fed with transgenic Synechocystis is fed to mice by gavage to study if the transgenic alga can influence animal fed with the fish through food chain. Results illustrate that the mice fed with experimental fish are not significantly different from the control mice in growth, blood cell count, haemoglobin content, serum aminotransferase activities, histological structure and reproductive function.Results of present study indicate that transgenic Synechocystis sp. PCC6803 containing flounder GH gene is an efficient growth promoter and a safe feed additive for fish. It could bring great benefits to fish farmers.更多还原