微囊藻毒素对3种淡水鱼肝脏HSP70基因转录水平的影响

论文摘要

随着社会工业化进程的加快,大量含氮、磷的污染物被排放入自然水体中,加速了水体的富营养化,引发全球性的环境污染问题。本文研究内容主要为微囊藻毒素（MC-LR）对淡水鱼类肝脏热休克蛋白（HSP70）基因诱导表达水平之影响。以三种淡水鱼:鲢鱼（Hypophthalmichthys molitrix）,草鱼（Ctenopharyngodon idellus）,及尼罗罗非鱼（Oreochromis nilotica）,对MC-LR的耐受性依次排列为尼罗罗非鱼、鲢鱼、草鱼,比较其处于微囊藻毒素造成的氧化压力下,肝脏HSP70的诱导表达水平。实验通过简并引物PCR从鲢鱼、草鱼及尼罗罗非鱼的肝脏克隆HSP70基因cDNA核心序列,并应用5′RACE和3′RACE技术分别扩增出它们的5′末端和3′末端序列而获得其cDNA全序列。序列分析表明,它们的HSP70基因cDNA全序列分别为2356bp、2346 bp及2242 bp,其中鲢鱼及草鱼的开放阅读框（ORF）均为1950 bp、编码649个氨基酸,而尼罗罗非鱼的ORF则为1917 bp,编码638个氨基酸。通过对上述三种淡水鱼（6-13）g活体腹腔注射亚致死量MC-LR(50μg kg-1 bwt)8h后,发现微囊藻毒素对尼罗罗非鱼肝脏HSP70基因表达有显著的诱导作用（P＜0.05）,然而鲢鱼肝脏HSP70基因mRNA表达水平虽出现明显的升势,但未出现显著的诱导作用（P＞0.05）,草鱼的则保持不变,即微囊藻毒素可能对草鱼肝脏HSP70基因mRNA的表达未能起到诱导作用。肝脏HSP70基因在微囊藻毒素的诱导作用下,其表达水平与对MC-LR的耐受性有着明显的相关性,对MC-LR有较高抵抗能力的鱼类（尼罗罗非鱼）其诱导表达水平最为显著,其次为抵抗能力一般的鱼类（鲢鱼）,而对毒素较感敏的鱼类（草鱼）则本身没有改变。因此我们认为在淡水鱼类中,肝脏HSP70基因mRNA之诱导表达水平可能与微囊藻毒素之耐受性有关。另外,本实验亦克隆出斜带石斑鱼（Epinephelus coioides）,鳙鱼（Aristichthysmobilis）,鲫鱼（Carasslius auratus）,白云金丝（Tanichthys albonubes）,中华鲟（Acipensersturio Linnaeus）,多鳍鱼（Polypterus senegalus Cuvier）,斑鳢（Channa maculata）等鱼类HSP70基因的部份cDNA序列。

论文目录

• Chinese abstract
• English abstract
• Chapter 1 Literature review
• 1.Microcystin pollution in China
• 2.Structure and acute toxicity of microcystins
• 2.1 Classification of Cyanotoxins
• 2.2 Structure and Toxicity of Microcystins
• 2.2.1 The Molecular Structure of Microcystins
• 2.2.2 The toxicology of microcystins
• 3.Heat Shock Protein
• 3.1 General Introduction to Heat Shock Protein
• 3.2 Heat Shock Protein 70
• 3.2.1 Structure of HSP70
• 3.2.2 Function and Regulation of HSP70
• 3.2.3 HSP70 in Environmental Monitoring
• 4.The background and significance of this paper
• Chapter 2 Transcriptional Responses of HSP 70 Genes in the Liver of Three Freshwater Fishes Intraperitoneally Injected with MC-LR
• 1.Introduction
• 2 Materials and Methods
• 2.1 Materials
• 2.2 Fish and Microcystin Exposure
• 2.3 Total RNA Isolation from Fish Liver
• 2.4 Cloning of Partial HSP70 cDNA Sequences of Fish
• 2.5 Full-length cDNA Amplification of silver carp,grass carp and Nile tilapia HSP70 Genes by 5' and 3'RACEs
• 2.6 Quantitation of Relative Silver Carp,Grass Carp and Nile tilapia Liver HSP70 mRNA Abundance after Exposure to MC-LR
• 2.7 Statistical Analysis
• 3 Results and Analysis
• 4 Discussions
• References
• Appendix-1 Detailed procedure for RNA isolation
• Appendix-2 Detailed procedure for reverse transcription
• Appendix-3 Detailed procedure for ligation and transformation
• Acknowledgements