【作者】 邵爱华；

【导师】 朱江；

【作者基本信息】 苏州大学 ， 细胞生物学， 2005， 硕士

【摘要】 线粒体DNA(mtDNA)是分子生物学研究的一个热门领域,已成为鱼类进化生物学和群体遗传学研究的重要遗传标志,mtDNA 分析技术在国外已被遗传学家用于鱼类种群遗传结构及品系鉴定的研究。暗纹东方鲀(Takifugu fasciatus)是具有极高经济价值的特种水产养殖对象,有关暗纹东方鲀mtDNA 的研究迄今未见报道。本论文在改进鱼类mtDNA 分离纯化方法的基础上,利用JGB(Janus Green B)能特异地对线粒体进行超活染色的特性来跟踪线粒体的提取,研究离心速度与线粒体回收率的关系。同时对常用的方法和技术进行了探索和改进,利用暗纹东方鲀组织较少取样,获取纯度好、得率高的mtDNA。利用分离纯化的暗纹东方鲀肝脏mtDNA 为模板,按照红鳍东方鲀(Takifugu rubripes ) mtDNA 序列设计合成特异引物进行PCR 扩增,首次克隆并测定了暗纹东方鲀mtDNA 的Cytb、COI 、COII、COIII、D-loop 基因及其侧翼的tRNA 基因。上述基因已在GenBank 登录。运用DNA 分析软件,对暗纹东方鲀mtDNA 测定的基因进行序列和结构分析,并选取GenBank 中十多个目近20 种鱼类的相应序列进行比较分析,结果显示暗纹东方鲀与这些鱼类具有较高的核苷酸和氨基酸同源性。利用分子生物学相关软件,对暗纹东方鲀与其他种类鱼的相关基因序列建立进化树,并与传统的分类地位进行比较,结果表明与传统的分类地位基本吻合。我们认为,对于鱼类,在采用线粒体基因序列进行分子进化分析时,应该综合考虑其繁殖模式及生态特点,将分子与形态、生态行为等多方面的证据结合起来,对生物多样性作出更客观的描述和解释。暗纹东方鲀线粒体的几个编码基因可能成为东方鲀属不同种间聚类分析与分子系统进化学研究的合适基因。推定的8 个tRNA 的二级结构表明:它们都具有典型的三叶草型结构,各臂的碱基配对率大多较高,稳定性较好。更多还原

【Abstract】 The study on mitochondrial (mtDNA) is a hot field in recent molecular biology research. With some special characteristics, mitochondrial DNA as an ideal genetic marker has been successfully applied to the research of population and evolutionary biology of fishes. Takifugu fasciatus is a special grage aquaculture object with huge economic value. Its mtDNA has not been reported by far. In this thesis, mtDNA from Takifugu fasciatus was isolated and purified by differential centrifugation and alkali lysed method. The isolation product was checked by Jenus Green B Staining Method step by step. It was found that the higher centrifugal speed, the more mitochondria were obtained. When the centrifugal speed reached 25000g~35000g, the mitochondria obtained rate raised to more than 90%. We have analyzed preliminarily that the molecular weight of mtDNA from Takifugu fasciatus is about 19.4kb. Mitochondrial DNA of Takifugu fasciatus was prepared as templates from the liver tissue. Several importent genes, such as cytochrome b (cytb) and cytochrome oxidase subunit I, II, III (COI,COII,COIII ) and D-loop, and their connected tRNA genes were amplified by PCR separately with a pair of specific primers which designed according to the sequence of Takifugu rubripes released from Genbank .The sequences with total length including the nucleotides of the important genes and partial sequences at both sides were cloned. All of them have been registered in GenBank . The cloned and sequenced genes of Takifugu fasciatus mtDNA were proved to have good homology with those of some species of fish in GenBank. The phylogenetic trees show that the phylogenetic relationships based on the homology of the mitochondrial genes of Takifugu fasciatus and the other kinds of fish are consistent with the morphological taxonomic results. We analyze the the phylogenetic relationships and consider that it was proposed when the molecular evolution is analysed based on mitochondrial genetic sequences, the factors of reproduction pattern and ecological characteristics should be involved. The genes of mtDNA of Takifugu fasciatus might be good ones of molecular evolution and phylogenetic marker among Takifugu. The tRNA genes have from 69 to 72 nucleotides. Deduced secondary structures for tRNA genes and suggest that most of them have classical cloverleaf structures with high complementary base rate of each arm and the structures have good stability.更多还原