Purification Of Mitochondrial DNA And Cloning And Analysis Of The Sequences Of Several Important Genes From Takifugu Fasciatus

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.