| As a unique cytoplasmic genetic system, mitochondrial DNA (mtDNA) is a kind of complete, small, self-replica ting and diverse genome. The study on mitochondrial genome is significant in the research of many biologic issues, such as gene structure and function, gene express ion regulation on time and space, nucleo cytoplasmic interaction, the dynamics of molecular evolution, the origin of mitochondrial, the origin and evolution of species, and so on. But the study on Bombyx mandarina mitochondria genome is still in the initial phase, and the study on B.mandarina in China still little reported.Some studies have proved that Bombyx mori is derived from B.mandarina in China. To the current time, mitochondrial genome complete sequences about four strains of Bombyx mori and a B.mandarina in Japan have published. But the mitochondrial genome complete sequence of B.mandarina in China is not reported still, and its phylogenetic analysis is little reported. The study on mitochondrial genome of B.mandarina in China has many advantages. Firstly, it can richen the database of mitochondrial genome. Secondly, comparison about mitochondrial genome sequences can illuminate the genome composition, gene order, structure and evolut ion about B. mandarina in China; important ly, phylogenet ic analysis can be of benefit to further study on the evolution relationship and gene marker characters between B. mandarina in China, different B. mori strains and B. mandarina in other countries.The study is most focalized on sequencing and analysis of the mitochondrial genome about B.mandarina in China; and also on evolutional character and phylogenetic analysis about different species or strains.1.Method to extract the mtDNA from B.mandarina in ChinaWild materials become fewer and fewer for surroundings alterated. It is difficult to gain the suitable materials in one place or at in a certain time .Therefore, it is of some importance to find a good way to get and store materials avoiding the limitation of space and time . In the study ,writer tried different methods to break cells and store material's , and also studied the influence of different methods on the extraction of the mtDNA from B.mandarina in China. At last, writer founded an optional method. The mtDNA obtained through this method can be used to amplify,endonuclease cleavage, clone and sequence.2. Screening and analysis primersThere are 37 kinds of insect mtDNA general primers and 81 percent of these primers which can be used to amplify and sequence. Then 12 kinds additional primers designed can amplify other mtDNA domains. All fragments, which are amplified, recoveried and purified, can help to finish the whole mitochondrial genome sequence of Rmandarina in China. 3.The result of mitochondrial genome complete sequence of B.mandarina in China (1)Genome composition and gene order.The whole length is 15688bps, The sequence is composed of 13 kinds of protein-coding genes, two kinds of ribosomal RNAs, 24 kinds of transfer RNAs and a 498bps AT rich region. Writer compared Rmandarina in China with different B.mori strains and B.mandarina in Japan, and found that 1.There are two kinds of transfer RNA genes which are only found in B.mandarina in China; 2.ther composition and order haven' t alternate. And writer also further found that there is difference more or less in different such as drosphilor, mosquito and human.(2)Map of the restrictive endonuclease siteThere are 52 sites can be cleavaged by 15 kinds of restrictive endonuclease. Among them, TaqI has 24 sites, but PstI has only one site. Compared with mtDNA of B.mandarina in Japan and B.mori strain Xia Fang", there are site number polymophism and length polymophism. (3)Comparison of gene mutationCompared wi th the mtDNA of B. mandarina in Japan and found that there are 24 coding-gene sequences mutated, the total mutation site number is 349, and 277 sites mutated in non-coding domains.Compared with mtDNA of Bombyx mori Xia Fang" and found that there are 25 coding-gene sequences mutated , the total mutati...
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