Cloning And Sequance Analyses Of Mitochondrial DNA Fragments Of Eri-silkworm (Philosamia Cynthia Ricine)

Mitoehondrial DNA(mtDNA) was extracted from pupae of Enilkworm variety uaLan. The pSK(+) (Ampr) was used as vector. The mtDNA and plasmid vector pSK(+) (Ampr) DNA were digested by EcoRI and XbaI, then linked up by T4 DNA ligase. The two recombinant clones are 1. 0 kb and 3. 0 kb of XbaI coRl fragments of Enilkworm mitoehondnial genome. The two fragments were partial sequenced. The 1. 0 kb clone includes complete genes of cytochrome c oxidase subunit 3 gene(cox3) , Gly transfer RNA (tRNAly) and partial sequances of oxido eductase NADH subunit 3 gene (ND3) (GenBank Accession NO. AF288145). The open reading frame of COX3 gene contains 789 nucleotides, encoding a putative protein of 262 amino acids. The tRNAly gene contains 66 nucleotides. The homologies between this gene and the mtDNA tRNAly gene of silkworm is 98. 48%. The start codon of mtDNA ND3 gene is ATT. The 3.0 kb clone includes partial sequences of oxidoeductase NADH subunit 6 gene (ND6),cytoehrome b gene(Cytb) and 16srRNA gene. The homologies between these sequences of these genes and the corresponding sequences of silkworm(Bombyx mori) are 64.9%,75.2% and 80.4% respectively. Based on the sequence of cox3 gene of Enilkworm and other 16 mtDNA cox3 genes which were published on GenBank, the comparison of homology with other COX3 of mtDNA was studied, and the molecular evolution was analysed. The results showed that the conservation of 3?termination was higher than 5ermination of COX3 gene, there were two conservative regions in C?termination of the putative protein of COX3. The homology between Apis me]lifera and other mtDNA COX3 genes is the lowest. The COX3 gene of mtDNA in Enilkworm is most closely related to that of silkworm (Bombyx mon.) with the identities of 80. 23% nucleotide and 85. 6% amino acid respectively. The identities of mtDNA cox3 gene between Enilkworm and 5 vertebrate animals is low. Except for Apis nzellifera ligustica, the percentage of each amino acid of cox3 is stable among different species. The percentage of Ala, Gly, His, lie, Glu, Phe, Sen and Thr is high, but Arg and Cys is low. The ratio of hydrophobic amino acid is 43.30% 48.47%.The composition of cox3 of Enilkworm and silkworm is identical basically. Based on the alignment of amino acid sequences and nucleotide sequences, the molecular phylogenetic trees of 12 invertebrate and 5 vertebrate COX3 genes were constructed. The two molecular phyiogenetic trees are identical basically except the percent divergence changing a little. There is little difference between the molecular phylogenetic tree based on mtDNA cox3 sequence and the species phylogenetic tree. But the results of Locusta nugratoria and Apis mellifera iigustica in molecular phylogenetic tree are different from those in species phylogenetic tree.The reproduction pattern and ecological characteristics should be considered when the molecular evolution was analysed based on mitochondnial gene sequences.