Cloning, identification and sequence analysis of Simulium rufibasis mitochondrial cytochrome oxidase subunit I(COI) gene

ObjectiveTo clone and identify the cytochrome C oxidase subunit I(COI)gene of Simulium rufibasis(S.rufibasis)in order to analyze the gene sequence.MethodsA pair of primers were designed and the template of adult S.rufibasis total DNA was used to amplify the gene with polymerase chain reaction and the amplification products were purified and cloned into the E.coli DH5α.The inserts of the positive clones were identified first with double digestion,then with colony PCR and further sequenced.The data were analyzed with nucleotide BLAST software of NCBI,and the features of COI gene sequence were determined.ResultsSequence analysis reveales that the open-reading frame of COI gene in S.rufibasis containes 1 542 bp,encoding 513 amino acids and the molecular weight is about 56 kDa and the isoelectric point is 5.84.Structural predication shows that the protein possesses a COI conservative domain and its composition(A,C,G and T)is 28.60%,36.90%,17.76%,and 16.74%,respectively.ConclusionThe COI sequence of S.rufibasis was cloned and identified.It will provide a base for further research.