Molecular Cloning And Bioinformatics Analysis Of An Unknown Function Gene From Musca Domestica

Housefly (Musca domestica L.) belongs to Diptera, extremely broad distribution in the worldwide. As an important resource insect, housefly was widely used in chemical industry, agriculture, medicine, food, cosmetics, environmental protection, printing and dyeing, paper making, and carrier of enzyme immobilization and other fields. At present, a lot of research on the housefly has done in the fields of morphology, biology, ecological characteristics, immune mechanism and breeding, however, basic research in the field of molecular biology is less. An EST sequences does not exist homologous sequences in the database, it may be an unknown functional gene that unique to the housefly, so cloning and analysis the cDNA sequences of the unknown functional gene of Musca domestica is necessary, that can prepare for further study of the mechanism and function to the gene.In this paper, based on an EST sequences from housefly, its cDNA sequence was cloned by RACE; using online bioinformatics software, the character, structure and function of protein was predicted, the main findings are as follows:According to the specific primer GSP1 for 5'-RACE reaction, the 5'-end of UFGMD (726bp) was cloned; then design specific primer GSP2 to clone the 3'-end of UFGMD (308bp) through 3'-RACE reaction. Obtain an 824bp full-length cDNA sequence through splice the 5'-end and 3'-end of UFGMD. It contains an open reading frame with 588bp (49bp-636bp), encodes a protein with 195 amino acids.UFGMD pro-protein does not have a signal peptide, so not belong to secreted protein; remove the N-terminal methionine (M), the amino acid sequence is the mature UFGMD protein. Using the online analysis software, its theoretical molecular weight and isoelectric point, molecular formula, amino acid composition, disulfide bonds, molar extinction coefficient, aliphatic index, instability index, grand average of hydropathicity, phosphorylation sites were predicted.Comprehend secondary structure prediction and hydrophobic cluster analysis, the UFGMD protein contains 5 a-helices and 11β-strands; using the SAM-T08 to predict tertiary structure of the UFGMD, that has 6 a-helixs and 11 P-strands on the secondary structure level, and similar to the result of secondary structure prediction, increased by only one a-helix.The blastn analysis showed that:There is no high homology sequence with UFGMD gene, but partial sequences are high homology to S6k and S6kⅡ. FASTA analysis showed that:there is no sequence that homology more than 40% with UFGMD protein, only the centromeric histone from Drosophila pseudoananassae (NCBI accession number: AAL78894.1) is insect protein, the homology is 27.6%. Combined with subcellular localization analysis, UFGMD protein may play a role in the nucleus and the Golgi apparatus; its specific function needs to be further studied.