Construction Of A SSH Library Of Musca Domestica Larva Upon Infection With Salmonella And Analysis Of The Differentially Expressed Genes

Various kinds of Musca domestica antimicrobial peptides that had already researched was a kind of viscous small molecule polypeptide,which had antibacterial activity,and the special properties made it become an important component of various organisms with innate immune defense system.The Musca Domestica that in the bacteria poor-living environment can behave surplus stronger adaptive survival ability was because of its relatively perfect innate immune system,forming a special mechanism against external harmful microbes invading defense.Therefore,the research of Musca Domestica Immune has become the hot spot and new direction.We constructed subtracted cDNA libraries between the infected Musca Domestica and uninfected Musca Domestica with the same age (three-day-old) and the same raising condition combined with suppression subtractive hybridization technology,PCR and bioinformatics method in order to clone and identify the differentially expressed genes.Using 5'RACE and 3'RACE to angle for Salmonella to induce Musca Domestica larvae library screening Musca Domestica defensin,aggression,no difference in homology cDNA fragments' full-length gene,and then,conduct bioinformatics analysis with the obtained full-length gene;The findings are as follows:(1)Using suppression subtractive hybridization technology to build the forward and reverse suppression subtractive hybridization library.Using induced Housefly larvae with Salmonella as tester(driver)and normal Musca domestica larvae as driver(tester).The forward and reverse suppression subtractive hybridization library of 500 clones were randomly selected by PCR,subtractive library of monoclonal recombinant rate was 92% and 91%,differential gene fragment was 0.2kb-1.Okb.(2)Using PCR amplification of secondary products of subtracted cDNA as a probe,The two subtractive cDNA library clones were positive for high-throughput screening.In the subtracted cDNA library,194 differentially expressed clones were sequenced,Further analyses showed they present 30 the antimicrobial peptide genes in Musca domestica,27 are unknown in the housefly,other 137 have a high similarity with Musca domestica and species genes,reversed subtractive cDNA library,71 differentially expressed clones were sequenced, including 66 with the housefly genes homologous sequences,5 are unknown sequence in housefly.(3)Using SMART cDNA RACE amplification technique,successfully cloned three full-length gene differentially expressed genes in Musca domestica:defensinI,full-length cDNA 489bp,ORF is 282bp; defensinΠ,full-length cDNA 474bp,ORF is 276bp;attacin,full-length cDNA 883bp,ORF is 719bp.(4)Using CLUSTALW and DNAman softwares,we analyzed the gene structure,protein structure and fuction of genes defensinI,defensinΠ,attacin and unhomologous. Musca domestica defensin gene and reported Musca domestica, Lucilia flies, stable flies sting relatively high homology with Drosophila melanogaster, Eristalis tenax defensin protein low homology, the encoded protein has the amino acid level highly conserved. attacin gene of Drosophila, gill tsetse flies, Harmonia axyridis, silkworm, Lucilia sericata and the gene encoding the amino acid green bollworm and other insects attacin Diptera similarity is low.