The Role Of TRAF6 In The Immunity And Reproduction Of House Flies

Musca domestica is a globally visible Diptera insect.Its living environment often breeds a large number of pathogenic microorganisms.The long-term evolution has made Musca domestica have a strong innate immune system.Therefore,Musca domestica has gradually become a good model for studying innate immunity.In this study,we used Musca domestica as the research object,and screened the homologous gene MdTRAF6 of TRAF6 through the transcriptome data of Musca domestica.Its expression was up-regulated by bacterial stimulation.We speculated that it might be involved in the anti-bacterial immune response of Musca domestica.To further validate the role of MdTRAF6 in innate immunity in housefly,we examined changes in the expression of MdTRAF6 after immunostimulation and detected changes in the expression levels of antimicrobial peptides and corresponding transcription factors after interference with MdTRAF6.At the same time,we unexpectedly found that using the RNAi method to knock down the expression of MdTRAF6 gene in adult flies,the egg production and larval survival rate of Musca domestica were significantly affected.In addition,the subcellular localization expression vector of MdTRAF6 gene was constructed and transfected into SF9 cells to analyze the distribution of MdTRAF6 in cells.The experimental research results are as follows:1.qPCR and Western blot showed that MdTRAF6 was distributed in different tissues of larvae,with the highest expression in intestinal tract,followed by adipose body and the lowest expression in blood cells.2.Bacterial challenge test showed that the expression of MdTRAF6 was significantly increased after bacterial prick.3.After RNAi interfered with the expression of MdTRAF6 in Musca domestica larvae,the expression level of antibacterial peptide Cecropin was significantly up-regulated.The expression of Muscin and Attacin was significantly down-regulated compared with the control group,while the expression of Diptericin was down-regulated but not significant.The detection of transcription factors on the relevant pathways revealed that the expression of Relish was up-regulated,the expression of Dorsal was down-regulated,and the expression of JNK was down-regulated but not significant.4.The expression levels of MdTRAF6 in different developmental stages of Musca domestica are relatively stable,and the expression level is relatively stable throughout the larval stage,but highest in the alfalfa and adult stages.5.Interference with MdTRAF6 gene expression in the adult fly stage resulted in a significant decrease in the cumulative fecundity of female flies compared with the control group,and the survival rate of the first generation larvae was also significantly reduced.6.Subcellular localization of MdTRAF6 in SF9 cells showed that TRAF6 gene was mainly distributed in the cytoplasm of Musca domestica.