Study On The Mechanism Of MiR-959 In The Immune Response Of Drosophila Toll

Drosophila melanogaster,as a model organism studying innate immune mechanism,has played a milestone role in the history of immunology.The innate immunity of drosophila melanogaster has been roughly understood by scientists.The innate immunity of drosophila melanogaster includes humoral immunity,cellular immunity and melanogenesis,among which Toll immune signal pathway and Imd immune signal pathway are the most important humoral immune modes in drosophila melanogaster.Toll pathway is one of the most important pathways in drosophila.The mechanism of action is to resist the invasion of grampositive bacteria and pathogens through a series of signal transduction and cascade reactions.A large number of studies have shown that microRNA(miRNA)plays an important regulatory role in the process of immune signal transmission in drosophila.miRNA is a small non-coding RNA widely distributed in eukaryotes.It is about 20 nucleotides in length and regulates the expression of genes at the post-transcriptional level by binding to specific target sites of target gene mRNA.Various physiological activities of the organism.However,at present,although the research reports on the mechanism of Toll signaling pathway have been thoroughly studied,the research on miRNA related to Drosophila Toll signaling pathway is not clear enough.Therefore,this thesis uses the combination of experimental biology and bioinformatics to design the following experiments to determine whether miRNA can affect the fruit fly Toll signaling pathway.It has been demonstrated in vivo and in vitro that Drosophila miR-959 can directly target the antibacterial peptide tube gene to regulate the Toll signaling pathway.The main experimental results obtained in this paper are as follows:1.Using Tub-Gal80ts/+;Tub-Gal4 system Drosophila hybridized with UAS-miR-959/960/961/962 Drosophila to obtain Drosophila strains with high expression of miR-959/960/961/962,detected by real-time fluorescence quantitative(qRT-PCR)High expression of antibacterial peptides in Drosophila after infection with Gram-positive M.luteus(0h,6h,12 h,24h,48 h and 72h)confirmed the immunological correlation.Subsequently,the expression levels of antibacterial peptide Drs at the RNA level of miR-959,miR-960,miR-961,and miR-962,respectively,were detected.The results showed that the expression level of Drs was significantly decreased at 12 h in miR-959.Furthermore,the miRNA expression of the miRNAs in the miR-959/960/961/962 group of Drosophila was detected in the high expression of Drosophila,and further confirmed by Western Blotting experiments to determine that miR-959 is immunologically relevant.2.Gram-positive bacteria were used to stimulate wild-type Drosophila,and the expression level of miR-959 in Drosophila was detected at the time points of RNA(0h,3h,6h,12 h,24h,48h),and expressed at 6 time points.The amount was up-regulated and the expression level of miR-959 was significantly increased at 12 h,reaching a peak value,indicating that miR-959 may be immunologically related to Toll signaling pathway in wildtype Drosophila after being stimulated by positive bacteria.3.To further investigate the mechanism of miR-959 affecting the Drosophila Toll pathway,we predicted that the gene that miR-959 might target was tube by TargetScan and miRanda two bioinformatics prediction software.4.The target relationship between miR-959 and tube genes was again verified by qRTPCR in Drosophila,further confirming that miR-959 inhibits the expression of antimicrobial peptides by targeting tube.5.The target relationship between miR-959 and tube gene was verified by qRT-PCR in Drosophila,further confirming that miR-959 inhibits the expression of antimicrobial peptide by targeting tube.6.Construction of a regenerative drosophila with high expression of miR-959 and miR-959 sponge,and further determination of miR-959 negative regulation of Toll pathway in Drosophila.In summary,by constructing a high-expression Drosophila strain of miR-959/960/961/962 cluster miRNA,we studied miRNAs that regulate Drosophila Toll immune signaling pathway,and elucidated that miR-959 can act through the target gene tube 3 ’UTR,which regulates the expression of the antimicrobial peptide Drs to negatively regulate the Toll signaling pathway.A related research reference has been added to explore the functional database of miRNAs in innate immunity.