| microRNA (miRNA) is a kind of small non-coding RNA, it widely exists in eukaryotic cells and plays an important role in the fine regulation of gene expression and biological growth process. Some recent studies have shown that microRNA involved in the immune regulation in multiple aspects, however, the innate immune regulation mechanism of miRNA is still not clear, it needs to be further study indeeply. Drosophila melanogaster is an important model organism, and plays a very important role in biological research. The study of drosophila immune related microRNA recognition, validation and regulating is important,it’s not only important in development and the response mechanism of Drosophila melanogaster immune system, but also helps to understand the origin and evolution of microRNA in vertebrates. At present, the miRNA related to the immune were miR-8, miR-let-7 and miR-125 have been reported in Drosophila melanogaster. so, in order to identify more immune related microRNA, in this paper, we first construst the UAS-GAL4 system that was high expression of microRNA, then we further verification the system of UAS-GAL4 high expression of miRNA with miR-8 and miR-let-7.On the basis, we use the UAS-GAL4 high expression of miRNA system validated and selected immune related miRNA. This paper obtained the following main results:(1) Firstly, Through the da-Gal4, ppl-Gal4 genotype Drosophila melanogaster hybridization respectively with different miRNA mution genotype Drosophila melanogaster, then collect the da-Gal4/UAS-miRNA genotype Drosophila melanogaster, but we have not find it. This suggests that Da-Gal4 driven gene high expression in whole body may lead to the fruit flies death. But we have collect little the ppl-Gal4/UAS-miRNA genotype Drosophila melanogaster, by fluorescence quantitative PCR analysis, we found that the miRNA in high expression of miRNA offspring (ppl-Gal4/UAS-miRNA) significantly raised compared with the control group, it suggest the ppl-Gal4 can drive genes expressed in Drosophila melanogaster fat body, but the collection efficiency is very low.(2) In order to construst a stable expression system, we futher explore the Gal4-Gal80ts drive system, we use different UAS-miRNA genotype Drosophila melanogaster hybridization with Gal4-Gal80ts genotype Drosophila melanogaster. we first put the Drosophila melanogaster in 18℃ environment, after the offerspring eclosion, we collect corresponding genotype offspring to 29℃ environment to activate the Gal4 protein expression for overexpress miRNA.we found Gal4-Gal80ts systerm can activate the overexpression of miRNA in Drosophila melanogaster in a specific period. This Tub-Gal80ts;UAS-miRNA/Tub-Gal4 system make Drosophila melanogaster miRNA overexpression successful, and the effection is Significant.(3) In order to further verify the Tub-Gal80ts; UAS-miRNA/Tub-Gal4 system, we used miR-8 and miR-let-7, in the Tub-Gal80ts;UAS-miR-8/Tub-Gal4 and Tub-Gal80ts;UAS-miR-let-7/Tub-Gal4 Drosophila melanogaster, the miRNA expression was raised significantly contrast with control. And we also research the Toll and Imd Pathway in the Tub-Gal80ts;UAS-miR-8/Tub-Gal4 and Tub-Gal80ts;UAS-miR-let-7/Tub-Gal4 Drosophila melanogaster. And we found the Drosomycin was reduced significantly in Tub-Gal80ts;UAS-miR-8/Tub-Gal4 contrast with control. It suggest that miR-8 was regulate Drosophila melanogaster Toll Pathway through Drosomycin to effct the Drosophila melanogaster immune. And this is consistant with the previous study. In Drosophila melanogaster S2 cells, it also confirmed miR-let-7 will influence Imd pathway through effect the expression of Diptericin. But this research found the miR-let-7 have not inhibit expression of Diptericin in the body of Drosophila melanogaster.(4) We then use Bioinformatics technology analysis the Drosophila melanogaster miRNA that may be associated with immune, in view of the above analysis, we selected miR-34, miR-375 and miR-958, then use the Tub-Gal80ts;UAS-miRNA/Tub-Gal4 systerm to overexpress this miRNA, then by fluorescence quantitative PCR detection the expression of miRNA, Drosomycin and Diptericin. The results show that miR-34, miR-375 and miR-958 expression were significantly higher, but only in Tub-Gal80ts;UAS-miR-958/Tub-Gal4 drosophila melanogaster strains, Drosomycin expression is lower contrast with the control. It suggest that miR-958 is associated with the Toll immune signaling pathway, it regulate the immune in Drosophila melanogaster. This preliminary validation the immune funclion of miR-958. At the same time, we also further proves the Tub-Gal80ts;UAS-miRNA/Tub-Gal4 system is feasibility.This Tub-Gal80ts;UAS-miRNA/Tub-Gal4 system provides help for the miRNA overexpression in Drosophila melanogaster. And it provides an important basis for further study the immune function of miR-958. |
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