Screening And Functional Study Of Key Genes Of Crosstalk In Drosophila Immune Pathway

The Drosophila melanogaster is one of the best-studied model animals in insects,and its immune system has been used as a model to study the innate immune systems of both insects and humans.Although a lot of achievements have been made in the study of drosophila immune response pathways,the physiological phenomena of innate immune system in response to immune stimulation cannot be fully and accurately reflected.One of the important factors is that these pathways that respond to immune stimuli are not completely isolated,but interact with each other through some genes or relationships between genes,or even with other non-immune pathways,forming a network of signaling and response.The change of one pathway will also affect other pathways,forming the crosstalk effect between pathways.Our research group used high-throughput experimental data after immune stimulation to drosophila and constructed the crosstalk network and the corresponding gene interaction network after immune stimulation in drosophila.On this basis,a number of candidate genes related to the innate immune pathway crosstalk in drosophila were screened,and 24 mutant strains of candidate genes were obtained from the Bloomington Drosophila database.In this work,Micrococcus luteus,a gram-positive bacterium,was used to microinject the mutant line of drosophila and detect the survival rate of drosophila at 24 hours after immune stimulation.The mutant strain of Drosophila melanogaster with significantly reduced survival after immune stimulation compared with the control strain wild type drosophila w¹¹¹⁸,and it was screened through repeated experiments.It was confirmed that the survival rate of apolipoprotein?apolpp?deficient drosophila was significantly lower than that of control strain w¹¹¹⁸.The defect type drosophila is by inserting the Minos transposable elements caused damage to DNA sequences,to detect apolpp gene is damaged,we amplified the Minos element sequence of the insertion sites by PCR amplification,and found that in the mutant strain apolpp genes encoding apolpp ? part of protein sequences appear early termination codon,the change would lead to apolpp ? protein cannot normal synthesis.In order to test the resistance of apolpp defective drosophila to bacterial infection,we conducted statistics on the bacterial colony load in apolpp mutant strains of drosophila after injection of Micrococcus luteus.It was found thatthe number of Micrococcus luteus in apolpp mutant strains was significantly higher than that of w¹¹¹⁸.After the immunodeficiency mutant drosophila apolpp strain was screened through survival rate experiment,we conducted transcriptome sequencing on the apolpp mutant strain and control strain w¹¹¹⁸ after stimulation by Micrococcus luteus and PBS,and conducted bioinformatics analysis on the transcriptome sequencing results.Through the identification of differentially expressed genes,significant differentially expressed genes were screened out among the four groups of samples,and functional annotation and pathway enrichment analysis of GO and KEGG were conducted respectively.It was found that w¹¹¹⁸ detected serine protease activity and "chitin binding" function after immune stimulation,but not in the apolpp mutant.In addition,the "folic acid biosynthesis" pathway in apolpp mutant after M.luteus infection was significantly activated,while the differentially expressed genes in the "starch and sucrose metabolism" and "galactose metabolism" pathways were both up-regulated in w¹¹¹⁸,but there was no significant change in apolpp mutant.By mapping differentially expressed genes into a previously constructed gene interaction network,we analyzed the genetic network changes in the immune response of apolpp defective flies and verified the results by q RT-PCR.The results showed that apolpp played an important role in the process of resisting Micrococcus luteus.We also observed abnormally high expression of the PGRP-SC1 receptor gene and significant inhibition of some of the anti-gram-negative peptides in the IMD pathway in the apolpp defective drosophila.This conclusion was demonstrated in a survival trial of the apolpp mutant strain injected with Escherichia coli.It indicated that the apolpp gene had an important effect on the activity of IMD.To further study the role of apolpp in the pathway crosstalk,we analyzed the transcriptome data and verified it by q RT-PCR experiment,and found that bacterial infection would significantly inhibit the expression of apolpp.To investigate whether the expression of apolpp gene is directly regulated by the innate immune pathway,we tested the changes in the expression of apolpp after immune stimulation of mutant strains of the three important transcription factors dorsal,dif and relish of Toll and IMD pathways.The expression of apolpp in all mutant strains decreased,which was consistent with the control w¹¹¹⁸.One possibility is that the Toll and IMD pathways may not directly regulate the expression of apolppthrough transcription factors.Another situation is that there is a redundant relationship between the three transcription factors.Only one mutation does not affect the regulation of apolpp.Our results indicate that apolpp is a key gene in crosstalk of drosophila innate immune pathway and plays an important role in drosophila response to bacterial infection.