Immune Response Of Different Pathogenic Microorganisms To The Main Immune Signal Pathway In Bombyx Mori

JAK/STAT, Toll, Imd and RNAi pathway are the major signaling pathwaysassociated with insect innate immune,To explore different responses in differentmicrobial infections to silkworm natural immune-related pathways.Inducing byE.coli,Sm,Bab,Beb,BmNPV,BmCPV,BmDNV and Nb, using real-time PCR to detectthe expression changes in Bmstat, Bmspz, BmPGRP-LB, BmPGRP-LE, Bmago2andBmdrc2,so that to further analysis the innate immune mechanism of Bombyxmori.Bombyx mori macula-like virus (BmMLV) is a kind of virus,which can recessiveinfection of Bombyx mori and cultured cells.The detection of Toll, Imd, JAK/STAT,RNAi four signaling pathway on immune response to BmMLV infection by real-timePCR,To study the mechanism of recessive infection of BmMLV,The main results are asfollows:1Inhibition of SOCS expression regulation on JAK/STAT pathwayIn order to investigate the silkworm BmSOCS2and BmSOCS6regulation effect onJAK/STAT signaling pathway, BmSOCS2, BmSOCS6specific siRNA was transfectedinto BmN cells of Bombyx mori, detection the influence of Bmstat expressionlevel,after BmSOCS2, BmSOCS6gene silencing.The results showed that,four kinds ofSOSC2siRNA silencing effect on BmSOCS2gene are not obvious, cause changes in theexpression level of Bmstat gene are not obvious.Cultured cells treat with socs6-48siRNA,the expression level of Bmsocs6gene decreased1.78times, while the expressionof Bmstat gene increased11.55times, suggesting that BmSOCS6is a negative feedbackregulator gene of Bombyx mori JAK/STAT pathway.2Immune responses of JAK/STAT pathway on bacteria, fungus and BmNPV In order to study immune response of silkworm JAK/STAT signal pathway inbacteria and fungus,5instar3days of silkworms were injected with inactivated E.coli,Sm and Bab, taken the experimental group and the control group of silkworm bloodafter6h,4instar silkworms were infection with Beb,taken the experimental group andthe control group of silkworm blood after96h, using real-time PCR to detect thetranscription level of Bmstat of the samples.The results showed that after differentbacterial and fungus infection, there are no significant changes of transcription levels inthe blood of Bmstat, speculated that the infection of fungus have response to theBombyx mori JAK/STAT signal pathway,but not the infection of bacteria.Blood cellsimmunohistochemistry results showed that, after BmNPV infection, the expression levelof Bmstat increased, the blood cells numbers increased, and coupled with the expressionof Bmstat,speculated that blood cell proliferation and differentiation are regulated byJAK/STAT pathway, BmNPV infection can activate JAK/STAT pathway.3Immune responses of Toll pathway on different microorganismsIn order to investigate the difference of immune response to different pathogens ofsilkworm Toll signaling pathway,different periods of Bombyx mori (Dazao) wereinduced by different microbial pathogens, the transcriptional level of real-time PCRdetection of Toll signal pathway receptor Bmspz.The results showed that,body injury byinjecting PBS may up regulate the transcription levels of Bmspz, after infection of Beband BmDNV, Bmspz expression level may up regulate, and the infection ofbacteria,BmCPV,BmDNV and Nb, the expression level of Bmspz did not obviouslyincreased, so we speculated that antifungal response by Toll signal pathway, and Tollpathway had no significant response on bacterial,some kinds of virus and Nb infection.4Immune responses of Imd pathway on different microorganismsIn order to investigate the difference in immune response to different pathogens ofsilkworm Bombyx mori Imd signaling pathway,different microorganisms were induced, the transcription level of real-time PCR detection of Imd receptors BmPGRP-LB andBmPGRP-LE. The results showed that, body injury by injecting PBS may up regulationtranscription level of BmPGRP-LB, Bombyx mori injection of G-bacteria E.coli andSm,the transcription level of BmPGRP-LB increased,silkworm infectedBmCPV,BmDNV and BmNPV, the transcription level of BmPGRP-LB increased, whileafter the Silkworm infected Nb,the transcription level of BmPGRP-LB had no obviouschange, but after the Nb infection, the expression level of BmPGRP-LE in the midgut ofsilkworm increased slightly.Infection of G+bacteria Bab, G-bacteria Sm and BmDNVcan upregulation transcriptional of BmPGRP-LE, but no significant changes inBmPGRP-LE transcription level in infected with other pathogenic micro-organisms. Sowe speculated that Imd signaling pathway is responsible for regulating the G-responseto bacteria, BmCPV, BmDNV and BmNPV infection may activated Imd signalingpathway by indirect method.5Immune responses of RNAi pathway on different microorganismsIn order to investigate the effect of RNAi signal pathway in response to infectionof different pathogens, silkworm by different microorganisms were induced, thetranscription level of real-time PCR detection of key RNAi signaling pathway genesBmago2and Bmdrc2. The results show, after injection of PBS, there is the significantchanges of the expression level of Bmdrc2,but not the Bmago2.after infected bydifferent virus, no significant change in the transcriptional level of Bmago2,significantwith the controls,after infection of BmCPV and BmDNV,no significant changes of theexpression level of Bmdrc2but not the BmNPV. BmCPV and BmDNV infection werefailed to improve the expression level of the Bmago2and Bmdrc2.Injection of E.colihad no significant change in the transcriptional level of Bmago2,but after induced bySm,Bab,Beb and Nb, the transcription level of Bmago2were4.79,1.52,2.08and1.56times,respectively.Induced by E.coli, Sm,Bab,Nb and Beb,the transcription level ofBmdrc2were2.95,5.86,5.10,2.03,1.42times, respectively.Showed that the virus infection could not activate RNAi pathway, some bacteria infection can activate RNAipathway.6BmMLV infection replication and host(cell) immune responseBombyx mori macula-like virus(BmMLV) is a kind of recessive infection ofBombyx mori and cultured cells of virus. The BmMLV genome is a6.5kb singlestranded+RNA. In order to study the mechanism of recessive BmMLV infection, thedetection of BmMLV infection on immune response to Toll, Imd, JAK/STAT andRNAi four signaling pathway by real-time PCR.The results showed that, after infectedwith BmMLV, the expression level of Bmstat, BmPGRP-LB, BmPGRP-LE, Bmago2,Bmdrc2increased significantly,showed that JAK/STAT, Imd and RNAi signal pathwayhad response to BmMLV infection, and the expression of Bmspz, gene had no obviouschange, speculated that Toll signaling pathway had no response to BmMLVinfection.Real-time PCR test results show that, BmMLV can be recessive infection Sf9cells, in mammalian cells (293N) are likely to maintain low levels of proliferation,andmaintain low in rats. Cells transfected with BmMLV RNA and full-length cDNA clone,can detect the virus proliferation, prove that the RNA and full-length cDNA cloneBmMLV have the ability of infection,but did not find the length of double and singlestranded cDNA fragments without obvious infection.37°C high temperature andmedium incorporation of mammalian cell medium in TC-100medium can promotevirus proliferation.