Relish Regulates ATG5 And Involves In The Anti-bacterial Process In Antheraea Pernyi

The innate immunity is the key process to defence pathogens infection in insect.The previous experiment results showed that as the component of Gram-negative bacteria's cytoderm,LPS up-regulated several autophagy related genes' expression during the induction of innate immune response.In the present study,ATG5 were cloned,expressed and analyzed to reveal its function in defending Gram-negative bacteria infection in Antheraea pernyi.Furthermore,the regulation between Relish,the key nuclear factor ?B(NF-?B)family transcription factors,and autophagy related genes were analyzed to investigate the regulation relationship in innate immunity and autophagy.The results were listed as follows:1.The A.pernyi ATG5 gene was cloned and prokaryotic expressed,and the ATG5 polyclonal antiserum was prepared.The full length of A.pernyi ATG5 gene was 5557 bp with five exons and four introns.The open reading frame(ORF)of ATG5 was 795 bp in length,which encoded a 264 amino acid protein and contained a APG5 functional domain.The amino acid sequence analysis indicated that A.pernyi ATG5 was conserved in insects which exhibited more than 90% similarity with other insects like Manduca sexta,Bombyx mori and Helicoverpa armigera.The prokaryotic expression vector pET-28a(+)-ATG5 was constructed,and the recombinant ATG5 protein was expressed and purified to prepare the rabbit polyclonal antiserum.The prepared ATG5 polyclonal antiserum was verified for the further study.2.The expression profiles of ATG6,ATG12 and ATG5 in the LPS induced innate immune response.After LPS injected to induce innate immunity in A.pernyi,the expression of ATG6,ATG12 and ATG5 were all significantly up-regulated.These autophagy related genes up-regulation may participate in the innate immune response to defence the Gram-negative bacteria infection in A.pernyi.3.The functions of A.pernyi ATG5 was investigated in response to defence the Gram-negative bacteria infection in innate immunity of A.pernyi.In the ATG5 interfered A.pernyi,the injection of Gram-negative and intracellular infection bacteria,Shigella flexneri,significantly increased the colony-forming unit(CFU)with dozens of times difference in A.pernyi hemocytes.Additionally,the survival rate of A.pernyi also obviously decreased after ATG5 RNAi following with S.flexneri injection.The over-expression vectors were constructed and transfected into Bm N cells to co-express ATG5-GFP and ATG12-mCherry proteins.Similarly to the results in autophagy activator Rapamycin treatment group,co-expression Bm N cells displayed accumulated and enhanced co-localized fluorescence dot(GFP and mCherry)and increased the number of co-localized dots.These findings suggest that LPS can trigger ATG5 and ATG12,promoting anti-microbial autophagy in response to Gram-negative bacteria infection.4.Relish regulated the transcriptional expression of autophagy-related gene and involved in innate immune response.The mRNA expression level of ATG6 and ATG8 were significantly reduced after Relish RNAi 24 h in A.pernyi.ATG5 mRNA and protein level both decreased significantly in Relish RNAi following with LPS injection.These results indicate that Relish,which activated by innate immune response,could regulate ATG5 transcription and involve in the pathogens defence process.5.The transcriptional regulation of ATG5 by the Rel domian in the N-terminal of Relish was verified.The four potential NF-?B transcription factor binding sites(630 bp,774 bp,1858 bp,and 2071 bp)in the ATG5 promoter were predicted by JASPAR,which is located upstream of the ATG5 locus.The Firefly luciferase report gene vector was constructed with ATG5 promoter,and the N-terminal Relish(Rel-N)was transfected and over-expressed in Bm N cells.The results of the dual-luciferase reporter assay revealed that cells co-transfected with the Rel-N and ATG5 promoter had significantly higher relative luciferase activity,indicating that the Rel domain in the N-terminal of Relish could bind to the ATG5 promoter and regulate its transcription.Our results revealed that the NF-? B transcription factor,Relish,regulates the ATG5 gene expression and activates the autophagy process to involve in the pathogen elimination and maintain self survival in A.pernyi defending the Gram-negative bacteria infection.The related study will enrich the research of insect innate immunity and deepen the understanding of the cellular degradation process involved insect immunologic homeostasis.