Immune Response Of Intestinal Reactive Oxygen Species And Peptidoglycan Recognition Protein Genes In Antheraea Pernyi

The Chinese oak silkworm(Antheraea pernyi Guérin-Méneville,1855)which belongs to Lepidoptera: Saturniidae,is well known wild silkworm for insect food and silk production with a long history of domestication.Owing to its feeding in the wild,the larval stage would be threatened with various pathogenic microorganisms,which can cause the outbreaking of the silkworm disease and restrict the development of sericulture.The gut lumen not only has the function of digesting and absorbing nutrients,but also as the immune barrier resisting the pathogen infection.Research on the interaction mechanism between intestinal immune system to microbial pathogens of A.pernyi,not only by helping us to clarify the immune regulatory mechanism,but also by providing theoretical foundation for insect immune molecular diversity.Based on the transcriptome database of A.pernyi midgut,combining with data resource advantages in Drosophila melanogaster and Bombyx mori,providing the basis for integrated research on A.pernyi intestinal immune system.RT-PCR strategies were adopted to clone the four peptidoglycan recognition protein genes,namely Ap PGRP-A,Ap PGRP-B,Ap PGRP-C,Ap PGRP-LE.The spatiotemporal and immune-induced expression profiles of four genes were analyzed by RT-q PCR.To investigate the effects of amidase-type PGRPs on the expression of antimicrobial peptides and its regulatory mechanism.Inspection analysis of ROS levels and the induction of NO concentration was conducted,as well the expression levels of Ap NOS1,Ap NOS2,Ap NOS3 and enzyme activity in response to microsporidium infection.The results are as follows:1.Four c DNA clones encoding PGRPs(Ap PGRP-A,Ap PGRP-B,Ap PGRP-C,Ap PGRP-LE)were isolated from the Chinese oak silkworm,Antheraea pernyi(Gen Bank accession number: KT725231.1,KT725232.1,KT725233.2,KT725234.1).The c DNA sequence of Ap PGRP-A,Ap PGRP-B,Ap PGRP-C,Ap PGRP-LE is 582 bp,597 bp,627 bp,1341 bp and its open reading frame(ORF)encodes 193,198,208,446 amino acids respectively.Bioinformatics analysis shows that the sequence of Ap PGRP-A,Ap PGRP-B,Ap PGRP-C contain signal peptides.The deduced amino acid sequences of Ap PGRP-B and-C share high identity with enzymatically active PGRP proteins and contain the amino acids required for amidase activity.Ap PGRP-LE has no signal peptide,exhibits as a intracellular protein.Four Ap PGRPs possess a conserved PGRP domain and share high identities of 51%-70% with homologous sequences from other lepidopteran insects.2.The spatiotemporal expression patterns of ApPGRPs and their response to immune stimulations were determined.Ap PGRPs are generally expressed in cuticle,Ap PGRP-B was predominantly expressed in midgut with a higher transcript,the transcript of Ap PGRP-C distributes in immune related tissues such as hemocyte,midgut and fat body,Ap PGRP-A,-C and-LE are expressed in the fat body.The expression level of Ap PGRPs exposure to microbial pathogens suggests that amidase-type Ap PGRP-B and-C act as a broad-spectrum patternrecognition protein and exhibit higher transcriptional levels in response to the Enterococcus pernyi immune challenge.Here,using an RNA-interference approach,we examined the function of amidase-type PGRPs in response to immune stimulations.We observed that RNAimediated silencing of Ap PGRP-A decreased the tested antimicrobial peptides(Attacin,Attacin 2,Ceropin,Gloverin,lysozyme and Lebocin)in response to E.pernyi challenge.However,reducing the Ap PGRP-B,-C m RNA levels led to a strong increase of the AMP genes(except for Lebocin).These results suggest that Ap PGRPs are necessary for pathway initiation complex formation,and activate AMPs generation.Our data also indicated that amidase-type PGRP-B and-C down-regulate AMPs generation in the immune response.3.In this study,we characterized the temporal and spatial expression of the Antheraea pernyi nitric oxide synthase gene and explored the role nitric oxide plays in response to microsporidia infection.The present findings demonstrated that challenge with the entomopathogenic Nosema pernyi increased nitric oxide and hydrogen peroxide production by 8.19-and 3.49-fold at 3 h PI.The gene expression of Ap NOSs induced by Nosema pernyi was investigated that Ap NOS2 elevated by 27.88-fold at 3 h PI,this initial intestinal response of the host to pathogen is rapid and transient.We speculated that the synthesis of nitric oxide was regulated by Ap NOS2.The spatial and temporal expression patterns indicated that Ap NOS1 and Ap NOS3 were constitutively expressed in hemocytes,malpighian tubule,midgut and gonad;Ap NOS2 expressed only in malpighian tubule,gonad and can be inducible in response to N.pernyi infection.4.Injection of exogenous nitric oxide donor SNP,which contributes to the generation of nitric oxide,without immune challenge significantly induced the m RNA levels of AMP genes in the gut.While the inhibitory treatment of a pharmacologic nitric oxide synthase inhibitor,L-NAME,alone did not influence the AMP expression.However,co-treatment of N.pernyi with L-NAME,suppressed the induction of AMP expression.When injected SNP with the N.pernyi infected silkworm,we found the expression of some antimicrobial peptides Defensin,Gloverin,Lebocin were elevated.Our investigation revealed that the elevated intestinal nitric oxide production in response to the microsporidia infection led to AMP gene transcription,which participates in the management of the systemic immune responses.Inhibit NO production could restrain the transcription of some antimicrobial peptide;conversely,increasing NO production could enhance the immune effect.Through this topic,the peptidoglycan recognition protein and nitric oxide synthase genes were identified,PGRP and NOS expression patterns of the oak silkworm in response to the immune challenge were studied at the molecular level,which provided theoretical basis and molecular basis to understand evolutionary status and functional diversity of pathogen recognition receptor genes in A.pernyi and even lepidoptera insects.In addition,it also provides favorable clues for effective prevention and control of agricultural pests.