Transcriptomic Analysis Of Hemolymph And Cuticle Of Antheraea Pernyi After ApNPV Infection And Effect Of RCL Length On Apserpin-6 Inhibitory Activity

Insects have evolved efficient innate immune systems to defend themselves against the invasion of pathogens and parasites.When pathogenic microorganisms invade,insects will produce an immune defense response to the attack of pathogenic microorganisms.Antheraea pernyi is an unique biological resource in China.They maybe infected by various pathogenic microorganisms during the breeding process,aslo cause illness and death.Therefore,studying the molecual defense mechanism of A.pernyi against pathogenic microorganisms is of great significance for elucidating the immune interaction mechanism between pathogenic microorganisms and insects,and for tapping disease-resistant germplasm resources of A.pernyi for genetic improvement.On the other hand,the insect innate immune response process requires the participation of multiple serine proteases(SPs)to ensure the transmission and expansion of immune signals,and serpins inhibit the activity of serine proteases(SPs)during this process.In order to make the immune response proceed rapidly and violently,but limit it to a certain degree and scope,to protect the host from harm.In this study,A.pernyi nucleopolyhedrovirus(Ap NPV)was used to infect A.pernyi larvae.Two database of A.pernyi larvae hemolymph and cuticle transcriptome were established to study the gene expression of A.pernyi infected with pathogenic microorganisms,to unravel the cellular and humoral immune responses of A.pernyi larval hemolymph and cuticle to Ap NPV.At the same time,the effect of systematic changes in Serpins reactive central loop(RCL)length on the inhibitory activity of recombinant serine protease inhibitor(Apserpin-6)was studied.The results are as follows.1.Comparison of gene expression levels revealed that a total of 3191 differentially expressed genes(DEGs),including 1107 up-regulated and 2084 down-regulated genes in Ap NPV-infected hemolymph.Of these,1416 and 982 DEGs were enriched in 3253 Gene Ontology terms and 258 KEGG pathways,respectively.DEGs involved in immunity related pathways were selected for further analysis.We screened 184 and 63 DEGs involved in cellular and humoral immune-related pathways,respectively,via KEGG analysis.Among the cellular immune-related pathways,autophagy,endocytosis,and lysosome were activated based on the expression profiles of several key regulatory genes,such as the up-regulation of Atg3 and LPS-induced TN factor,and the down-regulation of PIK3R4,HSC70 and V-ATPase.We concluded that phagosome,apoptosis,and ubiquitin-mediated proteolysis were suppressed by Ap NPV as a result of down-regulation of most related DEGs.The majority of the DEGs enriched in Toll,Jak-STAT,MAPK,NF-κB and Insulin signaling pathways,complement and coagulation cascades,and melanogenesis were down-regulated,demonstrating that Ap NPV infection greatly inhibited humoral immunity in the hemolymph of A.pernyi larvae.Our findings may serve as a basis for further research on the antiviral molecular mechanisms of A.pernyi.2.Baculovirus causes liquefaction of insect cuticle to enhance the dissemination of progeny virionsaway from the host cadavers for increasing viral transmission rates.Ap NPV infects A.pernyi larvae with circular pus blotches formed in cuticle in the early stage of liquefaction.To investigate the formation mechanism of those pus blotches,the transcriptome profile changes of the cuticles between Ap NPV-infected and non-infected A.pernyi larvae were analyzed using RNA-Seq.The transcriptome was de novo assembled using the Trinity platform.Comparison of gene expression levels revealed that a total of 2990 and 4427 unigenes were up-and down-regulated respectively in Ap NPV-infected cuticle,of which 2620 and 1903 DEGs could be enriched in different GO terms and KEGG pathways.In this study,we focused on chitin metabolism related DEGs,and screened 10 genes involved in chitin synthesis and degradation with down-regulated trends,indicating that the chitin metabolism pathway was inhibited by Ap NPV infection,which may promote liquefaction of A.pernyi cuticle.Besides,we also identified a large number of DEGs involved in immune related pathways via KEGG analysis,indicating that intense immune responses occurred in A.pernyi cuticle.Our research findings will serve as a basis for further researching the molecular mechanisms underlying cuticle liquefaction of A.pernyi induced by Ap NPV infection.3.This paper evaluated the effect of systematic changes in RCL length,N-terminal to the reactive center,on the inhibitory activity of recombinant Apserpin-6.Four Apserpin-6 variants proteins with RCL deletion or insertion residues were obtained,and their ability to inhibit pro PO activity in pernyi hemolymph was studied.The domain prediction results indicate that there is a reactive central loop(RCL)between 359-379 amino acid residues at the C-terminus of Apserpin-6.The predicted protein cleavage sites are between 374 serine(P1)and 375 serine(P1 ’).The N-terminal variable region P7-P1 of Apserpin-6 RCL was truncated or lengthened by base deletion or insertion through site-directed mutagenesis to construct four Apserpin-6 variants.The c DNA fragments of Apserpin-6 with different RCL lengths were cloned into p ET28a(+)expression vector by Bam HI/Xho I digestion,and named as p ET28a(+)-A+(one Ala insertion),p ET28a(+)-AA+(two Ala insertion),p ET28a(+)-F-(Phe deletion)and p ET28a(+)-GF-(Gly and Phe deletion),repectively.The recombinant protein of the Apserpin-6 variant was added to A.pernyi lymph fluid and incubated.Pro PO activity was detected by monitoring the absorbance at 490 nm by ELISA.Pro PO activity was compared at different recombinant protein concentrations and incubation times in hemolymph.The results indicate that the length of RCL affects the inhibitory activity of Apserpin-6.The recombinant Apserpin-6 with one or two residues addition in RCL had no effect on pro PO activity,whereas the deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6.The results of this study will facilitate the understanding of inhibition mechanism of RCL on pro PO activity.