Study On Immune Response Of Antibacterial Peptide Genes From Lasioderma Serricorne Under Parasitic Stress Of Anisopteromalus Calandrae(Howard)

As an important part of immune effector molecules,antimicrobial peptides play an indispensable role in innate immunity.The cigarette beetle,Lasioderma serricorne is a worldwide stored insect pest,which could cause devastating damage to a variety of storage materials.To study the innate immune function and the immune response in parasitic mode,four antimicrobial peptide genes,including LsAtt2,LsCole,LsDef1,and LsDef2 were cloned based on the transcriptome of L.serricorne parasitized by Anisopteromalus calandrae.It is of great significance for the study of parasitic mechanism and the development and utilization of antibacterial peptides.1.Sequencing of transcriptome and screening of immune related genes in L.serricorne parasitized by A.calandraeThe illumina Hiseq ^TM4000 high-throughput sequencing technology was used to obtain the differentially expressed genes after 6 h and 24 h parasitized by the late larvae,and to further explore the changes of immune related genes.Based on L.serricorne transcriptome,a total of 398.01 Mb clean reads and 123,820 Unigenes were finally obtained.Among them,81,600,25,234,67,820,62,249,51,155,62,806,and 62,911 Unigenes were successfully annotated in Nr,Nt,KEGG,Pram,GO,KOG and Swiss-Prot database,respectively.According to the homology analysis of unigenes annotated in Nr database,17.91%of unigenes showed the highest homology with A.glabripennis,while 15.45%of unigenes have higher homology with related genes of Tribolium castaneum.GO enrichment analysis showed that 51,155 Unigene were annotated to 58 functional groups in 3 categories.KEGG enrichment analysis showed that 67,820 Unigenes were annotated.Moreover,1,133 Unuigenes belonging to 51 classes of immune recognition,signal transduction,regulation and effector molecules were screened.The expressions of Attacin and two defense genes were significantly up-regulated,while expression of coleoptericin gene was significantly down-regulated after parasitism at 6 h and 24h.2.Cloning and sequence analysis of four antimicrobial peptide genes from L.serricorneThe full-length ORF sequence of four antimicrobial peptide genes,including LsAtt2,LsCole,LsDef,and LsDef2 were cloned and verified by RT-PCR.The length of these ORFs were 507 bp,354 bp,261 bp,and 273 bp,encoding 167,117,86,and90 amino acids,respectively.The calculated molecular weight of these predicted protein were 18.37 k Da,12.99 k Da,9.40 k Da,and 9.48 k Da,and the isoelectric points（PI）were 9.12,8.24,5.39,and 5.49,respectively.Four antimicrobial peptides have signal peptide regions at the N-terminal,conserved domains of antimicrobial peptide families at the C-terminal,All these four predicted amino acids were hydrophilic,and evenly distributed predicted phosphorylation sites.The predicted proteins of LsDef1and LsDef2 exist glycosylation sites at the 38th and 29th amino acids.Phylogenetic analysis showed that the four antimicrobial peptide genes of L.serricorne were clustered into a branch with the genes of their respective families,and the antimicrobial peptides of Coleoptera formed a relatively independent branch.Among them,LsCole has the closest genetic relationship（58%）with Td Cole C of Trypoxylus dichotomus;The amino acid sequence of LsAtt2 was 87%consistent with that of the Tc Att belonging to T.castaneum;The two defensins（LsDef1 and LsDef2）were clustered together,and the genetic relationship was 57% related.3.Expression pattern analysis in different developmental stage,tissues,and bacterial induction of four antibacterial peptide genesThe q PCR was used to investigate the spatiotemporal expression patterns of four antimicrobial peptide genes in L.serricorne,the results showed that four genes were expressed to varying degrees in the different developmental stages and tissues.The expression levels of LsAtt2 in late larvae and pupae were significantly higher than those in other tested stages.LsCole,LsDef1 and LsDef2 were highly expressed in the late larval stages.Tissue-specific expression showed that the highest expression of LsAtt2 in midgut was 14.1-fold of that in brain,LsDef1 and LsDef2 were mainly expressed in midgut and epidermis,respectively.The expression of LsCole in brain was significantly higher than that in other tissues.Expression of LsAtt2 and LsCole was induced exposure to Staphylococcus aureus and Escherichia coli at three time points.LsDef1 and LsDef2 were not induced by E.coli,but only responded to the injection of S.aureu,reaching the highest level at 12 h and 24 h respectively,which was 7.1-and 8.9-fold of the control.4.Functional analysis of four antibacterial peptide genesThe expression of four antimicrobial peptide genes in L.serricorne were detected within 7 days after the late larvae was parasitized by A.calandrae.Compared with the control,LsAtt2 was continuously highly expressed and the highest expression was observed at the first day.The expression patterns of LsDef1 and LsDef2 were similar,both two LsDefs were highly expressed from first to the fifth day,but the expression level of LsDef1 was higher than that of LsDef2,no significant change was observed in LsDef2 at the sixth day.LsCole was significantly decreased on the second and third day,and had no significant change at other times.The expression levels of four antimicrobial peptide genes were effectively inhibited by RNA interference technology.The sensitivity test results of the fourth-instar larvae of L.serricorne exposure to E.coli and S.aureu found that after knockdown of LsAtt2 and LsCole,the mortality of L.serricorne after exposure to E.coli and S.aureu was significantly higher than that of control group.The LsDef2 and LsDef1 treatment groups were similar,and only the S.aureu treatment group had a significant increase in mortality.The results of the parasitization experiment after knockdown of LsAtt2,LsDef1,and LsDef2,the death time of the late larvae was advanced to some extent,but the Lscole was not significant different from the control.