Identification And Physiological Functions Analysis Of Four Clip-domain Serine Protease Genes In The Cigarette Beetle,Lasioderma Serricorne

As essential components of extracellular signaling cascades,clip-domain serine proteases?CLIPs?play essential roles in insect development and innate immunity.In this study,the full-length cDNA sequences of four CLIP-encoding genes?LsCLIP1?LsCLIP2?LsCLIP3 and LsCLIP4?were cloned from the cigarette beetle,Lasioderma serricorne?Fabricius?.The molecular characteristics and functions of the four CLIP-encoding genes were explored.The results will provide a reasonable basis for the effective management of L.serricorne.The results are as follows:1.Identification and sequencing analysis of four LsCLIP genesBased on the L.serricorne transcriptome,four distinct cDNA fragments putatively encoding CLIP proteins,were identified by keyword searching.We subsequently cloned the full-length ORFs of four LsCLIP genes by RT-PCR.The ORFs of the four LsCLIP1,LsCLIP2,LsCLIP3 and LsCLIP4 genes were 1,194-bp,1,194-bp,1,173-bp and 1,158-bp in length,corresponding to 397,397,390 and 385amino acid residues,respectively.The calculated molecular weight and pI of LsCLIP1,LsCLIP2,LsCLIP3 and LsCLIP4 were 44.3,43.4,43.0 and 42.3 kDa,and 6.18,5.19,5.85 and 6.10,respectively.These results indicate that four LsCLIPs were secretory proteins.Domain analysis showed that four proteases contained a clip domain and a conserved trypsin-like serine protease domain?Tryp_SPc domain?at the C-terminal region,with six cysteine residues forming three disulfide linkages found in all clip domains.Multiple alignment of CLIP amino acid sequences from L.serricorn,Manduca sexta,Tribolium castaneum,and Anopheles gambiae conducted using DNAMAN version 6.03 showed high homology among the sequence,with several conserved motifs discovered.These included the six cysteine residues in the clip domain,the catalytic His-Asp-Ser triad,and the cysteine residues in the Tryp_SPc domain.The phylogenetic analysis was performed by MEGA version 6.06 using neighbor-joining method with 1000 bootstrap replicates and pairwise deletion.The result suggested that LsCLIP1 and LsCLIP2 were divided into the same subfamily C,LsCLIP3 was divided into the subfamily B,LsCLIP4 was divided into the subfamily D.2.Relative expression levels of temportal and spatial and 20E of the four LsCLIP genesTo further study the function of the four LsCLIPs,we examined the temportal and spatial expression patterns of the two identified genes.The results showed that four genes were expressed in different developmental stages,and that expression levels varied significantly throughout the five developmental stages.LsCLIP1 and LsCLIP2 had a similar expression patterns,with the highest levels of expression detected during the pupal stage,and the lowest levels observed at the late adult stage;LsCLIP3 and LsCLIP4 also had a similar expression patterns,with the highest levels of expression detected during the early adult stage,and the lowest levels observed at the late adult stage.qPCR analysis also showed that four LsCLIP genes were expressed in all tissues examined from the 5th instar larvae,with particularly high expression in the cuticle and low levels of expression in the gut.The four LsCLIPs transcripts were also abundant in the fat body and carcass,the lowest levels of expression detected in the tissue of gut. qPCR analysis was also carried out to determine the effect of 20E exposure and immune challenge on the mRNA levels of the four LsCLIP genes.Compared with the control,the mRNA levels of four LsCLIP genes were significantly increased following 20E treatment.Peak expression occurred at 8 h post-injection,with a13.68-fold increase for LsCLIP1 expression,a 43.51-fold increase in LsCLIP2expression,a 79.75-fold increase in LsCLIP3 expression and a 2.33-fold increase in LsCLIP4 expression observed at this time point.The results suggest that LsCLIP genes play an essential role in insect development.3.Relative expression levels of the four LsCLIP genes following peptidoglycan infection and after Anisopteromalus calandrae parasitized on Lasioderma serricorneqPCR was employed to explore the effect of the four LsCLIPs expression profiles after Anisopteromalus calandrae parasitized on Lasioderma serricorne.To larval,the results showed that LsCLIP1,LsCLIP2 and LsCLIP3 had a similar expression patterns,with the high levels of expression detected after parasitized,while there is little influence on LsCLIP4.The result is similar to pupa after Anisopteromalus calandrae parasitized on Lasioderma serricorne.To examine whether the four LsCLIPs are immune-responsive genes in L.serricorne,the transcript levels of four genes following PGN-SA or PGN-EB injection were detected by qPCR.LsCLIP1,LsCLIP2 and LsCLIP3 transcript level were significantly up-regulated PGN-SA or PGN-EB injection expect LsCLIP4.4.The physiological function analysis of LsCLIPs by RNAiRNAi was applied to explore the biological functions of LsCLIPs in L.serricorne molting development.Unique nucleotide regions in the molting transcripts,which were identified by comparing them with other CLIP-encoding sequences,were selected for dsRNA synthesis.To investigate the role of LsCLIPs in the molting development of L.serricorne,dsRNAs for LsCLIPs?dsLsCLIPs?and GFP?dsGFP,control?were synthesized in vivo and injected into 5th instar larvae.For the 5th instar larvae,the LsCLIPs mRNA levels were reduced after dsLsCLIP1,dsLsCLIP2,dsLsCLIP3 and dsLsCLIP4 injection,respectively,compared with the LsCLIPs mRNA levels for the control insects after dsGFP injection.In the control group,96.67%of larvae molted normally to pupae during the larvale-pupal transition,whereas the larvae injected with dsLsCLIPs showed obvious malformations,failed to complete the larvale-pupal molting,and eventually died.The accumulative survival rate of larvae injected with dsLsCLIPs was significantly lower from 2 to 5 days post-injection compared to the controls.The abnormal phenotypes could be divided into two types:?1?Individuals displayed light color but the insect body was strongly constricted by old cuticle,they failed to develop into pupae and finally died after dsLsCLIP1,dsLsCLIP2,dsLsCLIP3 and dsLsCLIP4 injection,respectively;and?2?Individuals displayed strong color but the insect body was strongly constricted by old cuticle,they failed to develop into pupae and finally died after dsLsCLIP1,dsLsCLIP2,dsLsCLIP3 and dsLsCLIP4 injection,respectively.To further investigate the role of LsCLIPs in the innate immunity of L.serricorne,dsLsCLIPs after LC₅₀ Escherichia coli 24 h and LC₅₀ E.coli?control?24h were injected into 5th instar larvae.For control,the mortality was 48.89%,and the motality increase 25.26%,25.26%,40%and 8.89%for LsCLIP1,LsCLIP2,LsCLIP3and LsCLIP4,respectively.The result suggests that LsCLIP1,LsCLIP2 and LsCLIP3play essential role in innate immunity of L.serricorne,LsCLIP4 probally is none of innate immunity concern.In summary,we identified and characterized four CLIP-encoding genes?LsCLIP1,LsCLIP2,LsCLIP3 and LsCLIP4?based on transcriptome database of L.serricorne.Different bioinformatics software were employed to analyzed these four sequences.Relative expression levels of temportal and spatial.Transcription of the four genes was significantly increased by exposure to 20E and following challenge with PGN-EB or PGN-SA and after Anisopteromalus calandrae parasitized.RNA interference technology was performed to investigate the functions of LsCLIP genes.These results provide valuable information for further study on the new strategies for pest control.