Identification Of Chitin Deacetylase 8 And Functional Analysis Of Response To BmNPV Infection In Silkworm,Bombyx Mori

The silkworm,Bombyx mori,is an important economic insect and an ideal model organism for the study of Lepidoptera.B.mori nuclearpolyhedrosis virus(BmNPV)is one of the most serious diseases affecting silkworm production,which severely restricts the development of sericulture.Peritrohic membrane(PM)is the first barrier for exogenous substances to enter the silkworm through the digestive tract.Chitin is an important component of PM,and chitin deacetyl ases(CDAs)are enzymes that catalyze the deacetylation of chitin.CDAs play an important role in regulating ecdysis physiology,tissue growth and disease resistance immunity during insect growth and development.The response of CDA to pathogen invasion in silkworm has not been reported.Therefore,the study of the relationship between the CDA and BmNPV is helpful to further complement the study of the mechanism of resistance to BmNPV in silkworm.In this study,BmNPV-sensitive strain P50 and BmNPV-resistant strain A35 were used as research materials,and a candidate protein BmCDA8 that may be involved in the response to BmNPV infection in silkworm was successfully screened through label-free quantitative proteomics study on the digestive fluid of the two strains infected with BmNPV and the control,and the related functions of this protein were further studied.The main results were as follows:1.Bioinformatics analysis of silkworm chitin deacetylase 8(BmCDA8)geneFirst,the cDNA nucleotide sequence and the encoded amino acid sequence of BmCDA8 were analyzed,and the positions of signal peptide in amino acids and ORF reading frame of BmCDA8 were determined.The homology analysis showed that BmCDA8 had high homology with the chitin deacetylase of Lepidoptera(Manduca sexta,Helicoverpa armigera,etc.).2.Spatiotemporal expression profiles of BmCDA8The expression level of BmCDA8 was detected by RT-qPCR and semi-quantitative RT-PCR.The results showed that BmCDA8 was present in foregut and hindgut of silkworm,and was not expressed in other tissues except head.In addition,BmCDA8 was only expressed at larval stage of silkworm,but not at egg,pupa and moth stage.The expression level of BmCDA8 increased significantly with the increase of instar.In addition,the protein was highly expressed after molting and is very low before and during sleep.It is speculated that BmCDA8 may be related to the renewal of peritrophic membrane in the larval stage of silkworm.3.Expression,antibody preparation and activity analysis of recombinant BmCDA8 proteinThe prokaryotic expression system was used for recombinant expression of BmCDA8,and the 49-331 amino acids of BmCDA8 were successfully obtained.The molecular weight of the protein was about 35 kDa.The protein was subsequently purified and used to prepare antiserum for subsequent experiments.The purified recombinant BmCDA8 protein was refolded in vitro to obtain soluble protein.The enzyme activity of recombinant BmCDA8 was 1.21 ±0.14U,which proved that recombinant BmCDA8 had good deacetylase activity.Then the chitin binding function of recombinant BmCDA8 was determined,and BSA was used as reference to prove that recombinant BmCDA8 had the ability of chitin binding.These results lay a foundation for further research on the function of this protein in response to BmNPV infection.4.Expression profile of BmCDA8 response to BmNPV infection in silkworm strains with different resistanceBoth transcriptional and protein levels,BmCDA8 expression levels in the midgut of sensitive strain P50 and resistant strain A35 were significantly increased from 12h to 48h after BmNPV infection.However,the expression of P50 was significantly down-regulated and the expression of A35 was still up-regulated at 72h.It is speculated that in the process of BmNPV infection,the resistant strain A35 could continuously up-regulate the expression of BmCDA8 to make the peritrophic membrane more complete,so as to better resist the infection of BmNPV.Further analysis of midgut,periophagous tissue and digestive juice by Western blot showed that the expression level of BmCDA8 in both susceptive and resistant strains was up-regulated after virus infection,and the up-regulated expression level of resistant strain A3 5 was more significant.5.The effect of BmCDA8 and its inhibitors on BmNPV replication efficiency was studiedAfter feeding recombinant BmCDA8 protein into silkworm strain P50,the infection ability of virus to silkworm was significantly reduced.It was speculated that recombinant BmCDA8 could bind to the perimetering membrane of silkworm,and enhance the modification effect of chitin,so that the perimetering membrane of silkworm could be more complete in resisting BmNPV infection,and could better prevent BmNPV infection.On the contrary,the feeding inhibitor(5-hydroxy-L-tryptophan)of silkworm strain P50 could enhance the infection ability of virus to silkworm individuals.In summary,a differentially expressed protein BmCDA8 in response to BmNPV was screened based on label-free proteomics.The results showed that the specific expression of this protein in foregut and midgut of silkworm had the functions of chitin binding and deacetylation.The expression of this protein was high in resistant strains,and the expression was continuously up-regulated after addition,and it could respond to BmNPV.Recombinant BmCDA8 protein could reduce the infection power of BmNPV to silkworm,and 5-hydroxy-L-tryptophan could enhance the infection power of BmNPV to silkworm.The results indicated that BmCDA8 may have antiviral activity,which provided theoretical support for further understanding the mechanism of response to BmNPV infection in silkworm resistant strains.