Studies On Immune-related Genes Of Silkworm Defensive To Beauveria Bassiana Infection

Beauveria bassiana is one of the important entomopathogenic fungi for silkworm,Bombyx mori.White muscardine of silkworm caused by B.bassiana infection is the most common type of fungal disease,always resulting in huge economic losses to sericulture industry.But at present,silkworm diseases,including white muscardine,are lack of effective treatments,so the disease prevention is critical.However,little is known about the molecular mechanism of silkworm response to fungi infection.In the present study,the expression profiling of BmcecA,Bmgloverin2 and BmLeb5 in the silkworm following infection of conidia of B.bassiana were investigated by real-time quantitative PCR.The immune genes were cloned and their sequence characteristics analysized by bioinformatics tools and molecular methodologies.Then their roles in the process of immune response to B.bassiana infection were studied by antifungal assays.Furtherly,the proteins in the hemolymph of normal and B.bassiana infected silkworm were also comparatively analyzed by Label free quantitative proteomic analysis and liquid chromatography-mass spectrometry.The cDNA encoding cecropin A(BmCecA)was cloned from the larvae of silkworm,p50,using RT-PCR.It encodes a protein of 63 amino acids,containing a 26 amino acid signal peptide and a 37 amino acid mat peptide of functional domain.The BmCecA secondary structure contains two typical amphiphilic ?-helixes.Real-time qPCR analysis revealed that BmCecA was expressed in all the tissues tested,including cuticle,fat body,hemocytes,Malpighian tubule,midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes.The gene expression of BmCecA was rapidly induced by B.bassiana challenge and reached maximum levels at 36 h after inoculation in third instar larvae.In the fifth instar larvae infected with B.bassiana,the relative expression level of BmCecA was upregulated in fat body and hemocytes,but not in cuticle,Malpighian tubule,midgut and silk gland.The cDNA segment of the BmCecA was inserted into the expression plasmid pET30 a to construct a recombinant expression plasmid.Western blot results revealed that his-tagged fusion protein was successfully expressed and purified.Then the mat peptide of BmCecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%.Mass spectrometry and SDS-PAGE showed its molecular weight to be 4.07 kDa.Antifungal assays indicated that the BmCec A had a high antifungal activity to entomopathogenic fungus B.bassiana both in vitro and in vivo in the silkworm larvae.Fluorescent quantitative real-time PCR analysis indicated that the relative expression level of Bmgloverin2 gene was up-regulated in the silkworm larvae infected by B.bassiana.The cDNA of Bmgloverin2 was cloned from the silkworm by RT-PCR and the DNA segment of the Bmgloverin2 peptide(without signal peptide sequemce)was inserted into pCzn1 expression plasmid and expressed in E.coli ArcticExpress(DE3).SDS-PAGE results revealed that soluble recombinant Bmgloverin2 was successfully expressed and purified.Polyclonal antibody against the Bmgloverin2 was successfully produced with the expressed recombinant protein.Western blot analysis indicated that Bmgloverin2 could be detected in the fat body of silkworm larvae infected with B.bassiana,suggesting that the expression of Bmgloverin2 could be induced by B.bassiana infection in silkworm.Antifungal assays indicated that the Bmgloverin2 had a synergistic antifungal activity with BmCecA to entomopathogenic fungus B.bassiana both in vitro and in vivo in the silkworm larvae.The full-length cDNA of lebocin 5(BmLeb5)was first cloned from silkworm by rapid amplification of cDNA ends(RACE).The BmLeb5 gene was 808 bp in length including a 39 bp 5'-untranslated region(UTR)and a 229 bp 3'-UTR with a polyadenylation signal sequence(AATAAA)and a poly(A)tail.The open reading frame of BmLeb5 encodes a 179 amino acid hydroxyproline-rich peptide,including a predicted signal peptide of 20 amino acids.Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins.Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins.The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified.Quantitative real-time PCR analysis revealed that BmLeb5 was expressed in all the tested tissues,but the highest in the fat body.In the silkworm larvae infected by B.bassiana,the expression level of BmLeb5 was up-regulated in the fat body and hemolymph which are the most important immune tissues in silkworm.Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defense B.bassiana infection.Furtherly,the proteins in the hemolymph of normal and B.bassiana infected silkworm were also comparatively analyzed by label-free quantitative proteome analysis.671 proteins were identified in the hemolymph,including 87 differential proteins.Relative to the normal silkworm,there were 36 proteins up-regulated and 40 down-regulated in infectied silkworm.There were another 6 proteins only in normal and 5 proteins only in B.bassiana infected silkworm.Among the 36 up-regulated proteins,serine protease,heat shock cognate 70,lysozyme,antibacterial peptide,cecropin B,storage protein,attacin,etc.may be closely related to immune responses.By contrasting and annotating the differential proteins to KEGG databases,the differential proteins mainly perform functions including amoebiasis,MAPK signaling pathway,Hippo signaling pathway,Toll and Imd signaling pathway,lysosome,regulation of actin cytoskeleton,viral carcinogenesis,PI3K-Akt signaling pathway,etc.These results lay a foundation for elucidating mechanisms of the silkworm immune response against fungal infection and the induced expression of antifungal genes,and provide a theoretical basis for breeding high resistant silkworm varieties.