Study On The Role Of Cytc In Response To BmNPV Infection In Silkworm,Bombyx Mori(Lepidoptera)

Bombyx mori Nucleopolyhedrovirus(BmNPV)is a blood-type pathogen and always found in silkworm rearing.The most typical symptom is the color of the hemolymph in white in the late stage of BmNPV infection,which brings huge economic losses to the sericulture every year.At present,many silkworm strains were reported with high resistance to BmNPV,such as Huakang No.1,Huakang No.2,etc.However,the antiviral molecular mechanism of silkworm is not clear.Based on our previous transcriptome data of different resistant strains following BmNPV infection,several apoptosis-related genes,including Bmcytc,Bmapaf-1,Bmcaspase-Nc and Bmcaspase-1 were found.Cytc is a key factor of the upstream of the mammalian mitochondrial apoptosis pathway,which will be released into the cytoplasm when cells face some kinds of stimulation that can change the permeability of the mitochondrial membrane changes.Once Cytc entry into cytoplasm,it will combine with the Apaf-1 to form apoptotic body that can activate the apoptotic effector Caspase-3.The active Caspase-3 eventually causes apoptosis by the Caspase cascade.Moreover,Bmcytc has been reported to be released into the cytoplasm after stimulation BmN cells with UV.Therefore,we speculated that Bmcytc was released into cytoplasm from mitochondria to active the mitochondrial apoptosis pathway in response to BmNPV infection,which was conformed at the cellular level using dsRNA and overexpression vectors in this study.The main results are as follows.To get the molecular characteristic of Bmcytc firstly,bioinformatics analysis was used.Results showed that the nucleotide sequence of Bmcytc contained a 122 bp 5’-UTR,a 159 bp 3’-UTR and a 324 bp open reading frame(ORF),encoding 108 amino acids,including a Cytc conserved structure domain(located at amino acids 8-107).Bmcytc amino acids had a high degree with its homology in other species.Phylogenetic tree showed Bmcytc was conservative in evolution.These results indicated that Bmcytc might have a similar function with other species,which was involved in the regulation of mitochondrial apoptosis pathway.The spatiotemporal expression profile showed the highest expression of Bmcytc was in the moth stage,and relative high expression level in the midgut and head.Besides,the expression profiles of Bmcytc were stable in selected tissues of AN resistant strain following BmNPV infection,but very disordered in p50.In order to further explore the role of Bmcytc in response to BmNPV infection,dsRNA and the overexpression vector pIZT/V5-His-mCherry-Bmcytc were used.After RNAi of Bmcytc,expressions of and Bmcaspase-Nc were all down-regulated,which was validated by the upregulation of the downstream genes after overexpression of Bmcytc.Moreover,green fluorescence signals of BV-EGFP(budded virus containing EGFP-tagged)were observed with microscope after RNAi and overexpression of Bmcytc at different time points.After knockdown of Bmcytc,fluorescence signals of experimental group were significantly stronger than that of the control group at 48 h and 72 h,and with the dissociation of BmN cells,but without difference at 24 h.The phenomenon was further validated by expression levels of vp39 under the same treatment.Briefly,the expression of vp39 in the RNAi group was significantly up-regulated at 48 h and 72 h,and without difference at 24 h.To further validate the RNAi result,BmNPV infection was analyzed after overexpression of Bmcytc in BmN cells.Results showed that fluorescent signals in the overexpression group were stronger than that in the control group at 48 and 72 h after virus treatment,but fluorescent signals in the overexpression group were less than that in the control group at 24 h.To further detect the proliferation of virus after overexpression of Bmcytc in BmN cells,qRT-PCR technology was used.Results showed that the expression of vp39 in the overexpression group was down-regulated at 24 h after virus treatment,indicating the proliferation of virus was suppressed in the early stage of infection.However,the expression of vp39 was significantly up-regulated in the experimental group at 48 h and 72 h after virus treatment,indicating the virus was promoted to proliferate in the late stage of infection.In order to verify whether Bmcytc induces the mitochondrial pathway apoptosis during viral infection.After overexpression of Bmcytc in BmN cells,virus proliferation was promoted after treatment with apoptosis inhibitor,indicating Bmcytc-mediated apoptosis pathway was inhibited.In order to verify this point,different resistant strains p50 and AN were infected with BV-EGFP after treatment with apoptosis inducer and apoptosis inhibitor.The virus proliferation was detected by analyzing the expression level of vp39.BmNPV proliferation was promoted in AN strain after suppression of apoptosis and was inhibited in p50 strain after induction of apoptosis,which was consistent with results obtained at the cellular level.The result in this study will provide data support for revealing the molecular mechanism of Bmcytc during BmNPV infection,completing the research content of the silkworm mitochondrial apoptosis pathway,and providing a theoretical reference for the breeding of silkworm resistant strains.