| Microsporidia are a group of eukaryotes with a broad host domain and specialized intracellular parasitism that infect most vertebrates and invertebrates,including humans.The Pěbrine disease caused by Nosema bombycis（Nb）infection of Bombyx mori is a serious and long-term untreated disease in sericulture production.As a transporter protein on the outer mitochondrial membrane,TSPO is widely involved in various biological functions related to mitochondrial function,such as membrane potential regulation,mitochondrial autophagy,immune response,calcium channel regulation,and apoptosis.In this study,we cloned and characterized the silkworm BmTSPO gene,analyzed its role in Nb infection,and identified downstream acting genes and immune signaling pathways.The main results are as follows:（1）BmTSPO has a nucleotide length of 486 bp,encodes a protein containing 161 amino acids,with a protein molecular size of 18 kDa and an isoelectric point of 10.26.The BmTSPO protein has no signal peptide and contains five transmembrane structural domains,one glycosylation site and 29 phosphorylation sites.Cell-Ploc predicted that BmTSPO was mainly distributed on membrane proteins in the mitochondria of the silkworm,and multiple sequence comparison showed that BmTSPO had high homology with TSPO of other species,especially with TSPO of insect origin,all of which were above 65%,proving that TSPO was relatively conserved in evolutionary terms.A recombinant baculovirus vBmEGFP-BmTSPOwas constructed using the Bac-to-Bac insect baculovirus expression vector system,and by observing the location of fusion expression protein fluorescence in the cell,it was found that the BmTSPO protein was mainly distributed in the cytoplasm in an aggregated punctate manner.（2）The tissue expression profile and developmental stage expression profile of BmTSPO in silkworm were mapped by qRT-PCR.The tissue expression profile showed that BmTSPO was expressed highest in the fat body,second in the midgut,third in the malleolus,and relatively low in the ovary,spermathecae and blood.The expression profile of developmental stages showed that BmTSPO expression was low in the embryonic stage and the pre-anthelmintic stage,high in the mid-5th instar and adult stage,and fluctuated greatly around the dormant stage.By overexpression and enzymatic assays,BmTSPO was found to enhance intracellular levels of ROS,Ca++,and ATP.（3）Gene timing expression profiles were mapped by qRT-PCR,and the results showed that BmTSPO expression was up-regulated after infection with Nb.Further silencing and overexpression of BmTSPO by RNAi technology and PIZ-mcherry vector,and Nb replication was subsequently examined.The results showed that the relative copy number of Nb genome was significantly increased after RNAi.The opposite was true for overexpression of BmTSPO.It was also found that the inhibitor PK11195 could promote the replication of Nb in BmN,which was consistent with the effect of RNAi.The agonist FGIN-1-27 could inhibit the replication of Nb in BmN,which was consistent with the effect of BmTSPO overexpression,indicating that BmTSPO has the function of anti-replication of Nb.（4）On the basis of RNAi and overexpression of BmTSPO,the changes of apoptosis level in the cells were investigated.The results showed that the apoptosis level of cells increased significantly after BmTSPO overexpression,while the overall apoptosis level of cells decreased after RNAi.While the overall apoptosis level of cells decreased significantly after Nb infection in both overexpression and RNAi groups,indicating that Nb infection could inhibit apoptosis of host cells,indicating that high expression of BmTSPO could induce apoptosis in host cells,but due to Nb has the ability to inhibit host cell apoptosis,so BmTSPO-induced apoptosis may not be the cause of reduced Nb replication,and thus the pathway of BmTSPO resistance to Nb needs to be further explored.（5）In order to find the downstream-acting genes of TSPO,we identified the downstream genes affected by BmTSPO overexpression using RNA-seq,and found that 131 genes were significantly changed in the BmTSPO overexpression state,among which 17 genes were up-regulated and114 genes were down-regulated in expression.GO enrichment analysis revealed that the main focus was on immune regulation,cellular processes,metabolic processes.The KEGG Pathway analysis revealed that the major changes were in oxidative phosphorylation,neurodegenerative diseases and other signaling pathways.On this basis,we analyzed the changes of some silkworm immune pathway-related genes after BmTSPO overexpression by qRT-PCR.The results showed that the expression of most genes involved in the Imd and JAK-STAT pathways were significantly up-regulated.It indicates that BmTSPO can activate the signal transduction pathway of innate immunity in the silkworm,which regulates microsporidia replication.In conclusion,our study provides preliminary evidence that BmTSPO plays an important role in Nb infection and inhibits Nb replication through activation of the Imd and JAK-STAT pathways in the silkworm. |
