Study On The Protein Transport Protein Sec61α、Sec61γ And Chaperone Protein CCTβ、CCTθ Of Nosema Bombycis

Microsporidian are a group of obligatory intracellular unicellular eukaryote with extensive parasitic domains,which can infect most vertebrates and invertebrates.Nosema bombycis was the first microsporidian identified by Carl N(?)geli in 1857.There have been more than 1,500 species of 200 genera of microsporidia reported.N.bombycis is the pathogenic microsporidian of the economic insect Bombyx mori,which brings great economic losses to the production of silkworm eggs and seriously affect the safety of sericulture.At present,there is little research on the protein folding and transport related proteins of N.bombycis.The main function of Sec61 complex is to participate in the transport of polypeptide chains across the endoplasmic reticulum,while the main function of CCT complex is to assist unfolded polypeptides to fold,assemble,transport or degrade misfolded polypeptides.In this thesis,the protein transport protein Sec61α,Sec61γ and chaperone protein CCTβ and CCTθ genes of N.bombycis were obtained by blast the genome database of N.bombycis,which were analyzed and identified from the aspects of physical and chemical characteristics of gene sequence,bioinformatics,subcellular location,transcription level,etc.The prokaryotic expression system was used to express the recombinant proteins Nb Sec61γ,NbCCTβ and NbCCTθ.Polyclonal antibodies were prepared for indirect immunofluorescence experiments to research the subcellular localization of Nb Sec61α,NbCCTβ,and NbCCTθ in N.bombycis.RNAi experiments were conducted to explore the transcriptional expression levels of Nb Sec61α and NbCCTβ genes.The research findings are as follows:1.Identification and localization of protein transport protein Sec61α and Sec61γ of Nosema bombycisWe blast the database and found that there are a variety of protein transport protein in N.bombycis.In order to research the function of these proteins in N.bombycis,we analyzed the two subunits of the Sec61 complex,Nb Sec61α(Gen Bank: EOB11550.1)and Nb Sec61γ(Gen Bank: EOB13474.1)for cloning identification.Sequence analysis showed that the full length of Nb Sec61α is 1422 bp,encoding 473 amino acids.The predicted molecular weight of Nb Sec61α protein is 53.3 k D,and its isoelectric point is 9.26.Nb Sec61α belongs to Sec61/Sec Y-α family,contains 10 hydrophobic transmembrane domains and a unique "plug" domain of Sec61 complex;the full length of Nb Sec61γ is 276 bp,encoding 91 amino acids.The predicted protein molecular weight is 9.9 k D and isoelectric point is 10.13.Nb Sec61γ contains a transmembrane domain.Sequencing results showed that the clone size of Nb Sec61α and Nb Sec61γ genes was correct and there was no mutation.Multiple sequence alignment showed that the sequence similarity of Nb Sec61α with most other microsporidian homologous proteins is more than 48%,which indicating that Sec61α gene is highly conserved in microsporidia.Phylogenetic analysis showed that N.bombycis was closely related to Nosema granulosis and Nosema apis,Nosema ceranae.Nb Sec61α polyclonal antibody was prepared by Nb Sec61α polypeptide antigen.The recombinant Nb Sec61γ protein is mainly expressed in the form of inclusion bodies,and the purified Nb Sec61γ protein is used as the antigen to prepare the Nb Sec61γ polyclonal antibody.Western blot detected the target band of the corresponding size in the total protein of N.bombycis.Indirect immunofluorescence results showed that Nb Sec61α protein was mainly distributed in the cytoplasm and plasma membrane of mature spores of N.bombycis.In the process of proliferation and development,Nb Sec61α is mainly distributed around the spore nucleus and partly in the cytoplasm.The results of real-time PCR showed that Nb Sec61α protein existed in the whole life cycle of Nosema bombycis,with high expression in the early stage and stable in the middle and late stage.After RNAi interference,the expression of Nb Sec61α gene was down regulated in each period,and the interference effect of 72 h was strongest.2.Identification and localization of the chaperone protein CCTβ and CCTθ of Nosema bombycisBy Blast the genome database of N.bombycis,we found that there are eight subunits of the annotated CCT complex and selected NbCCTβ(Gen Bank: EOB12444.1)and NbCCTθ(Gen Bank: EOB12136.1)for research.Sequence analysis showed that the full length of NbCCTβ is 1566 bp,encoding 521 amino acids.The predicted protein molecular weight is 57.6 k D and isoelectric point is 5.5.NbCCTβ has a transmembrane domain from 396 to 416 amino acids.The full length of NbCCTθ is 825 bp,encoding 274 amino acids.The predicted protein molecular weight is 31.5 k D and isoelectric point is 5.6.NbCCTθ has no signal peptide and transmembrane domain.Multiple sequence alignment showed that NbCCTβ and NbCCTθ sequences were conserved.Phylogenetic tree analysis showed that N.bombycis was closely related to N.granulosus and N.apis,N.ceranae.After removing the transmembrane domain of NbCCTβ,the NbCCTβ protein was expressed and purified to prepare polyclonal antibody;the recombinant NbCCTθ protein was mainly expressed in the form of inclusion body.After purification,polyclonal antibody against NbCCTθ was prepared by immunizing rabbits.Western blot detection showed that the prepared polyclonal antibody has good specificity.Indirect immunofluorescence results showed that NbCCTβ existed in the whole life cycle of N.bombycis and distributed in the cytoplasm.The distribution of NbCCTθ protein was different in different developmental stages of spores.In mature spores,NbCCTθ protein was mainly distributed in cytoplasm and plasma membrane.When spores germinated,NbCCTθ protein would eject from the polar tube and enter the host cells together with the sporoplasms.During the proliferative stage and early sporogonic stage,NbCCTθ protein was mainly distributed in cytoplasm,while in the late sporogonic stage,NbCCTθ protein was mainly distributed in nucleus.The results of real-time PCR showed that NbCCTβ was involved in the whole life cycle of Nosema bombycis,and its expression level was high at the later stage.After RNAi,the expression level of NbCCTβ was inhibited at all stages,and the interference effect was the strongest at 96 h.