Studies On The Putative Membrane Protein Ycf 1 And The General Transcriptional Repressor Ssn6 Of Nosema Bombycis

Microsporidia are obligate intracellular parasitic opportunistic pathogenic microorganisms that can infect almost all vertebrates and invertebrates,including important economic insects such as silkworms and honeybees.The Nosema Bombycis is the first discovered and identified microsporidia,which can infect the silkworm.Pebrine disease is an important disease in sericulture production,which causes huge economic losses to sericulture production,especially silkworm seed industry every year.After the N.bombycis infects the silkworm,it relies on the nutrients provided by the host for proliferation and finally completes a generation,producing many mature(dormant)spores and entering the next life cycle.In this process,many proteins related to important metabolic pathways are involved.This master’s thesis selects two proteins closely related to biological processes,namely a putative membrane protein Ycf 1 and general transcriptional corepressor ssn6 for research,to accumulate basic data for the analysis of the proliferation mechanism of N.bombycis.Membrane proteins are involved in many important metabolic pathways in cells,however,there are few reports on Microsporidian membrane proteins so far,while Ycf 1 is a putative membrane protein of N.bombycis and no relevant reports have been reported.In order to explore the mechanism of Ycf 1 in the proliferation of N.Bombycis,we did preliminary research on this protein.Bioinformatics analysis revealed that Ycf 1 has one transmembrane domain and signal peptide,thirty-six phosphorylation sites and one Oglycosylation site.The recombinant protein was expressed in a prokaryotic expression system,and monoclonal antibodies were prepared by immunizing mice.Indirect immunofluorescence analysis showed that Ycf 1 protein was mainly distributed on the plasma membrane of N.bombycis,which preliminarily proved that Ycf 1 is a membrane protein.The transcription level of Ycf 1 gene was detected by real-time quantitative PCR.The results showed that Ycf 1 gene was expressed in all developmental stages of N.bombycis,and RNA interference effectively inhibited the expression of Ycf 1 gene.Nbβ-tubulin is a housekeeping gene of N.bombycis,and its copy number can reflect the number of N.bombycis.After RNAi knocked down the expression of Ycf 1 gene,the copy number of Nbβ-tubulin was significantly reduced,indicating that Ycf 1 may be involved in proliferation of N.bombycis.TPR(Tetratricopeptide repeat)is a structural motif consisting of 34 amino acids,in eukaryotes,proteins containing TPR motifs are involved in many important biological processes.However,there are few reports of TPR motif-containing proteins in microsporidia,and the general transcriptional corepressor ssn6 from the genome database.Domain analysis showed that ssn6 has four TPR motifs.Prokaryotic expression of fusion protein was carried out using recombinant plasmid,and polyclonal antibody was prepared by immunizing rabbits after purification.Indirect immunofluorescence analysis showed that ssn6 existed throughout the life cycle of N.bombycis and was mainly distributed in the cytoplasm of N.bombycis.Real-time quantitative PCR analysis showed that the ssn6 gene was highly expressed in the early stage of N.bombycis proliferation and remained at a low level after 48 hours.After si RNA silencing,the expression of ssn6 gene was significantly inhibited,and the RNA interference efficiency was 76.08%,78.79%,52.08%,38.14% and55.87% at 24 h,48 h,72 h,96 h and 120 h,respectively,transcript levels were low throughout the proliferation of N.bombycis.In conclusion,this master’s thesis identified a membrane protein Ycf 1 of N.bombycis,which is mainly distributed on the plasma membrane;the subcellular localization of the general transcriptional corepressor ssn6 of N.bombycis was preliminarily studied.It is speculated that the two proteins are involved in the proliferation process of of N.bombycis,which provides a reference for the screening of chemical drug control targets and has a certain reference for the control of pebrine disease.