| Microsporidia,as a single-celled eukaryotic organisms,infect a broad range of hosts from almost all invertebrates and vertebrates,and some protists.Its proliferation and development mainly depend on host cells for nutrients and energy.Nosema bombycis,which the earliest identified microsporidium in human,is a great threat to the sericulture industry and the main object of quarantine in sericulture production.Due to intracellular parasitism,microsporidia’s genome is greatly reduced,but it still retains the inner membrane system used to transport cargo molecules.In the inner membrane system,coat protein complex Ⅱ(COPⅡ)mediates protein transport from the endoplasmic reticulum to the Golgi apparatus and is composed of Sar1,Sec23,Sec24,Sec13 and Sec31.Whether the microsporidia form COPⅡ vesicles to transports cargo proteins,and whether it plays other roles besides cargo transport,is not fully no data to prove it at present.In this study,NbSar1,Nb Sec23,Nb Sec24,Nb Sec13 and Nb Sec31 genes were successfully cloned and NbSar1 polyclonal antibody was prepared.The location of NbSar1 was explored by indirect immunofluorescence technique in different development stages of Nosema bombycis.The interactions between NbSar1,Nb Sec23,Nb Sec24,Nb Sec13 and Nb Sec31 were identified by yeast two-hybrid technology,and the regulatory relationships among NbSar1,Nb Sec23,Nb Sec24,Nb Sec13 and Nb Sec31 were analyzed by RNAi technology.The main results are as follows:Sar1,as a small GTPase,is a core participant in the regulation of endoplasmic reticulum COPⅡ vesicle formation.Bioinformatics prediction showed that NbSar1 encodes 215 amino acids with a molecular weight of 28 k Da and an isoelectric point of 9.26.The results of multiple sequence alignment showed that the amino acid similarity between NbSar1 and Sar1 of Nosema ceranae(XP＿024330127.1),Encephalitozoon intestinalis(XP＿003072817.1),Encephalitozoon cuniculi(NP＿597349.1),Encephalitozoon romaleae(XP＿0092644 37.1)were 63.89%,57.01%,56.56%,and 56.56%,respectively.Phylogenetic analysis showed that there was a close relationship between Nosema bombycis and Nosema ceranae.In this study,Sar1 gene sequence was searched through the Nosema bombycis database,and a complete ORF was cloned with a size of 648 bp without mutation.The recombinant expression vector PET-28a-NbSar1 was successfully constructed and the NbSar1 recombinant protein with molecular weight of 34 KDa was induced by prokaryotic expression in vitro.The purified NbSar1 recombinant protein was obtained by ultrasonic and elution with 200 mmol/L imidazole.Indirect immunofluorescence results showed that NbSar1 was mainly located around the nucleus of Nosema bombycis.In order to explore the interaction between COPⅡ proteins of Nosema bombycis,specific primers were designed according to the sequence of Nb Sec23-1,Nb Sec23-2,Nb Sec24-1,Nb Sec13,Nb Sec31-1 and Nb Sec31-2.A complete ORF of the genes was cloned and sizes are540 bp,648bp,648 bp,825bp,2463 bp,2721bp respectively.The prediction results showed that Nb Sec23-1 included Gelsolin domain,Nb Sec23-2 included zf-Sec23-Sec24 domain and Sec23-trunk domain.Nb Sec24-1 includes the zf-Sec23-Sec24 domain.Nb Sec13 had six WD40 domains.Both Nb Sec31-1 and Nb Sec31-2 contained three WD40 domains,and sequence alignment results showed that sec31-2 included sec31-1.According to the protein domain,the interaction between COPⅡ components was verified by yeast two-hybrid technique.The results showed that there were interactions between NbSar1 and Nb Sec23-2,Nb Sec23-2 and Nb Sec24,Nbsec23-1 and Nb Sec31-1,and Nb Sec13 and Nb Sec31-1.In order to explore the regulating relationship between COPⅡ proteins of Nosema bombycis,the q PCR was used to detect the silencing effect of RNA interference NbSar1.The results showed that there was obvious knock down effect at 24 h,48 h and 72 h after si RNA injection,but there was no knock down effect at 96 h.After RNA interference with NbSar1,the expression levels of Nb Sec23-1,Nb Sec24-1,Nb Sec13,Nb Sec31-1 and Nb Sec31-2 were detected by q PCR technology.The results showed that after RNAi for 24 h,48h and 72 h,Nb Sec23-1,Nb Sec24-1,Nb Sec13,Nb Sec31-1,Nb Sec31-2 significantly abnormal expression.These results suggest that NbSar1,Nb Sec23-1,Nb Sec24-1,Nb Sec13,Nb Sec31-1,Nb Sec31-2may work together to perform functions in the proliferation of Nosema bombycis.After RNA interference with NbSar1,NbSar1 protein wasn’t distributed in the perinuclear region of Nosema bombycis,but in the cytoplasm and the proliferation of Nosema bombycis was significantly inhibited.Therefore,NbSar1 protein is very important for the assembly of COPⅡ and the proliferation of Nosema bombycis.In conclusion,these results suggest that NbSar1,Nb Sec23,Nb Sec24,Nb Sec13 and Nb Sec31 may assemble to form COPⅡ in Nosema bombycis and the subunits of COPⅡ work together to perform functions in the proliferation of Nosema bombycis,while NbSar1 may play a key role in the formation of COPⅡ vesicles. |
