Identification And Characteristic Analysis Of Nb Septins From Nosema Bombycis

Microsporidia is a class of unicellular,obligate intracellular eukaryotic parasites,can infect many hosts from invertebrates to vertebrates,including economic insects like bees and silkworms,even patients with immune deficiency.Life of microsporidia generally divided into three stages: infection phase,proliferative phase and sporognic phase.The proliferative phase is the beginning phase of intracellular development,and the key stage of microsporidium rapid proliferation and propagation.Therefore,it is an important strategy for microsporidia prevention that blocking or limiting proliferation of Nosema bombycis.Septin is a highly conserved family of GTP-binding protein in most eukaryotes,including yeast,worms,and mammals,with the exception of plants.Septin was reported to participate in process of cytokinesis through forming cytoskeleton structures and recruiting proteins.Here,we studied septins as a pointcut to carry out research about the proliferation of microsporidium,in order to understand the forms and cycles of meront proliferation.In this study,the sequences of S.cerevisiae Septins as templates were used to blast the genome database of N.bombycis,and three septin were identifed.Characters of conservation,transcription,subcelluar localization and interation between NbSeptins were descript.The main contents are as following: 1.Identification and transcriptional characteristic analysis of Septins in Nosema bombycisIn this study,three Septin genes named NbSeptin1(GenBank NO: AGT97394.1,NbSPT1),NbSeptin2(GenBank NO: EOB13962.1,NbSPT2)and NbSeptin3(GenBank NO: AHN50233.1,NbSPT3)were obtained by bioinformation methods based on the genome database of N.bombycis and the sequences of S.cerevisiae Septins.Amino acid analysis sequence showed that the predicted molecular mass of NbSPT1 is 39.6 kDa and the theoretical isoelectric point is pI 5.82.The predicted molecular mass of NbSPT2 is 37.28 kDa,and the theoretical isoelectric point is pI 7.98.The predicted molecular mass of NbSPT3 is 40.75 kDa and the theoretical isoelectric point is pI 4.67.In addition,NbSeptins were predicted to have GTP binding domain,a conservative domain in Septin proteins,which suggested that the basic function of Septins in N.bombycis might be similar to septins in other species.Multiple sequence alignment revealed a large difference of three NbSeptins,suggesting that NbSPT1,NbSPT2 and NbSPT3 may play different functions.Evolutionary analysis of NbSeptins showed that Septins are conservative in microsporidian,and the homologous genes of NbSPT1,NbSPT2 and NbSPT3 were clustered together in one branch respectively,which indicated that homologous genes of Septin might play similar roles in microsporidian.According to the evolutionary tree,NbSPT1 and S.cerevisiae CDC11,SHS1,SPR28,CDC12 and SPR3 were clustered together.NbSPT2 and S.cerevisiae CDC3 were clustered together,NbSPT3 and S.cerevisiae CDC10 were clustered together.The result suggested that the roles of NbSeptins might be similar with its homologous genes in S.cerevisiae.To investigate the transcription pattern of NbSeptins,the mRNA from the infected Sf9 cells with Nosema bombycis at 1-4 dpi were extracted respectively.As RT-qPCR shown NbSeptins was detected in 1-4 dpi and have high levels of transcription at 2nd day post infection.The result indicated that NbSeptins might play role in proliferation of N.bombycis.2.Antibodies preparation and subcelluar localization analysis of NbSeptinsSpecific primers of NbSeptins were designed to clone NbSPT1,NbSPT2,NbSPT3 based on the gene sequence and recombinant plasmids of pColdI-NbSPT1,pColdI-NbSPT3,and pET30-NbSPT2 were constructed and transferred into E.coli stain Rosseta(DE3)for prokaryotic expression.Expression of proteins which were induced at 16 °C with 0.2 mM IPTG for 20 h.The recombinant NbSeptins proteins were purified by affinity chromatography.Mice and rabbit were immunized with purified protein to prepare antiserum against NbSeptins.Western blotting showed the band of 35 kDa,37 kDa,40 kDa was detected respectively by anti-NbSPT1,anti-NbSPT2 and anti-NbSPT3 antiserum in total protein of N.bombycis mature spores and Sf9 cells infected with N.bombycis.Subcelluar localization of NbSeptins were analyzed by IFA using the specific polyclonal antibodies.The results indicated that NbSeptins co-located at the membrane of meront,and a liner co-localizated signal of NbSPT2 and NbSPT3 between some diplokaryon of N.bombycis meront were observed.All the results suggested that NbSeptins play roles in the proliferation of N.bombycis,and NbSPT2 and NbSPT3 might function in the divsion of nucleus of N.bombycis.3.Interaction analysis of NbSeptinsSeptins were reported to function as homologous or heterologous polymers.Considering the co-localizated signal of NbSeptins at membrane of meront,interactions between NbSeptins were analyzed by yeast two-hybrid analysis and co-immunoprecipitation.Plasmids of pGBKT7-NbSPT1,pGBKT7-NbSPT2,pGADT7-NbSPT1 and pGADT7-NbSPT3 were constructed and transferred into S.cerevisiaec Y187 or AH109 yeast strains.Results of yeast two-hybrid displayed that Y187 [pGADT7-NbSPT1]/AH109 [pGBKT7-NbSPT2],Y187 [pGADT7-NbSPT3]/AH109 [pGBKT7-NbSPT1] and Y187 [pGADT7-NbSPT3]/AH109 [pGBKT7-NbSPT2] were able to activate the expression of downstream report genes,demonstrating that the interactions between NbSPT1,NbSPT2 and NbSPT3.Protein of N.bombycis infected Sf9 cells and mature spores were extracted and co-immunoprecipitation were used to verify the interactions between NbSeptins.Results suggested that NbSPT1,NbSPT2 and NbSPT3 could form polymers to participate in the proliferation of N.bombycis.