Identification And Localization Of Nucleoporins Nup170,Nup98 And Protein Transporter Sec23 Of Nosema Bombycis

Microsporidian are a group of obligatory intracellular unicellular eukaryote with extensive parasitic domains,which can infect most vertebrates and invertebrates.Since N?geli first identified the microsporidian Nosema bombycis in 1857,more than 1,500 species of 187 genera of microsporidia have been reported in the past 160 years.N.bombycis is the pathogenic microsporidian of the economic insect Bombyx mori,which can brings great economic losses to the production of silkworm industry.Nucleoporins(Nups)and protein transporters are important proteins involved in the transport of substances in eukaryotic cells.Nup170 and Nup98 are used as the important part of the nuclear pore complex(NPC),which is mainly involved in the transport of macromolecular substances between the nucleus and cytoplasm,and the Sec23 protein is necessary for the assembly of the coat protein complex ? ?(COP?)structural proteins ensure the orderly transport of macromolecules between the Golgi apparatus and the endoplasmic reticulum(ER).Nup170 is one of the important structural components of the nuclear pore complex and is necessary to maintain the structural stability of NPC.Bioinformatic analysis revealed that Nup170 protein was present in N.bombycis.The NbNup170 gene contains an ORF of 762 bp in length,encoding 253 amino acid residues.The predicted molecular mass of Nup170 protein of 29.38 k D and an isoelectric point of 8.28,with 1 N-glycosylation site and 2 phosphorylation sites,and the prediction results of subcellular location showed that the protein was distributed on the nucleus.The three-dimensional structure of the Nup170 protein in N.bombycis is predicted to be elliptical,while it is crescent-shaped in S.cerevisiae.Phylogenetic analysis results showed that N.bombycis and other microsporidia clustered on the same branch,and related to Nosema apis and Nosema ceranae closely.NbNup170 protein was mainly localized on the plasma membrane in dormant spores of N.bombycis,and as the spore germinated,it ejected together with the sporoplasm through the polar tube and enters the host cell.NbNup170 protein is distributed on the both sides of the nucleus and nuclear membrane during the proliferative phase of N.bombycis,and on the nuclear membrane during sporogonic phase of N.bombycis,which may be related to the assembly of NPC and the formation of the nuclear membrane.The q PCR results showed that the relative expression level of the NbNup170 gene maintained at a low level within 30 h to 78 h after infection with N.bombycis,and then quickly reached the highest value at 102 h,while the relative expression activity of NbNup170 remained at a low level after RNA interference,indicating that RNA interference significantly down-regulated the expression of the NbNup170 gene.Nup98 and Nup96 are two proteins with different functions encoded by a same gene,and is the main nucleoporin of NPC.Bioinformatics analysis revealed that NbNup98 protein was present in N.bombycis,and the NbNup98 gene contains an ORF with a length of 2 013 bp,encoding 670 amino acid residues.The NbNup98 protein has isoelectric point(p I)of 9.29 and 71 k D of molecular weight,with 64 phosphorylation sites,7 N-glycosylation sites.Immunofluorescence results showed that the the presence of NbNup98 in the mature spores of N.bombycis,and ejected together with the sporoplasm through the polar tube and enter the host cell as the spores germinated.NbNup98 protein was mainly distributed on the both sides of the nucleus and nuclear membrane during the proliferative phase of N.bombycis,which may be related to caryokinesis,and on the nuclear membrane during the sporogonic phase of N.bombycis,which may be related to the transport of protein and RNA.As an important component of COP?,Sec23 protein can closely connect the different components of COP? to ensure the orderly assembly of vesicles.This study identified the Sec23 protein of N.bombycis for the first time.Bioinformatics analysis revealed that the NbSec23 gene contains an ORF of 540 bp in length,encoding 179 amino acid residues.The NbSec23 protein has isoelectric point(p I)of 4.93 and 20.75 k D of molecular weight(MW),with 4 phosphorylation sites and 3 N-glycosylation sites,and the prediction results of subcellular localization showed that the Sec23 protein was distributed on the cytoplasm.Phylogenetic analysis indicated that the Sec23 protein of N.bombycis has high homology with other microsporidia,which proves that Sec23 protein may have the common evolutionary origin.In this paper,the NbNup170,NbNup98 and NbSec23 were successfully cloned for the first time,then identified and expressed the recombinant proteins,and studied the location of NbNup170 and NbNup98,in order to further explore the functions of NbNup170,NbNup98 and NbSec23,as well as laid a foundation for further researches of NPC and COP? in N.bombycis.NbNup170 and NbNup98 have been present throughout the life cycle of N.bombycis,but they are positioned differently at different developmental stages,this may be achieved through interaction with other proteins,which will be the focus of further research.