Colocalization And Interaction Of CCT Chaperones Subunits Of Nosema Bombycis.

Microsporidia are typical obligate intracellular parasitic unicellular eukaryotes.It can infect invertebrates and vertebrates,including humans.It has been more than 160 years since the first discovery of the Nosema bombycis from the silkworm.To date,200 genera and1500 species of Microsporidia have been identified.The pebrine disease is a serious infectious disease.It is infected by larvae or passed on to offspring through eggs,which causes serious economic losses to sericulture production.Therefore,pebrine disease is the only statutory quarantine object in sericulture.The entire life cycle of the microsporidia takes place in the host cell,which includes the synthesis and folding of a series of nascent peptides.When the eight subunits of the chaperonin CCT are used as monomers,they are called CCTα-CCTθ,and when they are used as complexes,they are called TRIC,also known as TCP-1 ring complex(chaperonin-containing T-complex polypeptide-1),which an prevent protein misfolding and aggregation.In this study,we preliminarily determined the colocalization of CCT complex subunits and the interactions between related subunits through gene cloning,colocalization analysis and interaction studies between subunits.The function of the complex and its role in the proliferation of N.bombycis have certain guiding significance for preventing pebrine disease in the sericulture industry.In this study,we cloned the CCTζ gene of N.bombycis.Bioinformatics analysis showed that the gene was 1206 bp in length,encoding 402 amino acids,and the predicted molecular mass and isoelectric point were 44 k Da and 9.15,respectively.NbCCTζ protein has no signal peptide and transmembrane domain,contains α-helix and random coil,contains thirty-two phosphorylation modifications and two N-glycosylation modifications,and has no O-glycosylation sites.The results of indirect immunofluorescence showed that the protein was distributed in the cytoplasm during the nuclear division of N.bombycis.characteristic,aggregated around the nucleus and spread to the periphery,presumably NbCCTζ protein is involved in the folding and assembly of actin and tubulin.The effect of NbCCTζ on the regulation of Nbssr RNA expression was examined by knocking down NbCCTζ gene by si RNA.The results showed that RNA interference down-regulated the transcriptional activity of NbCCTζ gene.After interfering with NbCCTζ,the expression of Nbssr RNA was inhibited,indicating that NbCCTζ may play an important role in the proliferation of N.bombycis.In order to explore the functions of CCT subunits,we conducted pairwise colocalization analysis between the eight subunits,and the results showed that NbCCTζ and NbCCTα,NbCCTζ and NbCCTη,NbCCTβ and NbCCTδ,NbCCTβ and NbCCTθ all have co-localization characteristics.Some subunits are completely overlapped,and some are unevenly distributed.The results show that when the CCT complex works,not all subunits are involved.To further explore the regulatory mechanism of the interaction between CCT subunits,NbCCTζ-p GADT7 and NbCCTα-p GBKT7 recombinant plasmids were successfully constructed,and the interaction between NbCCTζ and NbCCTα was verified by yeast two-hybridization.The RNAi results showed that after interfering with NbCCTα,the relative expression of NbCCTζ was down-regulated at all four time points;after interfering with NbCCTζ,the relative expression of NbCCTα was up-regulated at one time point,and down-regulated at the other three time points.We speculate that there is a synergistic regulatory relationship between NbCCTζ and NbCCTα in the process of CCT complex-assisted protein folding during the infection and proliferation of N.bombycis.