Localization And Functional Studies Of CCTα And CCTεsubunits Of Chaperone Proteins In Nosema Bombycis

NbCCTα and NbCCTε are two subunits of theCCT chaperone complex,which play an important role in the proliferation and development of N.bombycis.In order to investigate the functions of NbCCTα and NbCCTε in the development of N.bombycis,the main results of this study are as follows.The results showed that the NbCCTα gene contained a full length 1629 bp ORF,encoding 542 amino acid with molecular weight of 59.662 k D,isoelectric point of 5.81,and no signal peptide and transmembrane domain.Nosema bombycis,Nosema Apis and Nosema ceranaeCCTα are classified in the same cladum,suggesting that they are closely related to each other.The prediction of subcellular localization showed that NbCCTα was a cytoplasmic protein and also distributed in the nucleus and organelles.The cloned NbCCTε contains an ORF of 1596 bp with 99.25% similarity to the NCBI database,encoding 531 amino acids.The theoretical molecular weight of NbCCTε is58.392 k D and the isoelectric point is 5.12.Phylogenetic analysis showed that the Nosema bombycis and Nosema pernyiCCTε were classified into a larger cladistic group,indicating that the two species were closely related to each other.The prediction results showed that NbCCTε were mainly cytoplasmic proteins.The recombinant expression vectors p ET28a-NbCCTα and p ET28a-NbCCTεwere constructed by gene recombination technique,and were transferred into E.coli BL(DE3)for the expression of the recombinant proteins.NbCCTα and NbCCTε were successfully expressed in the prokaryotic expression vector.NbCCTα mainly exists in the supernatant in the dissolved form,while NbCCTε exists in the precipitate in the form of inclusion body.Purified protein for the preparation of polyclonal antibodies.The results of subcellular localization showed that NbCCTα protein was mainly localized in the cytoplasm and protoplasmic membrane of dormant spores.At the meront phase distributed to the cytoplasm and surrounding regions.NbCCTα was aggregated in the nuclear membrane during the sporoblasts cell stage.The results suggested that NbCCTα might be involved in cytoplasmic equipartition and nuclear division during the development of N.bombycis.NbCCTε was mainly found in the cytoplasm and protoplasmic membrane of the dormant spores.During the development process,NbCCTε was mainly distributed in the cytoplasm of the N.bombycis.During the sporoblasts stage,NbCCTε was mainly concentrated in the inner edge of the spore wall and the nuclear membrane,which may be related the formation of the spore wall to the division of the nucleus.RNAi inhibited the expression of NbCCTα,which was inhibited from 48 h after interference,and the inhibition effect was significant at 96 h after.Western Blot detection of NbCCTα protein expression,which was basically consistent with RT-PCR detection results.We detected the expression levels of Nbactin and Nbβ-tubulin genes,the results showed that the expression trends of Nbactin and Nbβ-tubulin were similar to the expression trends of NbCCTα gene,both of which began to inhibit at 48 h and showed significant inhibition at 96 h.The results showed that interference with NbCCTα inhibited the expression of Nbactin and Nbβ-tubulin genes.Four candidate proteins that may interact with NbCCTα were identified by immunoprecipitation:NbCCTγ,Actin family protein,ATP-binding cassette subfamily member B 7,ATPbinding cassette subfamily member G 1.