Functional Study On The Dispersed Centromere Protein Of Chromosome In Silkworm,Bombyx Mori

When the organism develops into an individual from the fertilized egg,it has been undergoing cell division.In the process of the cell division,the centromere plays an important role.There are two forms of centromere in eukaryotes: one form is single centromere,as known as local centromere;the other form is polycentromere,as known as dispersed centromere.Silkworm is the lepidopteran pattern insect,its centromere belongs to the dispersed centromere.In the process of cell division,genetic material is evenly distributed to two daughter cells.The centromere and spindle play an important role in this process.The function of the centromere is adhesion-replicating chromosome in the pre-mid-phase,attaching the spindle in the metaphase and participating in the migration behavior of the chromosome in the anaphase.Mammals is monocentric organisms.At present,the structure and the type of mammals centromere proteins have been relatively clear.But studies on dispersed centromere are relatively slow.For silkworms,the current research on centromeres only stays on the genome identification of its protein species.Recent studies have reported the structure of the outer centromere in silkworm,and its external structure of the centromere protein is relatively conservative,mainly composed of the Mis12 complex and the Ndc80 complex.The protein species and interaction mode of the complex have also been studied.However,the types of centromere proteins in the internal structure of centromere vary relatively,and there are different kinds proteins in different species.The CenpA protein plays an important role in mammals and can mark the location of the chromosomal centromere(the variant of histone H3 in centromere is called CenH3,which is named CenpA in mammals).CenpA is nonexistent in lepidopteran insects such as silkworm etc,and so does the CenpC protein interacting with the CenpA protein.The function of silkworm centromere protein in cell division and the specific process of silkworm centromere protein regulated by its interaction protein are studied.Through research,the following results are obtained:(1)Functional analysis on the structure of centromere protein in silkworm,Bombyx moriTo investigate the function of known centromere proteins and their role in mitosis.We first performed a subcellular localization analysis on the known centromere proteins,and found that the BmCenpI protein is located on the cytoplasm in the interphase of mitosis,located on both sides of the chromosome in the metaphase of mitosis,and also located on the chromosome in the anaphase;The BmCenpM,BmCenpL,and BmCenpN proteins are mainly located on the nucleus in the interphase,on both sides of the chromosome in the metaphase and on the chromosome in the anaphase.All the four proteins are located on the chromosome in the metaphase and anaphase of mitosis,suggesting that they have the function of centromere protein.To further investigate the function of these proteins,we silenced the expression of these genes using cellular interference techniques and found that in the absence of these centromere proteins in the metaphase of mitosis,the arrangement of tubulin is disordered and the chromosomes are not neatly arranged on the equatorial plate.These results indicate that centromere protein plays a very important role in cell mitosis.(2)Identification of CenpN complex of centromere protein in silkworm,Bombyx moriTo investigate the presence of other proteins involved in the function of the centromeres,we identified some proteins interacting with BmCenpN by LC-MS/MS.The complexes related to BmCenpN protein were enriched by immunoprecipitation,and then the samples after immunoprecipitation were detected by LC-MS/MS.34 proteins were identified in the control group and 166 proteins were identified in the experimental group,and 142 proteins have specific interaction with the BmCenpN protein.These proteins are mainly involved in cellular and metabolic processes in terms of biological processes,and mainly have binding functions of nucleic acid and protein in terms of molecular functions.Nine proteins involved in mitosis and cell cycle-associated were screened for interaction verification: ?-Tubulin,heat shock protein homolog abbreviated as HSC70,actin depolymerization factor abbreviated as ADF1,F-actin capping protein ? subunit abbreviated as FCPa,F-actin capping protein ? subunit abbreviated as FCPb,ubiquitin Ligase E2 abbreviated as Ube2,ubiquitin carboxy terminal hydrolase abbreviated as UchX1,Cdc2-related kinase abbreviated as CDK10,ubiquitin activating enzyme E1 abbreviated as Uba2.Cell colocalization and co-immunoprecipitation experiments were performed on 9 proteins and BmCenpN protein,respectively.Co-localization experiments showed that BmUba2,BmFCPa,BmUchX1,BmFCPb and BmCDK10 could co-localize with BmCenpN protein in the interphase,metaphase and anaphase,while BmADF1,BmUbe2,BmTubulin and BmHSC70 could colocalize in the metaphase and anaphase.Further,it was confirmed by immunoprecipitation experiments that 9 proteins can interact with BmCenpN protein.(3)Regulation of CenpN interaction protein in silkworm,Bombyx moriTo investigate the effects of 9 interacting proteins on the function of the CenpN protein.We first synthesized double-stranded RNAs of the target gene.RT-PCR results showed that the synthesized double-stranded RNA can specifically reduce the expression of the target protein,indicating that the synthesized double-stranded RNA can play a role in SID I cells.Furthermore,we established a cell line expressing EGFP-BmCenpN stably and interfered with genes of the interaction proteins.The results showed that the expression of EGFP-BmCenpN was significantly changed in the 9 experimental groups compared with the control,especially when the HSC70 gene was interfered and the EGFP-BmCenpN protein was localized in the nucleus and cytoplasm.Interestingly,when the HSC70 gene was interfered,the cells were treated with the proteasome inhibitor MG-132,and the expression of EGFP-BmCenpN was significantly restored.The results of Western Blot further confirmed the results of the fluorescence experiment.These results indicate that HSC70 protein can affect the subcellular localization and stability of the centromere protein BmCenpN in Bombyx mori..