| Centromere,as one of the vital structures of eukaryotic chromosomes,is an important functional element in the processes of mitosis and meiosis.It gets more and more attention in the studies.Centromere is involved in the precise separation of chromosomes and evenly divided them into daughter cells in the processes of mitosis and meiosis.It mediates the homologous chromosome pairing,acts as sites of sister chromatid cohesion,and regulates the anaphase separation.In recent years,the sequences,structures,and the related proteins of centromere have been hotspots due to its important functions for cell activities.The assembly of genomic sequences in centromere region is very difficult because this region is highly heterochromatinized and contains a large number of repeat sequences and retrotransposons.It is difficult for researchers to interpret its sequences.With the development of molecular and sequencing technologies,more and more centromere repeats have been decoded.In contrast to the function conservation,the length and DNA sequence composition of centromere regions are widely variable in different species,showing a trend of rapid divergence evolution.Such interesting phenomenon is called “centromere paradox”.A few active genes were also identified in the centromere region and the transcription processes of those genes might be important for the epigenetic modification for the centromere region.In contrast to the centromere repeats,centromere-specific histone CENH3 sequences have a similarity of about 60% among different species.The important functions of CENH3 have been reported by lots of studies.CENH3 is the basic skeleton of centromere structure and mediates the assembly of the kinetochore protein complex during cell division.The C-terminus of the CENH3 protein contains a highly conserved HFD domain,while the N-terminus is more variable,suggesting the adaption to the rapid evolution of centromere sequences.The previous studies showed that the functions of centromeres depend on the CENH3 protein rather than the centromere repeats and CENH3 protein has been used as a molecular marker for the active centromere.In recent years,researchers extensively used CENH3 protein to obtain centromere repeats because it can directly bind to centromere sequences.The sequencing of the Morus notabilis genome was completed in 2013.However,the sequences of centromere region are still unknown.The related studies of CENH3 proteins have not been carried out yet.In the present study,two MnCenH3 genes,MnCenH3-1 and MnCenH3-2,were identified for the first time from the Mulberry Genome Database using bioinformatic methods.In addition,H3.2 and H3.3 genes were also identified.Sequence alignment analyses of H3.2 and H3.3 genes indicated that the sequences from mulberry were identical to those in Arabidopsis,suggesting that H3 histones are extremely conserved in eukaryotes.MnCenH3-1 encodes 199 amino acids,while MnCenH3-2 encodes 129 amino acids.Alignment of protein sequences revealed that MnCENH3-1 was 70 amino acids longer than that of MnCENH3-2 at the N-terminus and only 4 amino acids were different at the C-terminus.Further analysis of the gene structure revealed that MnCenH3-1 contains 7 exons.Compared with MnCenH3-1,MnCenH3-2 lacks the first and second exons and the translation started from the end of the third intron.MnCenH3-1 is the gene with the full-length and MnCenH3-2 is a truncated alternative splicing product.There is a 59 bp duplication in the N-terminal region of MnCenH3-1 gene.The length of the common CENH3 protein is about 160-180 amino acids,while the MnCENH3 is 199 amino acids.It is speculated that the prolongation of proteins is related to this repetitive structure.Alignment of protein sequences of CENH3 with other species and the prediction of tertiary protein structure showed that both MnCENH3-1 and MnCENH3-2 have typical CENH3 protein structure: the N-terminal sequence is significantly different,the C-terminal sequence is highly conserved with 4 α helix,2 circular structures,and a CATD region directly bound to the centromere repeats.The phylogenetic tree was constructed by NJ method with the CENH3 protein sequences of Morus notabilis and 23 other species.The results showed that Morus notabilis and Rosaceae are on the same branch,indicating that they are closer with each other in evolution.MnCENH3 expression vector was constructed and the expression was verified by Western blot.The recombinant MnCENH3 protein was successfully purified.DNA Immunoprecipitation(DIP)experiments were performed using the purified protein.Non-specific amplification of the enriched Morus notabilis DNA fragment was carried out.After being sequencing,no centromere region sequences were obtained even if several improvements to the experimental system have been tried.It is speculated that the expressed fusion protein may not be active due to the incorrect folding/modification of the prokaryotic expressed protein system.It failed to bind to the centromere sequence in DIP experiments.It may also be caused by inadequate binding between the protein tag and the affinity medium.Subsequent researches should be improved by using the eukaryotic expressed protein systems,changing protein tags and affinity media,and optimizing the DIP experimental system.To investigate the possible chromosomal fusion and break in M.notabilis,Anti-MnCENH3 polyclonal antibody was also prepared using the 11-amino acid peptide at the N-terminus of MnCENH3 protein as the antigen.The centromeres were tracked by protein immunofluorescence.The signal was detected only in the positive control group.In the experimental group,no centromeric fluorescence signal was detected on the stippled,medium-long,and long metaphase chromosomes.The reason may be the uses a peptide sequence as the antigen to make the Anti-MnCENH3 antibody.So the antigen sequence may not be exposed on the slide,resulting in the antibody not being able to bind.In the future,the related studies should be improved using different antibodies.In the present study,the MnCenH3 gene of M.notabilis was identified for the first time.The antibody against M.notabilis specific centromere marker protein MnCENH3 was prepared and the sequence of M.notabilis centromere was preliminarily explored.It provided some information and experimental basis for future studies on the centromere sequence and protein of M.notabilis. |
