Studies On NnCenH3 Protein And Centromeric DNA Of Sacred Lotus (Nelumbo Nucifera Gaertn.)

Centromeres, which are responsible for accurate chromosome pairing /segregation and chromosome stabilization during meiosis and mitosis, are significant markers of the eukaryote chromosomes. The centromeric paradox between the conserved centromeric function and the highly divergent of CENH3 (Centromeric histone H3) and centromeric associated DNA epigenetically defines centromeres. Selective constraints are responsible for the highly rapid evolution in repeat-rich heterocentromeric regions. Natural centromeres degeneration and neocentromeres emerging make centromeric structure dynamic. These features hamper the studies of centromere evolution and formation mechanism.Centromeres have been extensively studied among monocots and eudicots. However, information concerning centromeric sequences structure of basal eudicots is limited. Sacred lotus (Nelumbo nucifera Gaertn.) is an aquatic perennial basal dicot, which is significant in the position of evolution. Here, we have characterized the centromeric marker protein NnCenH3, and then used ChIP-seq technology and anti-NnCenH3 antibody for centromeric sequences identification in sacred lotus. The main results are as follows:1. The CDS (Coding sequence) of NnH3.3, NnCenH3-A and NnCenH3-B were obtained using the method of homologous cloning. NnCenH3-B is the alternative splicing variants of NnCenH3-A. Multiple alignments among the primary structures of these proteins and CenH3 of other plants indicated these sequential variations in N-terminal regions and they were conserved in C-terminal regions. This result revealed that NnCenH3 (NnCenH3-A and NnCenH3-B) had a longer N-terminal and one extra amino acid residue in loop 1 region compared with the amino acid sequence of NnH3.3. In addition, NnCenH3-B lacked a portion of CATD that was responsible for centromere targeting, which suggested that the centromere recognition function of NnCenH3-B might be interrupted. The similar truncated transcripts were discovered in human CENPA (the CenH3 homolog).2. An anti-NnCenH3 polyclonal antibody was raised against 13 residues in the N-terminal region, then verified by western blot analysis of NnCenH3 expressed in vitro and vivo, and the antibody was used to observe the distributions of NnCenH3 in sacred lotus chromosomes. The results indicated that NnCenH3 was an authentic centromere-specific protein in the cells and tissues of sacred lotus.3.700,000 random Illumina Hiseq 2000 reads were used for the reconstruction of sacred lotus genomic repeats by similarity-based sequence clustering approach. The repeats occupied approximately 43%of the sacred lotus genome size, and Tyl /copia LTR retrotransposon was prevalent in the sacred lotus genome.4. To clarify the centromeric DNA sturcture in the sacred lotus genome, we have obtained the centromeric associated DNA sequences through ChIP (Chromatin immunoprecipitation) technology with anti-NnCenH3 antibody. And using the FISH (Fluorescence in situ hybridization) method, we have found the signals of ChlPed-DNA probes were specifically located at the centromere regions. Interestingly, an extra pair of weak ChIPed-DNA signals was observed at the q-arm of chromosome 1. Subsequently, the ChIP-seq data revealed the featrures of low density and activity for the genes expression in NnCenH3-nucleosomes. Furthermore, The alignment between CBS (NnCenH3 binding scaffolds) and sacred lotus repeat database revealed that Tyl/copia class became the predominant TE in CBSs of sacred lotus, although Ty3/gypsy class was commonly the major centromeric TE constituents in other angiosperms. These results showed several features of the centromere DNA structure in the sacred lotus genome.5. We combined ChIP-seq and the similarity-based sequence clustering approach, and then identified several centromeric repeats that were tested by FISH. These identified centromeric repeat clusters showed different distribution patterns on chromosomes, although the majorities were located on the primary constriction of the sacred lotus chromosomes specifically. These patterns were described as follows:1) Chromosome 1 contained two domains with CL6 signals, which were similar with ChIP-FISH signals. However, NnCenH3 was mapped on centromeres specifically. This could be explained by the presence of dicentric chromosome with one active centromere.2) The telomere-like centromere repeats were located on both centromeres and telomeres.3) Some clusters showed dispersed distribution along chromosome arms.In the present study, we have developed the anti-NnCenH3 antibody, which could contribute to the studies of sacred lotus centromere proteins and DNA sequences. In addition, we have first described the centromere profile of sacred lotus, the ancient basal eudicot, which might provide new insights of centromeric sequence structure and centromere evolution. In addition, the identification of centromeric DNA sequences is of great significance for the construction of fine-scale genetic and physical maps in sacred lotus.