棉花着丝粒DNA组成与结构分析及染色体空间分布规律研究
发布时间：2019-07-04 14:32
【摘要】：着丝粒作为真核生物染色体上必备的功能元件,参与动粒的组建、染色体的分离、姊妹染色单体的粘合、同源染色体的配对等重要生物学过程。着丝粒序列主要是由反转座子以及卫星重复组成,但在不同的物种间甚至是在近缘物种间呈现高度变异,因此,难以克隆分析。本研究通过基于着丝粒上具有的特异组蛋白CENH3(centromeric histone H3;亦称CENP-A)的染色质免疫沉淀和高通量测序分析(ChIP-seq),开展了四倍体棉D基因组祖先种雷蒙德氏棉着丝粒DNA的发掘与分析。结果显示:(1)该棉种着丝粒内具有的重复序列属于LTR类反转座子,归属于植物CRM反转座子家族。(2)比较分析后发现,这些序列在四倍体棉A基因组祖先种中棉(AA)中不存在或只有极低拷贝数,但在四倍体陆地棉(AADD)中则出现大量扩增。因此,暗示棉花多倍化后D组着丝粒中的重复序列发生了爆发式增长。(3)结合转录组数据分析,发现着丝粒区仍然存在转录活性基因,但在密度以及表达水平上较染色体其它区域显著降低。(4)此外,在现有的参考基因组上对着丝粒区域进行了标定,同时,也结合着丝粒定位结果指出部分染色体在着丝粒区的组装存在错误。这一工作的开展对解析着丝粒结构和功能之间的关系及其演化机制提供了有效的数据支持,对未来人工染色体构建以及在棉花育种中的应用奠定了基础。染色体在细胞核内并不是随机分布的,而是会占据特定的核空间,形成染色体领域(chromosome territory,CT)。有研究证明,基因组的这种有序组织形式对于基因的表达调控以及基因组功能的正常行使来说至关重要。因此,近些年基因组三维结构的解析已经成为了功能基因组研究的重要内容。通过对陆地棉(AADD)细胞有丝分裂中期染色体分布行为的探究。我们发现:(1)陆地棉的A、D组染色体在空间上发生了分离,并呈现出了典型的径向(Radial)排布模式,即染色体形态较小的D组染色体会聚集在细胞核的中心部位,而形态较大的A组染色体倾向于散布在核的外围。(2)在对人工六倍体棉花(AADDGG)染色体分布行为的观察中发现,由于G组染色体的进入,A组与D组染色体间所具有的有序排布会被扰乱。但该六倍体与陆地棉不断回交后,随着G组染色体的不断丢失,A、D组染色体间的径向分布模式又重新建立。(3)另外,在人工合成的四倍体棉花(AAGG)中也发现了相似的基因组分离现象。这些结果表明,基因组分离模式的建立是在四倍体棉花形成后很短的时间内完成的,并且能在四倍体棉花内稳定的遗传。结合其它动植物研究,我们推测基因组的分离在不同基因组间应普遍存在。这一发现为阐明植物细胞内基因组的构象规律及其功能提供了证据和借鉴。
[Abstract]:Centromere, as a necessary functional element on eukaryotes, is involved in the formation of motilla, chromosome separation, sister chromatid bonding, homologous chromosome pairing and other important biological processes. The centromeric sequence is mainly composed of inverters and satellite repeats, but it shows a high degree of variation among different species and even among related species, so it is difficult to clone and analyze. In this study, based on the chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis of the specific histone CENH3 (centromeric histone H 3 (also known as CENP-A) on the centromere, the DNA of Ramonde cotton centromere, the ancestor of the D genome of tetraploid cotton, was excavated and analyzed. The results showed that: (1) the repetitive sequences in the centromeres of the cotton species belonged to LTR inverters and belonged to the plant CRM invertor family. (2) after comparative analysis, it was found that these sequences did not exist or had a very low copy number in the (AA) of tetraploid cotton A genome ancestor species, but a large number of copies were amplified in tetraploid upland cotton (AADD). Therefore, it is suggested that the repetitive sequences in group D centromere increased explosively after polyploidy. (3) combined with the analysis of transcriptional group data, it was found that there were still transcriptional active genes in centromeric region, but the density and expression level were significantly lower than those in other regions of chromosome. (4) in addition, the centromeric region was calibrated on the existing reference genome. Combined with the results of centromere localization, it is pointed out that there are errors in the assembly of some chromosomes in the centromeric region. The development of this work provides effective data support for the analysis of the relationship between centromere structure and function and its evolution mechanism, and lays a foundation for the construction of artificial chromosomes and their application in cotton breeding in the future. Chromosomes are not randomly distributed in the nucleus, but occupy a specific nuclear space, forming the chromosome domain (chromosome territory,CT). It has been proved that this orderly tissue form of genome is very important for the regulation of gene expression and the normal exercise of genome function. Therefore, in recent years, the analysis of three-dimensional structure of genome has become an important part of functional genome research. The chromosome distribution in mitotic metaphase of (AADD) cells in upland cotton was studied. We found that: (1) Group D chromosomes of upland cotton were separated in space, and showed a typical radial (Radial) arrangement pattern, that is, the staining of group D with small chromosome morphology gathered in the center of the nucleus, while the larger group A chromosomes tended to be scattered on the periphery of the nucleus. (2) it was found in the observation of the distribution behavior of (AADDGG) chromosomes in artificial hexaploid cotton. Due to the entry of group G chromosomes, the orderly arrangement between group A and group D chromosomes will be disturbed. However, after continuous backcross between the hexaploid and upland cotton, with the continuous loss of G group chromosomes, the radial distribution pattern between group D chromosomes was re-established. (3) in addition, similar genomic segregation was found in synthetic tetraploid cotton (AAGG). These results suggest that the establishment of genome segregation model was completed within a short period of time after the formation of tetraploid cotton, and it can be inherited stably in tetraploid cotton. Combined with other animal and plant studies, we speculate that genome isolation should be common among different genomes. This discovery provides evidence and reference for illustrating the conformational regularity and function of plant intracellular genomes.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562
[Abstract]:Centromere, as a necessary functional element on eukaryotes, is involved in the formation of motilla, chromosome separation, sister chromatid bonding, homologous chromosome pairing and other important biological processes. The centromeric sequence is mainly composed of inverters and satellite repeats, but it shows a high degree of variation among different species and even among related species, so it is difficult to clone and analyze. In this study, based on the chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis of the specific histone CENH3 (centromeric histone H 3 (also known as CENP-A) on the centromere, the DNA of Ramonde cotton centromere, the ancestor of the D genome of tetraploid cotton, was excavated and analyzed. The results showed that: (1) the repetitive sequences in the centromeres of the cotton species belonged to LTR inverters and belonged to the plant CRM invertor family. (2) after comparative analysis, it was found that these sequences did not exist or had a very low copy number in the (AA) of tetraploid cotton A genome ancestor species, but a large number of copies were amplified in tetraploid upland cotton (AADD). Therefore, it is suggested that the repetitive sequences in group D centromere increased explosively after polyploidy. (3) combined with the analysis of transcriptional group data, it was found that there were still transcriptional active genes in centromeric region, but the density and expression level were significantly lower than those in other regions of chromosome. (4) in addition, the centromeric region was calibrated on the existing reference genome. Combined with the results of centromere localization, it is pointed out that there are errors in the assembly of some chromosomes in the centromeric region. The development of this work provides effective data support for the analysis of the relationship between centromere structure and function and its evolution mechanism, and lays a foundation for the construction of artificial chromosomes and their application in cotton breeding in the future. Chromosomes are not randomly distributed in the nucleus, but occupy a specific nuclear space, forming the chromosome domain (chromosome territory,CT). It has been proved that this orderly tissue form of genome is very important for the regulation of gene expression and the normal exercise of genome function. Therefore, in recent years, the analysis of three-dimensional structure of genome has become an important part of functional genome research. The chromosome distribution in mitotic metaphase of (AADD) cells in upland cotton was studied. We found that: (1) Group D chromosomes of upland cotton were separated in space, and showed a typical radial (Radial) arrangement pattern, that is, the staining of group D with small chromosome morphology gathered in the center of the nucleus, while the larger group A chromosomes tended to be scattered on the periphery of the nucleus. (2) it was found in the observation of the distribution behavior of (AADDGG) chromosomes in artificial hexaploid cotton. Due to the entry of group G chromosomes, the orderly arrangement between group A and group D chromosomes will be disturbed. However, after continuous backcross between the hexaploid and upland cotton, with the continuous loss of G group chromosomes, the radial distribution pattern between group D chromosomes was re-established. (3) in addition, similar genomic segregation was found in synthetic tetraploid cotton (AAGG). These results suggest that the establishment of genome segregation model was completed within a short period of time after the formation of tetraploid cotton, and it can be inherited stably in tetraploid cotton. Combined with other animal and plant studies, we speculate that genome isolation should be common among different genomes. This discovery provides evidence and reference for illustrating the conformational regularity and function of plant intracellular genomes.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562
【参考文献】
相关期刊论文 前7条
1 韩金磊;吴为人;王凯;;染色体构象及其研究方法—3C技术[J];分子植物育种;2016年12期
2 苑宝文;王秀杰;;染色质高级结构——基因组调控的重要形式[J];生命科学;2015年03期
3 ;国际棉花基因组测序计划[J];国际学术动态;2008年02期
4 ;Establishment of a Multi-color Genomic in situ Hybridization Technique to Simultaneously Discriminate the Three Interspecific Hybrid Genomes in Gossypium[J];Journal of Integrative Plant Biology;2008年03期
5 丁戈;姚南;吴琼;刘恒;郑国，
本文编号：2509987
本文链接：https://www.wllwen.com/shoufeilunwen/zaizhiyanjiusheng/2509987.html
