棉花著絲粒DNA組成與結(jié)構(gòu)分析及染色體空間分布規(guī)律研究
發(fā)布時(shí)間：2019-07-04 14:32
【摘要】：著絲粒作為真核生物染色體上必備的功能元件,參與動(dòng)粒的組建、染色體的分離、姊妹染色單體的粘合、同源染色體的配對(duì)等重要生物學(xué)過(guò)程。著絲粒序列主要是由反轉(zhuǎn)座子以及衛(wèi)星重復(fù)組成,但在不同的物種間甚至是在近緣物種間呈現(xiàn)高度變異,因此,難以克隆分析。本研究通過(guò)基于著絲粒上具有的特異組蛋白CENH3(centromeric histone H3;亦稱CENP-A)的染色質(zhì)免疫沉淀和高通量測(cè)序分析(ChIP-seq),開展了四倍體棉D(zhuǎn)基因組祖先種雷蒙德氏棉著絲粒DNA的發(fā)掘與分析。結(jié)果顯示:(1)該棉種著絲粒內(nèi)具有的重復(fù)序列屬于LTR類反轉(zhuǎn)座子,歸屬于植物CRM反轉(zhuǎn)座子家族。(2)比較分析后發(fā)現(xiàn),這些序列在四倍體棉A基因組祖先種中棉(AA)中不存在或只有極低拷貝數(shù),但在四倍體陸地棉(AADD)中則出現(xiàn)大量擴(kuò)增。因此,暗示棉花多倍化后D組著絲粒中的重復(fù)序列發(fā)生了爆發(fā)式增長(zhǎng)。(3)結(jié)合轉(zhuǎn)錄組數(shù)據(jù)分析,發(fā)現(xiàn)著絲粒區(qū)仍然存在轉(zhuǎn)錄活性基因,但在密度以及表達(dá)水平上較染色體其它區(qū)域顯著降低。(4)此外,在現(xiàn)有的參考基因組上對(duì)著絲粒區(qū)域進(jìn)行了標(biāo)定,同時(shí),也結(jié)合著絲粒定位結(jié)果指出部分染色體在著絲粒區(qū)的組裝存在錯(cuò)誤。這一工作的開展對(duì)解析著絲粒結(jié)構(gòu)和功能之間的關(guān)系及其演化機(jī)制提供了有效的數(shù)據(jù)支持,對(duì)未來(lái)人工染色體構(gòu)建以及在棉花育種中的應(yīng)用奠定了基礎(chǔ)。染色體在細(xì)胞核內(nèi)并不是隨機(jī)分布的,而是會(huì)占據(jù)特定的核空間,形成染色體領(lǐng)域(chromosome territory,CT)。有研究證明,基因組的這種有序組織形式對(duì)于基因的表達(dá)調(diào)控以及基因組功能的正常行使來(lái)說(shuō)至關(guān)重要。因此,近些年基因組三維結(jié)構(gòu)的解析已經(jīng)成為了功能基因組研究的重要內(nèi)容。通過(guò)對(duì)陸地棉(AADD)細(xì)胞有絲分裂中期染色體分布行為的探究。我們發(fā)現(xiàn):(1)陸地棉的A、D組染色體在空間上發(fā)生了分離,并呈現(xiàn)出了典型的徑向(Radial)排布模式,即染色體形態(tài)較小的D組染色體會(huì)聚集在細(xì)胞核的中心部位,而形態(tài)較大的A組染色體傾向于散布在核的外圍。(2)在對(duì)人工六倍體棉花(AADDGG)染色體分布行為的觀察中發(fā)現(xiàn),由于G組染色體的進(jìn)入,A組與D組染色體間所具有的有序排布會(huì)被擾亂。但該六倍體與陸地棉不斷回交后,隨著G組染色體的不斷丟失,A、D組染色體間的徑向分布模式又重新建立。(3)另外,在人工合成的四倍體棉花(AAGG)中也發(fā)現(xiàn)了相似的基因組分離現(xiàn)象。這些結(jié)果表明,基因組分離模式的建立是在四倍體棉花形成后很短的時(shí)間內(nèi)完成的,并且能在四倍體棉花內(nèi)穩(wěn)定的遺傳。結(jié)合其它動(dòng)植物研究,我們推測(cè)基因組的分離在不同基因組間應(yīng)普遍存在。這一發(fā)現(xiàn)為闡明植物細(xì)胞內(nèi)基因組的構(gòu)象規(guī)律及其功能提供了證據(jù)和借鑒。
[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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S562
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