麂属动物以及六带犰狳DNA重复序列家族的克隆、序列分析和染色体定位
文献类型：学位论文
| 作者 | 刘妍 |
| 学位类别 | 硕士 |
| 答辩日期 | 2007-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 杨凤堂 |
| 关键词 | 麂属 六带犰狳 重复序列 克隆 序列分析 荧光原位杂交定位 |
| 其他题名 | Cloning, characterization and FISH mapping of repetitive DNAs in muntjacs and the six-banded armadillo |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 1．黑麂和费氏麂卫星DNA的克隆、序列分析和染色体定位 麂属动物在很短的时间内经历了快速的物种辐射，并且种间染色体数目存在巨大差异，是研究动物核型进化和物种起源的理想模型。近二十年来的分子细胞遗传学研究已基本上证实染色体串联融合(端粒－着丝粒融合)是麂属动物核型演化的主要染色体重排方式。尽管染色体串联融合的分子机制仍不清楚，但研究提示着丝粒区域的卫星DNA可能介导染色体的非同源重组。因此，着丝粒卫星DNA的克隆、分析序列以及染色体定位研究不仅有助于阐明麂属染色体核型演化规律，还可能揭示染色体串联融合的分子机制。迄今为止，上述研究工作已经在赤麂、小麂和小麂台湾亚种开展过。但是，尚无有关黑麂、费氏麂和贡山麂卫星 DNA 克隆、序列分析以及染色体定位研究的报道。 在本研究中，我成功地克隆了黑麂的卫星DNA I、II和IV，分别命名为BMC5、BM700和BM1.1k，并且从费氏麂中克隆了卫星DNA II，命名为FM700。对这些卫星DNA克隆进行序列分析，并将这些克隆探针分别与黑麂、费氏麂、贡山麂和小麂的染色体杂交。研究结果表明： 1)黑麂的卫星DNA I(BMC5)与小麂卫星DNA I(C5)序列高度相似，并且在小麂、黑麂、费氏麂和贡山麂染色体上的大部分串联融合位点处均有分布，因此卫星DNA I可能代表着染色体发生串联融合后保存下来，来源于麂属动物祖先染色体着丝粒的一种卫星DNA。卫星DNA I在这四种麂属动物染色体上的分布也表明黑麂、费氏麂和贡山麂与赤麂的核型演化过程相似，很可能从一个2n ＝ 70的共同祖先通过一系列的串联易位进化而来。 2) 将卫星DNA II（BM700和FM700）克隆探针分别杂交到黑麂和费氏麂的染色体上，只检测到几对间隔分布的信号。这提示在核型进化过程中不同卫星DNA间可能发生了广泛的重组，从而导致卫星DNA II大量丢失。大部分重组断裂位点可能位于卫星DNA I 与卫星DNA II之间，或者在卫星DNA II 区域内。 2．六带犰狳重复序列家族的克隆、序列分析和染色体定位 六带犰狳属于犰狳科、贫齿目，是六带犰狳属中唯一的一个代表物种。系统发育研究认为贫齿目与非洲兽总目是有胎盘哺乳动物中最原始的两个类群。C显带结果揭示六带犰狳30%的基因组是由组成性异染色质构成的，并且C带分布的位置也较复杂，提示在六带犰狳基因组中存在多种重复序列元件。 为了研究六带犰狳异染色质的组成，我从六带犰狳的基因组中克隆了七种位点特异性的重复序列。根据测序结果以及它们在染色体上的分布，将这些重复序列分为五个重复序列家族。其中AMD-EcoRI 837与AMD-BglII 811的序列相似，都是由大小约116 bp的单位组成，分布在大多数染色体的着丝粒区域，同时在一些染色体臂也有分布。AMD-EcoRI 832，AMD-EcoRI 836和AMD-EcoRI 934是特定染色体的重复序列，并且都分布于着丝粒区域。另外，AMD-BglII 634，AMD-EcoRI 731两个克隆都属于长散在分布重复序列(L1)，倾向于分布在G带阳性、富含AT碱基的区域，并且这两种重复序列在染色体上的定位与C带阳性的非着丝粒的异染色质区域很相似。本研究提供了六带犰狳异染色质区域的部分基因组信息，并且这些重复序列家族也可以用于研究六带犰狳及其近缘物种的系统发育关系。 |
| 英文摘要 | 1． Cloning, sequencing and FISH mapping of four satellite DNAs in the M. crinifrons and M. feae. The genus Muntiacus has experienced rapid and radical chromosome evolution in a short time, and displayed an extreme diversification in chromosome numbers, thus constituting an ideal model for studying karyotypic evolution. Recent molecular cytogenetic studies demonstrate that the extensive centromere-telomere fusions are the main chromosomal rearrangements underlying the karyotypic evolution of extant muntjacs. Although the molecular mechanism underlying tandem fusions remains unknown, satellite DNAs are believed to have involved in non-homologous chromosome recombination. Therefore, in addition to a better understanding of the molecular mechanism of tandem fusions, the isolation, characterization and FISH mapping of centric satellite DNAs may facilitate the study of karyotypic evolution in muntjacs. So far, some studies on satellite DNAs have been carried out in M. reevesi, M. muntjak vaginalis and Muntiacus reevesi micrurus. However, there is no report on the cloning, sequencing and chromosomal mapping of satellite DNAs in the M. crinifrons and M. feae. Here I have successfully isolated four satellite DNAs from the genomes of M. crinifrons and M. feae. Satellite DNA I, II, IV clones of M. crinifrons were isolated and named as BMC5, BM700 and BM1.1k respectively. M. feae satellite DNA II clone named as FM700 was also obtained. These satellite DNAs clones have been hybridized to metaphases of M. crinifrons, M. fea and M. gongshanensis. The mapping results indicate: 1) Satellite I element (BMC5) are present at most tandem fusion sites along the chromosomes of M. crinifrons, M. fea and M. gongshanensis, previously defined by M. reevesi painting probes. Chromosome distribution of this satellite DNA suggests satellite DNA I is a sort of centric satellite DNAs inherited from the ancestral acrocentric chromosomes. On the other hand, the results also suggest that the karyotypic evolution in these three species is very similar to that of M. muntjak vaginalis, and that the karyotypes of extant muntjacs have evolved from an ancestor with a 2n = 70 karyotype. 2) Only a few interstitial signals from satellite DNA II (BM700 and FM700) probes were observed, suggesting that intragenomic rearrangements have occurred among satellite DNAs leading to the loss of satellite DNA II during the process of chromosome fusions. Furthermore, chromosomal breaks accompanying tandem fusions at centric regions may have occurred mostly at the regions between Sat DNA I and Sat DNA II or among Sat DNA II regions in M. crinifrons and M. feae. 2．Cloning, characterization and FISH mapping of seven repetitive DNAs from six-banded armadillo (E. Sexcinctus) Six-banded armadillo as the only representative species of genus Euphractus belongs to family Dasypodidae, Xenarthra. The Afrothria and Xenarthra are the most basal clades in placental mammals. At present, only several studies on repetitive DNA families in Xenarthra have been reported. C band staining revealed that at least 30% genomic DNA of six banded armadillo chromosomes are composed of constitutive heterochromatin. The distribution patterns of various C-positive heterochromeric blocks indicate complex repeated elements exist in armadillo genome. To investigate the molecular composition of heterochromatin of six-banded armadillo, I isolated seven site-specific repetitive DNA sequences, which were further classified as five repetitive DNA families according to their chromosome distribution. AMD-EcoRI 837 and AMD-BglII 811 are composed of 116-bp length monomer units, and the two repetitive elements are localized at centric regions of most of chromosomes and coincided at some interstitial heterochrometic blocks along chromosome arms. AMD-EcoRI 832, AMD-EcoRI 836 and AMD-EcoRI 934 are chromosome specific repetitive DNAs that localize at centric regions. Besides, AMD-BglII 634 and AMD-EcoRI 731 belong to L1 element. These two elements seem to have accumulated at the G-positive, AT- rich regions that overlap the interstial heterochromatic blocks on the sex chromosomes and autosomes. In addition to providing genomic information on the composition and organization of heterochromatin of six-banded armadillo, the repetitive DNA families that we have cloned could serve as markers for investigating the phylogenetic relationship of closely related species. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6119]  |
| 专题 | 昆明动物研究所_其他 |
| 推荐引用方式 GB/T 7714 | 刘妍. 麂属动物以及六带犰狳DNA重复序列家族的克隆、序列分析和染色体定位[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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