【作者】 李俊；

【导师】 李立家；

【作者基本信息】 武汉大学 ， 遗传学， 2010， 博士

【摘要】 端粒是真核生物线性染色体的末端结构元件,具有保护染色体、防止染色体末端融合、保持遗传信息稳定等重要作用。端粒与生物的衰老、肿瘤发生等也具有重要的关联。在生物中端粒结构比较保守,一般分成几个部分,包括串联重复的端粒区域、亚端粒区域和中间的连接部分。与其它类型的染色质相比,端粒染色质显示出一种特殊的结构特征。本论文分别以玉米和茼蒿为材料,对植物端粒的结构进行了一些研究,结果如下：亚端粒序列是端粒结构的重要组成部分,多种已报道的亚端粒序列都是基于不同的PCR策略扩增得到的。在玉米中,我们设计出一种简单的亚端粒序列克隆的方案,即以端粒C链引物和G链引物分别进行单引物PCR反应来克隆亚端粒序列。当用端粒C链引物扩增时,得到一条特异性扩增带；当用G链引物扩增时却得到的是一个弥散的扩增带型。对特异性扩增条带克隆和测序结果表明：个412 bp长的克隆序列(被命名为MTAS1)与一个已报道的382bp长的玉米亚端粒序列具有很高的同源性,这说明MTAS1是一个亚端粒序列。MTAS1序列显示出一个新奇的结构特征,它的两端由一个正向的端粒序列和一个倒置的端粒序列构成。Southern杂交结果显示MTAS1的信号呈ladder状,这说明MTAS1是一个串联重复序列。荧光原位杂交(fluorescence in situ hybridization, FISH)实验结果表明：强的MTAS1信号主要分布在一些长染色体的短臂末端上,这证实MTAS1是一个亚端粒序列,同时高亮度的信号表明MTAS1是玉米基因组中的一个多拷贝的串联重复序列。以MTAS1和端粒序列作为探针,在玉米中期染色体和染色质纤维上的双色荧光原位杂交的结果表明：MTAS1信号与端粒信号紧密相连或者相互重叠,在很多染色体的亚端粒区域MTAS1信号呈现多态性分布特征,甚至在同一染色体的两个亚端粒区域MTAS1信号分布都是不同。有研究报道,很多生物的核仁组织区(nucleolar organizer regions, NORs)都定位于染色体的端粒区域,并且在细胞学水平与端粒具有紧密的结构联系。在本研究中,我们偶然在茼蒿这个物种中发现,其所有的rDNA都定位于染色体的端粒区域,并且rDNA信号在不同的中期分裂相中表现出不同的信号类型。在染色质纤维上的双色FISH实验结果表明,端粒序列与rDNA序列在结构上紧密连接或分布于rDNA序列中。鉴于这些观察到的rDNA与端粒之间的紧密细胞学关系,我们想到如果用rDNA引物和端粒引物的组合引物对去扩增基因组DNA,可能会得到两端具有rDNA序列和端粒序列的序列片段。PCR实验结果证实了这一假设,我们得到了多个克隆序列。一个代表性克隆CHS2具有紧密相连的rDNA和端粒序列结构,这一结构说明端粒序列插入到了保守的rDNA序列中。另外,其它一些克隆序列的两端仅含有rDNA单引物或端粒单引物。这些实验结果都表明在染色体末端串联重复的rDNA序列和端粒序列之间可能发生了重组事件。在脊椎动物中,研究发现端粒染色质与近着丝粒异染色质一样,都会发生转录活动,产生含有端粒重复单元的RNA序列(telomeric repeat-containing RNA, TERRA或TelRNA)。TERRA被认为是端粒染色质的重要结构组分之一,并可能在表观遗传学水平上对端粒染色质结构进行调节。在本研究中,我们研究探索不同玉米组织中的TERRA差异性表达情况。用端粒引物进行反转录PCR (reverse transcription polymerase chain reactions, RT-PCR)无法检测TERRA的表达情况,因此我们以临近端粒序列的亚端粒序列设计引物,进行反转录PCR,以此来探测端粒染色质的转录状况。以上面经过深入分析的亚端粒序列MTAS1设计引物。将反转录PCR的产物进行克隆和测序。所得到的克隆都含有一部分与端粒同源的序列和一部分亚端粒同源序列,这证实了端粒染色质发生了转录并证实了玉米中TERRA的存在。荧光定量PCR实验结果显示：具有不同分化状态的各组织中,其TERRA的表达存在数量上的差异,这说明TERRA的表达可能在发育水平上受到调控。此外,通过端粒酶活性检测实验(telomeric repeat amplification protocol, TRAP)来探测这些组织中的端粒酶活性。结果表明,具有最高TERRA表达水平的组织中,其端粒酶活性也是最高的,这暗示着TERRA的表达水平可能对端粒酶的活性具有某种调节作用。更多还原

【Abstract】 Telomere is the terminal part of eukaryotic chromosome and can serve as a protective cap for stabilization of chromosomes and prevention of chromosomal end fusions. Telomere may be also associated with the organism aging and tumorigenesis. The telomere structure shows conservation between organisms, which constitutes the tandem repetitive telomeric region, the subtelomeric region, and the linkage region between them. Telomeric chromatin shows a specific feature, compared with other types of chromatin, which has unique chromatin modifications and specific packaging of nucleosomes. In this research, we study the structure of plant telomeres, using maize and Chrysanthemum segetum L as materials. The results are as follows:Subtelomeric sequence is an important part of the telomere structure and different PCR strategies were used to isolate subtelomeric sequences. In this study, a simple method was developed to isolate the subtelomeric sequence in maize, in which two complementary telomeric primers were used respectively in the single primer amplification of genomic DNA for isolating the subtelomeric sequence. A specific amplification band was obtained with telomere C-strand primer. A smear amplification pattern was obtained with telomere G-strand primer. The specific amplified band was cloned and sequenced. A 412 bp clone (designated as MTAS1) showed a high homology with a reported 382 bp maize subtelomere sequence, suggesting that MTASl is a subtelomeric sequence. The MTAS1 showed a novel sequence structure feature that it was flanked by a forward telomeric sequence and an inverted telomere sequence at the two ends. A ladder pattern of MTAS1 signals was seen in the Southern blot hybridization, which demonstrated that MTAS1 was a tandemly repetitive sequence. Fluorescence in situ hybridization (FISH) results revealed that MTAS1 was mainly distributed at the ends of the short arms of some long chromosomes, which confirmed that MTAS1 was a subtelomeric sequence. The brightness of MTAS1 signals suggested that this sequence was a highly repetitive sequence in maize. Dual-FISH with telomere and MTAS1 probes on the chromosomes and genomic fibers revealed that MTAS1 signals were located adjacent to or overlapped with telomere signals and distributed heterogeneously in subtelomeric regions of several chromosomes, even at two ends of a single chromosome.Several reports revealed that the nucleolar organizer regions (NORs) were located at the telomeric regions of chromosomes and exhibited a close structure connection at cellular level with telomere in many organisms. In Chrysanthemum segetum L., we found by accident that all the 45S rDNAs were located at the telomeric regions of chromosomes. The rDNA showed different signal patterns among the observed chromosome metaphases. Fiber-FISH with rDNA and telomere probes revealed that the telomere signals were closely connected with or interspersed into rDNA signals on the fibers. Based on these cytogical observations, we assumed that specific sequences flanked by rDNA and telomere sequences might be obtained using a combination of telomere and rDNA primers. One representative clone designated as CHS2 showed a close connection between rDNA and telomere sequences, which suggested that the telomere sequence was inserted into the conservative rDNA sequence. Seveal other clones were found to be flanked by the single telomere primer or rDNA primer. Our data implied that homologous recombinations occurred at the chromosomal terminals between the tandem repeated rDNA sequences or telomeric tandem repeats.In vertebrates, some studies revealed that telomeric chromatin similar to percentric heterochromatin was transcribed and generated telomeric repeat-containing RNA (abbreviation:TERRA or TelRNA). TERRA is considered to be an important component of telomeric chromatin and plays a unique role in epigenetically regulating the telomeric chromatin structure and telomerase activity. In this study, we investigated differential expression of telomeric repeat-containing RNA (TERRA) in different maize tissues. Reverse transcription polymerase chain reactions (RT-PCR) with telomere primers could not detect the TERRA, so the subtelomeric sequence primers close to the telomere were employed to detect the transcription of telomeric chromatin. The subtelomeric primers were designed from the maize subtelomeric sequence MTAS1. The product of RT-PCR was cloned and sequenced. These clones contained both a part of a telomere homologous sequence and a part of a subtelomere homologous sequence, which confirmed that the telomeric chromatin was transcribed and the TERRA existed in maize. The real-time PCR results showed that the expression of MTAS1 was quantitatively specific between tissues with different developmental states, suggesting that the transcription of TERRA was developmentally regulated. In addition, the telomerase activity was detected by a telomeric repeat amplification protocol (TRAP) among those tissues and the results showed that the highest telomerase activity was detected in the tissue with the highest expression level of TERRA, suggesting the possible regulation of telomerase activity by the level of TERRA.更多还原