植物细胞核仁动态结构的研究

【作者】 施寒清；

【导师】 焦明大；

【作者基本信息】 东北师范大学 ， 遗传学， 2004， 硕士

【摘要】 核仁是真核生物细胞的rRNA合成加工和核糖体装配的场所。核仁的超微结构主要有三种基本结构组分：FC、DFC和GC。核仁内rDNA的分布、转录定位、rRNA前体的加工及核糖体的装配和核仁的蛋白质组学等都是细胞研究中的重要问题和方面。关于rRNA基因的转录位点是在FC还是DFC，到目前为止还存在争议。本文通过采用常规电子显微镜方法和柠檬酸铽单链RNA特异性染色法，以洋葱为材料对核仁周期中核仁的形态结构变化规律和核仁中的单链RNA的分布进行了研究。由于新合成的RNA短链可能还没有形成二级结构，一些单链RNA的分布区，代表了新合成的RNA的分布区，这有利于转录位点争议的解决。本研究得出如下结论：从洋葱分生组织可以得出如下规律：在细胞周期中，核仁在有丝分裂末期开始出现，在G1期的较早阶段核仁很小，还没有形成FC和DFC结构，随着细胞的发育，核仁逐渐变大，FC和DFC结构开始明显，后来核仁开始合并，在G1期的中后期阶段一个细胞中一般为1—2个核仁；核仁的大小在G1期的中后期、S期和G2期无明显变化，在S期和G2期，DNA的加倍，可能对核仁的体积无明显影响。在S期、S与G2的过渡时期，核仁的边缘出现了毛边现象，在形态学上可见核仁与其周边染色质的联系增多。在G1期、G2期的典型阶段和前期的早期阶段则无此现象。核仁FC、DFC的变化及分布与细胞周期变化不是同步的。在G1期、 S期和G2期都存在较大的FC和较小的FC，一般具较大的FC的细胞，FC的数量较少；FC较小的细胞，核仁内FC的数量较多；但也有一些细胞的一个核仁<WP=4>中同时存在大的和小的两种FC。DFC从G1期的中后期开始清晰，直至有丝分裂的前期的较早阶段为止都在核仁中清晰可见，其大小和比例在此一段区间内无明显的变化。核仁中存在的是较大的FC还是较小的FC及其分布状态与单个细胞有关，而与细胞的核仁周期可能无相关性。FC是异质性的还是同质性的与环绕其的DFC的大小和比例可能无必然的联系。在一些细胞中核仁内FCs呈区域性集中分布，这反映了rDNA的串联性，同时也反映了多个NOR在组织同一个核仁时，各自组装FC和DFC的区域性。核仁在有丝分裂前期开始消失，这是一个过程。早期阶段核仁内的FC和DFC结构无明显变化，后来核仁开始解体为几个小核仁，此时FC和DFC 结构仍然存在，随后FC和DFC结构逐渐消失，在核仁转变成NOR前已没有FC和DFC结构。核仁如果有核仁腔隙，该结构可能发生于S期，其发生可能是多位点的。二、在洋葱核仁中单链RNA分布于DFC及其周围区域，涉及到GC区，在S期和G2期细胞中没有发现FC中存在单链RNA，因此核仁rDNA进行转录时新合成的RNA应定位于DFC，rRNA基因的转录发生于DFC中。在核仁的DFC中单链RNA的分布有区域性，这可能反映了在DFC中rDNA的分布是呈区域性的。更多还原

【Abstract】 Nucleoli are the domains in which transcription and processing of rRNA and assembly of ribosomes take place. The nucleolus has three basic ultrastructural subcompartments: fibrillar centers (FCs), dense fibrillar components (DFCs), and granular components (GCs). The studies on nucleolus focus on the sites of transcription and distribution of rDNA, assembly of ribosomes, and proteomics of nucleolus. Up to now, there are still controversial documents on whether the transcription of rDNA takes place in either FCs or DFCs. By using routine EM and terbium citrate single-stranded RNA (ssRNA) special staining, we investigated the morphological changes of the nucleolus and the distribution of ssRNAs in it. In the nucleolus cycle, due to newly synthesized rRNAs cannot form a secondary structure, the distribution of some ssRNAs represents the distribution of newly synthesized ssRNAs, which is beneficial for solving the controversy about where are the transcription sites of rDNAs. The results from this study are as follows:First, by conventional EM, we found that:In the cell cycle, the nucleoli begin to emerge at the end of mitotic telophase. At the earlier stage of G1, the nucleoli are very small, and they don’t form FCs and DFCs. With the cell grow up, the nucleoli become bigger gradually, and the structures of FCs and DFCs turn obvious and then the nucleoli begin to fuse. In the mid- and late-G1, the cell usually has one or two nucleoli. In the time of the mid- and late-G1, S and G2, the size of nucleolus has no obvious change. DNA doubling in S and G2 phases has no obvious effects on nucleolus volume. In S and S to G2, the nucleolus rim shows hairy and thus more linking of the nucleolus rim to the peripheral chromatins become apparent. In the typical G1 or G2, the morphology changes of the nucleolus rim and the increased linking to the peripheral chromatins are not observed.3. The changes and the distributions of FCs and DFCs in nucleoli are not synchronous to the changes of the cell cycles. In the phase of G1, S and G2, there exist both bigger FCs and smaller FCs in the nucleoli. Generally, cells whose nucleoli have bigger FCs but in lower number and cells whose nucleoli have smaller FCs but in higher number But in <WP=6>some cells, we can find that one nucleolus has both bigger and smaller FCs. DFCs become clear at the mid-G1 and exist in the nucleolus until the early mitotic prophase. Whether the nulceolous has bigger or smaller FCs and the FCs’ distributions may be related to a single cell, but not to the nucleolus cycle of the cell. Whether FCs are heterogeneous or homogeneous cannot be necessarily related to the sizes and ratios of DFCs around it. In some cells, FCs show regionalized and centralized distribution, suggesting the tandem characters of rDNAs and reflecting the regionalism of FCs and DFCs when many NORs organizing one nucleolus.4. It is a process that the nucleoli begin to disappear at prophase. At the early stage, the structures of FCs and DFCs are not obviously changed in the nucleoli. And then, the nucleolus begins to disintegrate in to several small nucleoli. At this moment, structures of FCs and DFCs still exist. Later, the structures of FCs and DFCs disappear. Before the nucleoli turn into NORs, there have been no structures of FCs and DFCs.5.If nucleoli have the nucleolus lacunae, a nucleolus has generally only one. The lacunae structures appear in S and they originate from multiple sites.Second, in Allium cepa, with the ion terbium (III), we found that ssRNAs mainly distribute in DFCs and their peripheral regions, and these distribution further extents in the regions of GCs. ssRNA are not found in FCs S and G2. So when nucleolus rDNAs are transcribed, newly synthesized RNAs should be located in DFCs. That means that the transcription sites of rDNAs take place in DFCs. In DFCs, distribution of ssRNAs shows regional, reflecting the distribution of rDNAs is also regional in DFCs.更多还原