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题名	贾第虫的“双等核”及其保持等同机制的研究
作者	秦燕
学位类别	博士
答辩日期	2008-01
授予单位	中国科学院研究生院
授予地点	北京
导师	文建凡
关键词	贾第虫 双核 机制 基因转换
其他题名	The Two Equal Nuclei of Giardia and the Mechanism of Keeping Them Equal
学位专业	动物学
中文摘要	贾第虫是一类寄生于肠道的单细胞原生动物，也被认为是目前已知的最原始的真核细胞。它的一个十分奇特和令人感兴趣的特点是：具有在形态和大小上都很相似的左右对称的两个细胞核。已有一些证据表明贾第虫的这样的两个核在其它一些方面也都很相似甚至完全相同，如两核的DNA含量相等，两核都含有rDNA和至少一套染色体，并且在DNA复制、转录和核分裂等功能活动方面也基本都是同步的。但是，这两个核在基因的组成方面是否完全一致，甚至两核之间的对应基因（等位基因）的序列是否完全相同呢？若是，贾第虫为什么需要这样两个完全“等价”的细胞核（“双等核”）？这两个核又是如何长期保持一致而不会因两个核内发生不同的基因变异而产生差异呢？此外，目前普遍认为贾第虫至少是四倍体，即每个核至少为二倍体。那么同一个核内的等位基因是否序列也一致？保持其一致的机制又是什么？这些问题不仅饶有趣味，而且对于揭示贾第虫特殊的遗传机制乃至真核细胞基因组倍性的起源进化等问题具有重要意义。 本文首先对其两个核内是否有相同的基因组成进行了检验。我们选择了三个执行不同功能蛋白的基因为代表以检验贾第虫的两个细胞核是否都含有这些基因。这三个基因分别是：DNA拓扑异构酶II基因（topII）, 核仁蛋白KRR1基因（krr1）和目前贾第虫中报道的极少数含有内含子的基因之一的铁硫蛋白基因（fes）。利用荧光原位杂交(FISH)技术在两个核中进行了定位分析。结果表明这几个代表性的基因在贾第虫的两个细胞核中都同时存在。这提示着贾第虫的两核中具有相同的基因组成，也表明贾第虫的这两个核可能在功能方面也是 “等价”的。 其次，我们对其两个核中的等位基因是否具有一致的序列进行了检验，并对其两核之间以及同一核内的等位基因保持一致的机制进行了研究。选择前人文献报道的存在多态位点（即这些位点可能是易变位点）的两个基因：fen1和pdi为研究对象，在自己建立的一个贾第虫克隆培养系上进行单细胞PCR和测序的跟踪分析。跟踪分析了18个月（约分裂1600代）后，检测到如下结果：尽管在fen1基因上尚未发现位点变异的规律，但在pdi基因上发现存在几个位点会间歇出现套峰。据此我们推测套峰的出现可能是由于其中一个等位基因发生了突变，而后套峰消失则可能是突变被恢复。这种等位基因的修复机制很可能是基因转换（gene conversion）。进而我们在贾第虫基因组中找到了参与基因转换的很多酶基因的同源基因，RT-PCR的结果也表明这些基因在贾第虫中是活跃转录的。这进一步提示了贾第虫中发生基因转换的可能性。因此以上结果不仅表明贾第虫的两核之间和同一核内的等位基因的序列都是一致的，同时还说明基因转换可能是同一核内等位基因保持一致的机制。至于两个核之间保持等位基因序列一致的机制，我们采用了两种染核的方法对大量的贾第虫细胞进行了观察，以期能从中找到两核保持等同的证据。结果发现在大量细胞群体中存在一定比例的单核细胞和一侧含有两个核的细胞。据此我们推测：贾第虫可能通过发生一侧细胞核的丢失或萎缩，然后由另外一侧的核进行复制，经过核的重排恢复正常的左右核的状态。这可能就是贾第虫消除两核基因出现的差异，保持两核之间等位基因一致乃至两个核完全等同的机制。
英文摘要	Giardia lamblia is a flagellated unicellular parasite and one of the earliest diverging eukaryotic cells. Of all the cellular features within the trophozoite, the most puzzling and intriguing is the presence of two nuclei in mirror symmetry, which are identical or similar in several respects. They replicate at nearly the same time, they are equivalent with respect to the amount of DNA harbored in each nucleus, the presence of ribosomal DNA sequences, and the transcriptional activity，and genes from each of the five chromosomes are found in both nuclei, confirming that each nucleus has at least one complete copy of the genome. However, whether each nucleus of Giardia contains the same DNA sequences, and whether the allelic genes in the same nucleus are uniform is not known. If the sequences are uniform, why Giardia needs two equal nuclei? How the Giardia to keep the sequences in the two nuclei equal even though the spontaneous gene mutation? Also, various data indicate the trophozoite of Giardia is tetraploid, that is each nucleus is haploid. Then, How the Giardia to keep the allelic genes in the same nucleus equal? The research into these questions is intriguing, and it can also reveal the unusual genetic mechanism of Giardia and provide useful clue to the ploidy evolution of eukaryote. In the present work, we first determined the two nuclei of Giardia contain the same gene. We randomly selected three genes: topII、krr1 and fes, which functioned differently. We used fluorescence in situ hybridization (FISH) to demonstrate that these genes are found in both nuclei, confirming they are both present in each nucleus. We can also infer the two nuclei maybe functioned uniformly in these respects. Secondly，we determined the sequences in the same nucleus is uniform. Based on this, we attempted to elucidate the mechanism of Giardia keeping the allelic genes of the same nucleus and of the two nuclei equal. In order to see whether the gene sequences were still identical after many generations, we selected genes fen1 and pdi which had been reported having polymorphic sites and tracked their dynamic variation in our established single-cell-cloned line. In the gene fen1, we found no variation after 18 months long. However, we found some sites that changed intermittently in pdi1, which indicated gene conversion maybe occurred. So we searched the Giardia genome and did find the homologous genes of gene conversion. We concluded the gene conversion maybe occurred as an efficient mechanism to eliminate the allelic sequence heterozygousity of Giardia. The another question we have addressed in the present work is how Giardia eliminate sequence difference between nuclei. We stained the nuclei of the Giardia expecting to reveal a clue. Surprisingly, trophoziotes with single nucleus and two nuclei in one lateral were found. The potential reason for this could be intermittent loss of one nucleus, and another nucleus replicated and the two nuclei repositioned to the normal pattern. So the homozygosity of the two nuclei was maintained.
语种	中文
公开日期	2010-10-14
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6097]
专题	昆明动物研究所_真核细胞进化基因组
推荐引用方式 GB/T 7714	秦燕. 贾第虫的“双等核”及其保持等同机制的研究[D]. 北京. 中国科学院研究生院. 2008.

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