原生动物核糖体RNA基因拷贝数及多态性的研究

The ribosomal RNA operon is one of the most important biomarker in molecular taxonomy, evolutionary and molecular ecological studies. Recently, for the first time, the applicant has found that: 1) the copy numbers and sequence polymorphisms of rRNA gene in ciliated protozoa (unicellular eukaryotes) are generally higher than these in all other eukaryotes including plants, animals and fungi; 2) the sequence polymorphism is significantly correlated with the copy number, and 3) thus provide evidence that the relative abundance of ciliates in environmental samples could be overestimated due to their high rDNA copy number per cell, and that the estimation of species richness and phylogeny of ciliates in environmental samples could be problematic because of their high sequence polymorphism in a single cell. Despite these advances, little is known about the situation at the RNA level, and more data from diverse ciliate taxa are needed to strengthen these new findings..Following the line of the previous study, this project proposes to determine the variations of the number and polymorphisms of rDNA copies among individuals, populations, species and higher taxa, by using single-cell based quantitative PCR and deep sequencing technologies. By doing so, the source of main variation can be identified. As the ciliates unlikely need so many rDNA copies (mostly over 100,000) for faster transcription, some rRNA gene must not be actively expressed. Therefore, how many and which rDNA copies are expressed under certain conditions are interesting questions, which are also hot topics on epigenetics of rRNA genes. In this project, we are going to generate such important data by comparing the numbers of rRNA transcripts and rDNA in newly formed duaghter cells and mature individuals, using reverse-transcription PCR and pyrosequencing. With these data, the hypothesis of high copy number – high sequence polymorphism can be further tested, and the effect of high polymophosims of rDNA on rDNA-based phylogenetic inference can be investigated.

核糖体RNA基因是原生动物分子鉴定、进化与生态研究中一个极为重要的分子标记。申请人近期的研究首次发现纤毛虫原生动物基因组中rRNA基因的拷贝数与多态性总体上高于其他真核生物类群、多态性与拷贝数呈正相关，并指出该现象可能影响原生动物分子生态、种类鉴定及系统演化推断的准确度。本项目拟在此工作基础上，应用单细胞定量PCR与焦磷酸测序测序，分析不同类群纤毛虫基因组中rDNA拷贝数与多态性在个体、种群及种间水平的变异，弄清rDNA拷贝数及序列变异的主要来源；明确不同营养及环境（温度、盐度、溶氧）条件下rRNA基因表达的比例及基因型；评估rDNA多态性对原生动物物种鉴定、系统进化推断及分子多样性研究的影响。研究成果将为原生动物分子分类、生理与生态适应机制、rRNA基因的表观遗传学、分子生态研究提供第一手数据，也为rDNA非协同进化类群的系统进化提供重要参考。

核糖体RNA（rRNA）基因被广泛用于构建生命进化树、揭示环境中微生物类群及群落结构、评估它们的丰度与活性，是原核及真核微生物（原生生物）分子鉴定、进化与生态研究中一个极为重要的分子标记。rRNA基因在原生生物基因组中通常以串联、重复的结构存在，具有多个拷贝，也可能具有一定程度的变异。在不清楚rRNA基因的拷贝数及序列多态性在细胞中变异的情况下，基于rRNA基因来推测细胞个体数量及不同类群之间的比例等生态学特征将可能发生误判。在此背景下，申请人以常见的纤毛虫原生生物细胞为研究对象，应用单细胞定量PCR与焦磷酸测序测序，分析不同类群纤毛虫基因组中rDNA拷贝数与多态性在个体、种群及种间水平的变异。以扇形游仆虫、具沟急游虫、肾形虫等为研究对象的结果表明：指数生长期的单细胞rDNA与rRNA拷贝数随着培养温度的升高而降低，但此时细胞体积也变小，回归分析表明rDNA与rRNA拷贝数与细胞体积符合异速生长模型，呈拟合度很高的幂函数关系；rRNA:rDNA与细胞生长速率无关，但随细胞体积的增大而增高。对肾形虫迟滞期、指数期、平台期、包囊期的研究同样支持了上述rDNA与rRNA拷贝数与细胞体积的幂函数关系，进一步表明此关系基本不依赖于细胞个体所处的生长阶段。然而，与其他生长阶段不同，包囊期时rRNA拷贝数明显降低，表明利用此rRNA拷贝数与细胞体积的“二元性”进行估算环境中包囊比例的可能性。此外，对单细胞rDNA与rRNA的高通量序列表明，这些分子在细胞内部具有较高的多态性，序列差异可高达到9%，远高于克隆文库的已有结果。研究了不同抗生素浓度或者重金属浓度条件下，几种模式纤毛虫原生动物单细胞拷贝数与序列多态性的变化情况，发现总体上在抗生素与重金属胁迫条件下，细胞rRNA与rDNA、序列多态性均有增加的趋势。研究成果为原生生物分子分子生态数据的生态学意义与解释提供依据与重要参考。

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