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环境变化对纤毛虫原生生物核糖体RNA基因SNP、拷贝数及其表达的影响
付娆
学位类型博士
导师龚骏
2017-05-21
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词纤毛虫原生生物 温度 环境污染物 生长速率 细胞大小 18s Rdna/rna分子标记 拷贝数变化 序列多态性
摘要核糖体小亚基RNA基因(SSU或18S rDNA),由于其广泛存在性和物种特异性,是生物进化与生态学研究中最理想和最常用的分子标记之一。基于rDNA分子标记进行生物进化和生态学研究时,细胞中rDNA拷贝数及其序列变异程度将对研究结果产生直接影响。纤毛虫原生生物rDNA拷贝数与多态性总体上高于其他真核生物类群,且多态性与拷贝数呈正相关,该现象可能影响纤毛虫分子生态、种类鉴定及系统演化推断的准确度。不同个体,不同生长阶段或环境条件的变化都可能引起动、植物或原生生物细胞内rDNA量或拷贝数的变化,但环境因素引起rDNA拷贝数差异或序列多态性的原因,环境变化对rDNA及其表达产物的数量及其序列多态性的影响以及rDNA/RNA拷贝数与生物体水平的表型性状之间的关系还不清楚。另外,之前的研究多数是基于细胞群体水平或多细胞生物体进行的,缺乏基于单细胞水平的相关研究。
本工作中,我们选取三种单细胞异养纤毛虫作为模式生物,进行了温度、重金属铜离子(Cu2+)和四环类抗生素土霉素(OTC)三种环境条件下的处理实验,在单细胞水平上研究环境变化对纤毛虫原生生物细胞大小,核糖体基因拷贝数、表达量和序列变异程度的影响。得出的主要结论如下:
(1)温度升高造成纤毛虫原生生物生长速率增加,细胞体积减少;而重金属和抗生素胁迫造成纤毛虫生长速率降低,细胞体积增加。纤毛虫细胞在温度变化的返回实验组中可恢复生长速率和细胞大小,具有表型可塑性,而在重金属和抗生素胁迫中,则不能恢复到原始水平,为有不可逆的毒性作用。
(2)基于单细胞水平的qPCR结果显示,本研究涉及的三种纤毛虫细胞均具有极高的rDNA/RNA拷贝数,且同一个细胞中,rRNA拷贝数显著多于rDNA拷贝数。温度升高造成rDNA/RNA拷贝数的显著减少,而重金属和抗生素胁迫则造成拷贝数的增加。将细胞内核糖体拷贝数和多态性与表型(细胞大小、生长速率)相关联,发现rDNA/RNA拷贝数与细胞大小显著正相关。此外,最大生长速率与细胞rRNA:rDNA比值并不良好相关,表明rRNA:rDNA比值并不是原生生物活性的良好指标。
(3)rDNA和rRNA分子标记种内单核苷酸多态性分析发现,AT→GC类型的碱基转换比GC→AT类型的碱基转换更常见。温度升高或重金属和抗生素胁迫情况下,rDNA/RNA中AT→GC突变率增多,导致序列GC含量的增加,用于提高核酸分子的灵活性或弯曲性,保证基因组稳定性。
(4)三种纤毛虫rDNA/RNA分子标记的种内变异度极高,基于97%的相似性水平,单个体内可得到多个rDNA/RNA序列可操作分类单元(OTU),温度升高引起OTU数目的增加,而重金属和抗生素胁迫则造成数目的减少。
本研究在前期纤毛虫原生生物单一个体内rDNA拷贝数及序列多态性的研究工作取得重要进展的基础上,主要深入分析环境变化对纤毛虫rDNA/RNA拷贝数和序列变异的影响,并解析分子生物学特征与表型(细胞大小、生长速率)的相关性。研究结果对基于rRNA与rDNA的原生生物分子生态学研究具有指导意义,为从rDNA角度理解原生生物生理生态适应机制提供了重要数据。
其他摘要Small subunit (SSU or 18S) ribosomal RNA gene (rDNA) is one of the most important biomarkers in phylogenetics and ecological studies as its extensive occurrence in public reference databases and its expected species specificity. Knowing the copy numbers and the variations of rDNA sequences within individuals of single-celled eukaryotes is important for interpreting evolution and ecology of organisms when rDNA sequence-based methodologies are used. rDNA copy numbers and intra-individual polymorphism in protists are higher than those in other eukaryotic groups, and sequence polymorphism is correlated significantly with copy numbers. These may cause inaccuracy in molecular ecology, species identification and phylogeny. Different levels of cellular rDNA contents or copy numbers have been found among isolates and strains, or in different growth phases, of plants, animals and protozoa, or in different environmental factors. However, the causes and effects of environmental factors on the number of rDNA and its expression and sequence polymorphism, and the linkage between rDNA/RNA copy number and phenotypic traits at an organism level are not well understood. Furthermore, previous researchers mainly used pooled cells and multicellular organisms, a fundamental understanding of copy number variations and sequence polymorphism of rDNA/RNA at the single-cell level are still lacking.
Here three kinds of heterotrophic ciliated protozoa were selected as models to determine the effect of three environmental factors of temperature, heavy metal copper (Cu2+) and tetracycline antibiotics oxytetracycline (OTC) on growth rate, cell size, rDNA/RNA copy numbers and sequence polymorphism by using single-cell analysis. The main conclusions in this study are as follows:
(1) Growth rate increased and cell volume decreased with higher temperature, while growth rate decreased and cell volume increased with heavy metal and antibiotics. In addition, we observed that growth rate and cell volume could return to original levels when culturing temperature recovered, highlighting the phenotypic plasticity in ciliates. However, growth rate and cell size could not return to original levels in the returning tests of heavy metal and antibiotics treatments, indicating irreversible toxicity of contaminants on protists.
(2) Extremely high rDNA/RNA copy numbers and different numbers between different ciliated protozoan species basing on single-cell qPCR were found, and rRNA content per cell was generally much higher than rDNA. The rDNA/RNA copy numbers significantly decreased with increasing temperature, while increased significantly with heavy metal and antibiotics. Linking cellular ribotype copy numbers and polymorphisms to biological phenotype (growth rate, cell size), we found that rDNA/RNA copy numbers were positively correlated with cell size. Furthermore, our study also demonstrated that maximum growth rate was not positively correlated with rRNA: rDNA ratio, indicating that rRNA:rDNA is not a good indicator for protistan activity.
(3) Analysis of single nucleotide polymorphisms in rDNA and rRNA molecular markers showed that AT→GC transitions were more common than GC→AT transitions. Higher temperature and heavy metal and antibiotics tended to increase the probability of AT→GC mutations in rDNA and rRNA, which resulted in the increase of GC-content, used to improve the flexibility or bendability of nucleic acid molecules, to ensure the stability of the genome.
(4) Our results showed that intraspecific variability of rDNA and rRNA molecular markers in ciliates could be particularly high. At 97% identity level, several operational taxonomic units (OTU) were obtained within individuals. In addition, OTU numbers of rDNA/RNA sequences increased with higher temperature, while decreased with heavy metal and antibiotics.
Based on rDNA copy numbers and sequence polymorphism data from individual ciliated protists, this study mainly analyzed the environmental impact on rDNA/RNA copy numbers and sequence variation of ciliates, and detected the correlation between molecular biology and phenotype (cell size, growth rate). These individual-based findings may have important implications for population and community ecology of protists using rDNA and rRNA as molecular markers, and provide important data for understanding the physiological and ecological adaptation mechanism of protists from the perspective of rDNA.
学科领域海洋生物学
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/21995
专题中科院烟台海岸带研究所知识产出
作者单位1.中国科学院烟台海岸带研究所
2.中国科学院大学
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付娆. 环境变化对纤毛虫原生生物核糖体RNA基因SNP、拷贝数及其表达的影响[D]. 北京. 中国科学院大学,2017.
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