基于RNA-Seq 技术研究环境污染物对河蚬的毒性和毒理作用机制
文献类型：学位论文
| 作者 | 陈辉辉 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 王子健 ; 査金苗 |
| 关键词 | 河蚬，RNA-Seq 技术，毒性效应评价，作用机制，分子标志物， Asian clam （Corbicula fluminea）, RNA-Seq, Toxicity assessment Molecular biomarkers |
| 其他题名 | Toxicity assessment and mechanisms investigation of environmental pollutants to Asian clam based on RNA-Seq |
| 学位专业 | 环境科学 |
| 中文摘要 | 本研究以我国本土淡水底栖动物河蚬作为实验生物，建立了可用于水生态毒理学的河蚬生物毒性测试系统，包括河蚬的实验室驯养和养殖方法、河蚬毒性测试方法和RNA-Seq 表达谱测试方法，并基于该方法对新型环境污染物药品和个人护理品（PPCPs）中的污染物卡马西平和氟西汀进行了毒性效应和作用机制研究。 对我国主要流域的河蚬分布进行了调查，选择洪泽湖河蚬，建立了河蚬实验室驯养和长期养殖的方法和规范，并对河蚬的实验室繁殖方法进行了初步摸索；利用第二代测序技术RNA-Seq 首次对河蚬转录组进行了测序和生物信息学分析。测序共产生62,250,336 高质量的reads，组装后获得134,684 条转录本，平均长度为791 bp，并对这些转录本进行了生物信息学分析，包括基因功能注释、GO 功能分类，KEGG 信号通路注释，开放阅读框和SSRs 鉴定等。选择具有注释信息并与毒理学研究相关的转录本，鉴定和克隆了河蚬抗氧化酶系统、ABC 蛋白家族、细胞色素P450 酶家族、神经系统和热休克蛋白家族等多条功能基因的片段。河蚬转录组的测序和分析丰富了河蚬的生物背景资料，为利用河蚬进行生物毒性测试提供了基础。 对卡马西平进行毒性效应评价和作用机制研究表明，卡马西平在环境浓度水平下对河蚬没有急性毒性，长期暴露能够抑制河蚬的虹吸行为和造成氧化损伤，造成亚慢性毒性。对卡马西平暴露后的河蚬内脏团进行RNA-Seq 表达谱测序和分析，鉴定出9,821 个差异表达转录本，对这些差异转录本进行GO 功能KEGG信号通路富集分析，表明卡马西平主要影响河蚬甘氨酸代谢通路、药物代谢-细胞色素P450 酶、谷胱甘肽代谢通路、细胞调亡和癌症相关等通路。结合基因定量指标重点研究了卡马西平对河蚬细胞应激反应系统的影响；卡马西平同时在转录水平和蛋白水平对河蚬热休克蛋白系统通路的调控具有干扰作用，造成了河蚬组织的分子损伤。这些结果同时证明所建立的河蚬生物毒性测试系统可适用于PPCPs 的毒性评价和毒性作用机制研究。 利用RNA-Seq 技术，结合生长指标、行为学指标、生化指标、抗氧化系统和MXR 系统相关基因表达，对氟西汀的毒性效应及其作用机制进行了多指标和多终点的研究，证明了氟西汀在环境浓度下对河蚬没有急性毒性；氟西汀对河蚬虹吸行为具有抑制作用，能够造成氧化损伤，对河蚬具有亚慢性毒性效应。对氟西汀暴露后的河蚬内脏团组织进行RNA-Seq 表达谱测序和分析，鉴定出9,383个差异表达转录本，对这些差异转录本进行GO 功能和KEGG 信号通路富集分析，表明氟西汀主要对河蚬酪氨酸代谢通路、细胞凋亡通路、细胞自噬作用通路、谷胱甘肽代谢、类固醇合成代谢通路和ABC 跨膜蛋白通路等通路有影响；对河蚬MXR 系统信号通路相关组分的进行分析，氟西汀暴露激活了河蚬细胞内解毒作用机制，同时抑制了ABC 转运蛋白基因表达，造成了氟西汀在细胞内的聚集，产生了毒性效应。 |
| 英文摘要 | In this study, we developed a water ecotoxicology toxicity test method used the local freshwater clam Corbicula fluminea. The methods include the laboratory domestication and breeding methods of the clam, toxicity testing methods and RNA-Seq expression profiling test methods. Then, the toxicity and mechanisms of two unclarified pharmaceuticals and personal care products （PPCPs），carbamazepine and fluoxetine were assessed by the methods. The clams from Hongze Lake were selected for the domestication and breeding in the laboratory. We used a next-generation high-throughput DNA sequencing technique RNA-Seq, to analyze the transcriptome from the whole bodies of Asian clam C. fluminea. More than 62,250,336 high-quality reads were generated based on the raw data, and 134,684 unigenes with a mean length of 791 bp were assembled. Partial cDNA sequences of 30 functional genes were cloned and identified based on the assembled sequences. The C. fluminea transcriptome advances the underlying molecular understanding of this freshwater clam, provides a basis for further exploration of C. fluminea as an environmental test organism and promotes further studies on other bivalve organisms. The filtration rates were significantly decreased by carbamazepine treatment,indicating a negative impact on C. fluminea health. Moreover, Up to 9,821 differentially expressed transcripts (DETs) were identified in the digestive gland after carbamazepine exposed besed on RNA-Seq. The transcript expression fold changes determined by these two methods were highly correlated with a significant coefficient of 0.807. Carbamazepine exposure affected the transcriptome of the digestive gland from C. fluminea encompassing several pathways and processes, including Glycine, serine and threonine metabolism, Metabolism of xenobiotics by cytochrome P450,Glutathione metabolism, Lysosome, Apoptosis and Pathways in cancer, et al. These findings provide a basis for further characterization of the molecular response to carbamazepine stress in C. fluminea. In aggregate, these results confirm that environmentally relevant concentrations of carbamazepine can exert a negative effect on C. fluminea tissue at the molecular and protein level. To investigate the toxicity of fluxetine on the Asian clam, many toxicological endpoints were examined. These endpoints included siphoning behavior, the levels of three stress biomarkers, the expression of the MXR genes in gills and digestive gland. Siphoning behavior was inhibited by treatment with 50 μg/L fluxetine. These findings suggest that fluxetine affects behavior and induces oxidative stress in C. fluminea. Additionally, a total of 9,383 DETs were identified between the control group and fluxetine exposed libraries based on RNA-Seq method. Numerous candidate genes associated with immune response, detoxification, apoptosis pathway were identified. Several Pathways involved in response to fluoxetine stress were implicated by mapping the DETs to reference pathways in the KEGG database,includeing Tyrosine metabolism, Drug metabolism - cytochrome P450, Steroid hormone biosynthesis,Glutathione metabolism, Apoptosis, ABC transporters, et al. These findings will provide a basis and valuable gene information for further characterization of the molecular response to fluxetine stress in the clam |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/34117]  |
| 专题 | 生态环境研究中心_环境水质学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 陈辉辉. 基于RNA-Seq 技术研究环境污染物对河蚬的毒性和毒理作用机制[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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