利用高通量RNA-seq深度测序探讨β-双酮类抗生素痕量复合污染对斑马鱼慢性致毒的分子机理

β-diketone antibiotics (DKAs) is one kind of false persistent pollutants, and its main source causing water environmental pollution is from the trace residue at the end of wastewater treatment plant. Therefore, its toxicological effect is easily neglected due to trace level in aquatic environment. In earlier experiment, we found that the compound exposure of DKAs to zebrafish resulted in the significant effect on its behavior and some markers. This proposal aims to reveal the molecular mechanism of chronic toxicity after the compound exposure of DKAs to zebrafish by the following researches: (1) The transcriptome information analysis at different developmental stages will be carried out by high throughput RNA-seq deep sequencing technique. Based on the previous results, the transcript database was then established, their differences were compared, and finally the relationship between the differentially expressed gene and disease occurrence was analyzed; (2) The significantly dynamic changing sRNA will be excavated from the view of post transcriptional regulation, and then its target gene functional classification and metabolic pathway analysis involved in the regulation are performed. The platform for sRNA identification and functional study will be established, which is used to probe the new regulation mode hidden in the gene information and their differences. (3) After integrating the 2-DE data, the novel markers will be explored according to the discovery and validation of markers in relation to medicine source disease in the regulation center. This study can reveal the chronic toxicity molecular mechanism of the compound pollution of DKAs to zebrafish, establish systems toxicology model system, explicit systematically their ecological risk and provide the basis for the diagnosis and treatment of medicine source disease.

β-双酮抗生素（DKAs）是一类假持久性新型污染物，其污水处理末端的痕量残留是水环境二次污染的主要源头，人们最易忽视其环境效应。申请人前期研究发现：痕量浓度DKAs复合污染可引起斑马鱼行为毒性和某些标志物的明显变化。本项目拟以污染源头的实际浓度和种类复合暴毒斑马鱼，利用高通量RNA-seq深度测序技术，完成斑马鱼不同发育期高度动态的转录组信息分析，建立转录本数据库并比较其差异，分析差异表达基因与疾病发生关系。并从转录后调控角度，挖掘显著动态变化的sRNA,进行sRNA靶基因功能分类和参与调控的代谢途径分析，建立sRNA鉴定和功能研究平台，探讨隐藏在基因信息层中全新的调控模式及其差异。整合已完成的2-DE数据，发现和验证一批调控枢纽中与药源性疾病相关的新型标志物。揭示其痕量浓度复合污染下慢性致毒的分子机理，建立系统毒理学模型体系，深入而系统地明确其生态风险，为药源性疾病的诊断与治疗提供依据。

β-双酮抗生素（DKAs）是一类新型”假持久性”污染物，其污水处理末端的痕量残留是水环境二次污染的主要源头，低剂量抗生素长期作用于水生态系统引发的慢性致毒效应是对水生生物和人类健康产生隐患的最大杀手，也是人们最易忽视的环境威胁。课题组在DKAs 复合急性暴露斑马鱼形态畸形初探的基础上，展开了环境浓度下 DKAs 复合暴露斑马鱼分子水平上的毒性效应与致毒机制。首选利用利高通量 RNA-seq转录组 和2-DE蛋白质组学技术，完成DKAs暴露下斑马鱼不同发育期高度动态的转录组和蛋白组的差异分析，建立差异转录本和差异蛋白质的数据库，并在NCBI上递交了数据共享。其次，在生物信息分析的基础上，整合转录水平和翻译水平上的差异，借助于经典的分子生物学的方法及斑马鱼遗传操作和用于毒理学研究的优势，对DKAs暴露的差异基因展开了实验验证和分析，表明DKAs环境浓度的长期暴露对生殖系统、神经行为、内分泌干扰与免疫方面都有不同程度的影响。探讨了相应系统的关键基因和标志物的异常表达代谢与疾病发生的相关系，辅助生化分析、行为学、和病理组织佐证揭示药物的致毒效应。第三，本项目又从转录后调控角度，挖掘显著动态变化的、起主导调控作用的差异miRNA和lnc-RNA，实验证实了20 items miRNA在神经毒性、生殖能力、脂肪代谢紊乱方面的调控作用，及3 items lnc-RNA在肝脏和脾脏等重要组织发异常变化调控免疫相关靶基因的生物学功能，及这些靶基因参与的信号通路和代谢途径分析，建立ncRNA的功能鉴定平台，完善系统毒理学模型体系。该研究系统地从转录—转录后调控---蛋白表达---生物功能层层阐明DKAs致毒的分子机制，揭示该类药物的环境污染潜在的生态风险，探讨其慢性致毒的分子机理，为某些污染性疾病的卫生预防、早期干预提供理论依据。