摘要：抗生素滥用导致的抗性基因（Antibiotic resistance genes，ARGs）污染是当今亟待解决的环境热点问题之一。为探究四环素污染的水体对反硝化菌工艺中的反硝化菌群的影响，本课题采用定量PCR、克隆文库、高通量测序等分子生物学技术探究四环素添加后，反硝化菌群数量、结构及其四环素类抗性基因（Tetracycline resistance genes，TRGs）的变化。实验表明：抗性基因的丰度随四环素添加浓度的增加而增加，四环素浓度变化对反硝化菌群中的外排泵机制类抗性基因（tetA、tetC和tetG）、核糖体保护机制类tetQ和酶灭活机制类tetX的丰度影响明显。此外，随着四环素浓度增加，可移动遗传因子intI1和功能基因的丰度均增加。intI1丰度的上升显著提高了TRGs在环境转移的风险，相关性分析结果显示intI1与TRGs均呈正相关，说明两者间具有较强的关联性。研究成果将为进一步深入研究减少甚至消除TRGs的反硝化脱氮工艺提供理论基础和技术支持。89786

Abstract  In recent years, The antibiotic resistance genes(ARGs) pollution caused by antibiotic abuse that is one of the hot environmental problems to be solved urgently。 In order to investigate the effect of tetracycline contaminated water on denitrifying bacteria community in denitrification processes, molecular biological techniques liking quantitative PCR, clone library and high-throughput sequencing were taken to explored the changes of numbers and structures of denitrifying bacteria community, as well as Tetracycline resistance genes(TRGs)。 The experiment results indicated that the abundances of tetracycline resistance genes (TRGs) were raised with the increase of antibiotics concentration。 The change of tetracycline concentration apparently impacted abundance of mechanism of efflux pump resistance genes (tetA, tetC and tetG), mechanism of ribosomal protection resistance gene (tetQ) and mechanism of enzyme inactivation resistance gene (tetX) of denitrifying bacteria。 Not only the abundance of the TRGs, but also those of intI1 and function genes were increased while tetracycline concentration increasing。 The abundance increase of intI1 evidently raised the risk of TRGs transfer in the environment。 Correlation analysis proved that intI1 was positively correlated with both TRGs, and there was a strong correlation between them。 The achievements of this project will lay a theoretical foundations and technical supports to explore denitrification processes for TRGs reducing and removal

毕业论文关键词：四环素； 反硝化菌群； 四环素抗性基因

Keyword: tetracycline; denitrifying bacteria; tetracycline resistance genes

目    录

1 材料和方法 5

1。1 材料 5

1。2 方法 5

1。2。1 反应器运行设计 5

1。2。2 样品采集 5

1。2。3 DNA提取 5

1。2。4 高通量源Q于W优E尔A论S文R网wwW.yOueRw.com 原文+QQ75201,8766 测序 6

1。2。5 qPCR分析 6

1。2。5。1 PCR扩增 6

1。2。5。2 回收并纯化目的片段 7

1。2。5。3 目的片段连接 7

1。2。5。4 热击法转化连接产物 7

1。2。5。5 蓝白斑筛选 7

1。2。5。6 qPCR 8

1。2。6 RT-qPCR分析 8

1。2。6。1 RNA提取