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目的研究细菌对地塞米松类甾体激素的降解作用及其分子机制,为构建清除水环境医源性地塞米松类药物污染的工程菌奠定基础。方法采用富集培养法从医院废水中分离降解地塞米松的细菌,采用固相萃取-高效液相色谱(HPLC)法检测细菌对地塞米松的降解效能,通过16S r DNA序列测定进行鉴定。用Illumina Hiseq4000结合第三代测序技术对降解菌的全基因组测序,并进行序列组装、注释和分析,对降解地塞米松相关基因进行RT-q PCR验证。结果分离到1株对地塞米松有较高降解作用的细菌,经鉴定属于伯克霍尔德菌属(Burkholderia),命名为Burkholderia sp.CQ001。该菌对地塞米松磷酸钠及地塞米松的降解率分别为84.8%和77.11%。全基因组测序表明Burkholderia sp.CQ001包含2条染色体和4个巨型质粒,与代谢相关基因大部分集中在2号染色体上,共3 260 157 bp。生物信息学分析表明,该菌含有甾体代谢通路中许多重要酶类的编码基因,其中与地塞米松降解相关的有ABC转运子,3-甾酮-9α-脱氢酶等,这些基因在以地塞米松磷酸钠为碳源的培养条件下表达量不同程度地高于以蔗糖为碳源的培养条件。结论 Burkholderia sp.CQ001是一株具有强大的代谢功能和代谢途径丰富的细菌,具有降解地塞米松类甾体激素的性能,该菌株为后续研究甾体激素的降解机制和构建清除水环境医源性地塞米松类药物污染的工程菌提供了菌种。
Objective To study the degradation of dexamethasone-like steroids by bacteria and its molecular mechanism, and lay a foundation for the construction of engineering bacteria that can remove i-dexamethasone-contaminated water in aquatic environment. Methods The bacteria that degrade dexamethasone were isolated from hospital wastewater by enrichment culture. The degradation efficiency of dexamethasone by bacteria was detected by solid-phase extraction and high performance liquid chromatography (HPLC) and identified by 16S r DNA sequencing. Whole genome sequencing of the degrading bacteria was carried out with Illumina Hiseq4000 combined with the third generation sequencing technology, and the sequences were assembled, annotated and analyzed. RT-q PCR was used to verify the genes related to dexamethasone degradation. Results A strain of bacteria with high degradation of dexamethasone was isolated and identified as belonging to the genus Burkholderia named Burkholderia sp. The degradation rate of dexamethasone sodium phosphate and dexamethasone was 84.8% and 77.11%, respectively. Whole genome sequencing showed that Burkholderia sp. CQQ001 contains two chromosomes and four giant plasmids, and most of the genes related to metabolism are concentrated on chromosome 2, a total of 3 260 157 bp. Bioinformatics analysis showed that the bacteria contain many important enzymes in the steroid metabolic pathway encoding genes, which are associated with dexamethasone degradation of ABC transporters, 3-ketosteroid -9α-dehydrogenase, these genes in the Dexamethasone sodium phosphate as a carbon source under the conditions of the expression of different levels higher than sucrose as a carbon source of culture conditions. Conclusion Burkholderia sp.CQ001 is a bacterium with strong metabolic and metabolic pathways and degrades dexamethasone-like steroid hormones. This strain is the basis for subsequent studies on the degradation mechanism of steroid hormones and the construction of scavenging water environment Engineering bacteria contaminated with dexamethasone drugs provide bacteria.