反硝化反应器喹啉降解相关基因多样性与定量分析

Diversity and quantification analysis of functional genes in a lab scale denitrifying quinoline-degrading bioreactor

摘要:

摘要：【目的】本文旨在探讨喹啉驯化的反硝化反应器微生物群落中苯甲酰辅酶A还原酶基因(bcrA)和8-羟基-2（1H）喹喏酮基因的加氧酶组分(oxoO)基因的多样性与分布。【方法】根据GenBank数据库oxoO基因序列的保守区设计了oxoO基因专一性引物。扩增采用机械击打与酚-氯仿抽提相结合的方法从反应器生物膜样品中提取微生物总DNA，对oxoO基因和bcrA基因进行基因扩增，并构建oxoO和bcrA基因克隆文库。采用荧光实时定量PCR方法对反应器微生物群落中bcrA和oxoO基因进行定量分析。【结果】定量分析结果表明，在反应器运行过程中，bcrA基因数量逐渐增多，而oxoO基因数量逐渐减少。克隆文库基因序列的系统发育分析表明，bcrA基因克隆文库中部分序列与Thauera等菌株的bcrA基因的相似性为97%以上，其余序列与已知bcrA基因序列的相似性为74%-86%；oxoO基因克隆文库中部分序列与恶臭假单胞菌(Pseudomonas putida)的oxoO基因相似性为99%，而其余序列和已知的oxoO基因的序列相似性较低。【结论】喹啉驯化的反硝化反应器系统中，bcrA和oxoO基因的多样性较丰富，且具有一些新的未知的类型。bcrA和oxoO基因的数量随反应器的运行状况而发生变化，显示出其与反应器中微生物种群构成及功能之间的密切关系。这两个基因可以作为一种潜在的生物分子标记，用于监测含喹啉废水反硝化反应器的运行状态。

Abstract:

Abstract:[Objective] In order to study the diversity and distribution of Benzoyl coenzyme A reductase (bcrA) and oxygenase components of 1H-2-oxoquinoline 8-monooxygenase(oxoO) gene in a lab scale denitrifying bioreactors for treating quinoline-containing wastewater. [Methods] Genomic microbial DNA was extracted from the biofilm samples of bioreactor. Based on the known oxoO genes sequences in GenBank, we designed primers for oxoO gene amplification. Using our designed oxoO gene primers and a pair of degenerate primers of bcrA gene obtained from literature, we amplified the DNA samples. And the amplicons were used for constructing clone libraries of oxoO and bcrA gene. We also performed quantification analysis of these two genes in bioreactor by using Real-time qPCR method. [Results] The quantification analysis showed gradual increase of the bcrA gene abundance and reduction of the oxoO gene abundance along with time. The clone library analysis of these two genes indicated that most clones in bcrA gene clone library having more than 97% sequence similarities to the known bcrA gene of Thauera bacteria and others only having 74%~86% sequence similarities to bcrA gene sequences. Whereas, only a few clones in the oxoO gene clone library have 99% sequence similarities to that gene of Pseudomonas putida. But the sequences of most clones only distantly related with known oxoO genes. [Conclusions] This study showed a high bcrA and oxoO gene diversity, with some new gene sequences, in the lab scale denitrifying bioreactors. The abundance of bcrA and oxoO gene changed remarkably during the running of bioreactor, and closely related with the performance of bioreactor. Therefore, bcrA and oxoO genes have potential to be used as molecule markers to monitor the process of treating quinoline containing wastewater.