废水处理系统中降解基因的克隆新技术研究及多样性分析
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摘要
杂环芳香族化合物是工业废水处理过程中的主要污染有机物。目前已有很多相关的高效去除这些污染物的研究报道,但是人们对参与生物处理的主要功能菌以及降解相关基因知之甚少。本研究主要采用微生物分子生态学技术及基因克隆技术,对降解喹啉和焦化废水的生态系统中的主要功能菌进行研究,并进行克隆降解基因的方法探索。
喹啉是含氮杂环化合物的典型代表,用途极其广泛,由于这类化合物水溶性较强,已成为环境中普遍存在的污染物。我们建立了一个实验室规模反硝化喹啉降解生物反应器,经过约12个月的驯化后,喹啉去除率达到约80%以上。
研究显示在本实验室生物反应器190-600天的运行过程,反应器中bcrA和oxoO两个功能基因的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。说明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。带有oxoO基因的功能菌数量随反应器的运行逐渐减少,带有bcrA基因功能菌数量逐渐增多。间接说明bcrA基因可能参与了喹啉的厌氧降解。由于它们数量变化与反应器的降解过程及运行状态密切关联,因此这两个基因可以作为一种潜在的分子标记,用于监测含喹啉废水处理系统的状态。
本研究中构建了一套启动子捕获质粒系统。初步摸索了该系统的筛选方法,并尝试利用该系统,以底物诱导的基因表达筛选方式,从焦化废水处理系统中克隆具有不同启动子活性的基因片段。研究显示通过筛选系统的筛选,8121个克隆的文库中筛选获得两种类型的克隆,其中一类克隆由于在载体的致死基因sacB中插入了IS-2转座子导致基因的失活突变,从而被筛选系统筛选到。由于转座子IS2在E.coli K-12菌系的菌株中普遍存在,本研究的发现为今后类似的以致死基因进行克隆的研究工作中提供了借鉴,用于克隆的宿主E.coli菌株的选择将是一个重要的考虑因素。另外一类克隆中具有启动子活性的基因片段,但克隆获得的启动子为未知序列,还需后续的深入研究。
Heterocyclic aromatic hydrocarbons are major pollutants in industrial wasterwater treatment. Many efficient pollutant treating techniques have been reported, but we know little about the microbial major functional species and degrading-related genes. In this study, molecular microbial ecology technology and genomic technology were used to analyze the microbial communities in heterocyclic aromatic hydrocarbons (such as quinoline) containing industrial wasterwater treatment. Functional species were studied and a promoter trap plasmid system was constructed for cloning degrading-related genes.
Quinoline is the typical pollutants in industrial wasterwater treatment. Due to its strong water solubility, it has become widespread pollutant. We had set up a lab scale denitrifying Quinoline-degrading bioreactor. After 12 months of acclimation, it reached high quinoline removal efficience of about 80%. The study showed that in the process of bioreactor, there are high diverse bcrA and oxoO genes at the bioreactor. The number of bacteria with oxoO gene decreased while the bacteria with bcrA gene increase gradually during the running of the bioreactor. It indirectly proved that bcrA gene participates in the anaerobic degradion of quinoline. Because the bundance of these functional genes closely related with the degradation rate in the process of bioreactor, we can use these two genes as a biomarker to monitor the quinoline degradation in wastewater treatment system.
In order to intensively investigate the functional genes related to heterocyclic aromatic hydrocarbons degrading, a promoter trap plasmid system was constructed. The screening method was initially explored for cloning gene fragments with different promoter activity from the wasterwater treatment system based on the substrate-induced gene expression screening. Two types of clones were screened out from the clone library containing 8121 clones. One type of clone was screened due to the IS2 insertion fragment which has destroied the suicide gene function of plasmid. Because the IS2 from E.coli K-12 strains, the result indicate the choose of E.coli strain will be a important factor should be considered for the similar research in future. Another type of clone had the insertion and showed promoter activity.But the structure of promoter in this insertion fragment still unknown due to the limit of software for promoters searching.
喹啉是含氮杂环化合物的典型代表,用途极其广泛,由于这类化合物水溶性较强,已成为环境中普遍存在的污染物。我们建立了一个实验室规模反硝化喹啉降解生物反应器,经过约12个月的驯化后,喹啉去除率达到约80%以上。
研究显示在本实验室生物反应器190-600天的运行过程,反应器中bcrA和oxoO两个功能基因的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。说明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。带有oxoO基因的功能菌数量随反应器的运行逐渐减少,带有bcrA基因功能菌数量逐渐增多。间接说明bcrA基因可能参与了喹啉的厌氧降解。由于它们数量变化与反应器的降解过程及运行状态密切关联,因此这两个基因可以作为一种潜在的分子标记,用于监测含喹啉废水处理系统的状态。
本研究中构建了一套启动子捕获质粒系统。初步摸索了该系统的筛选方法,并尝试利用该系统,以底物诱导的基因表达筛选方式,从焦化废水处理系统中克隆具有不同启动子活性的基因片段。研究显示通过筛选系统的筛选,8121个克隆的文库中筛选获得两种类型的克隆,其中一类克隆由于在载体的致死基因sacB中插入了IS-2转座子导致基因的失活突变,从而被筛选系统筛选到。由于转座子IS2在E.coli K-12菌系的菌株中普遍存在,本研究的发现为今后类似的以致死基因进行克隆的研究工作中提供了借鉴,用于克隆的宿主E.coli菌株的选择将是一个重要的考虑因素。另外一类克隆中具有启动子活性的基因片段,但克隆获得的启动子为未知序列,还需后续的深入研究。
Heterocyclic aromatic hydrocarbons are major pollutants in industrial wasterwater treatment. Many efficient pollutant treating techniques have been reported, but we know little about the microbial major functional species and degrading-related genes. In this study, molecular microbial ecology technology and genomic technology were used to analyze the microbial communities in heterocyclic aromatic hydrocarbons (such as quinoline) containing industrial wasterwater treatment. Functional species were studied and a promoter trap plasmid system was constructed for cloning degrading-related genes.
Quinoline is the typical pollutants in industrial wasterwater treatment. Due to its strong water solubility, it has become widespread pollutant. We had set up a lab scale denitrifying Quinoline-degrading bioreactor. After 12 months of acclimation, it reached high quinoline removal efficience of about 80%. The study showed that in the process of bioreactor, there are high diverse bcrA and oxoO genes at the bioreactor. The number of bacteria with oxoO gene decreased while the bacteria with bcrA gene increase gradually during the running of the bioreactor. It indirectly proved that bcrA gene participates in the anaerobic degradion of quinoline. Because the bundance of these functional genes closely related with the degradation rate in the process of bioreactor, we can use these two genes as a biomarker to monitor the quinoline degradation in wastewater treatment system.
In order to intensively investigate the functional genes related to heterocyclic aromatic hydrocarbons degrading, a promoter trap plasmid system was constructed. The screening method was initially explored for cloning gene fragments with different promoter activity from the wasterwater treatment system based on the substrate-induced gene expression screening. Two types of clones were screened out from the clone library containing 8121 clones. One type of clone was screened due to the IS2 insertion fragment which has destroied the suicide gene function of plasmid. Because the IS2 from E.coli K-12 strains, the result indicate the choose of E.coli strain will be a important factor should be considered for the similar research in future. Another type of clone had the insertion and showed promoter activity.But the structure of promoter in this insertion fragment still unknown due to the limit of software for promoters searching.
引文
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