具有氨氧化功能大肠杆菌工程菌的构建

发布时间：2018-05-08 19:26

本文选题：氮素污染 + 氨氧化细菌　；参考：《陕西科技大学》2016年硕士论文

【摘要】：水污染是一个重要的环境问题,氮素是水体的主要污染源之一。目前采用硝化-反硝化工艺去除污水中的氮素,氨氧化细菌是氮素去除中起主要作用的微生物,可将氨态氮转化为亚硝态氮。但因其生长速率缓慢,较难培养且不易分离到纯种菌株,仍然是污水脱氮处理的一个限制性因素。因此,构建快速生长、易培养的生物脱氮菌株将促进生物脱氮技术的发展。本研究从土壤富集液中克隆了氨氧化细菌的两个功能基因(amoA基因和hao基因),并将这两个基因共同表达于大肠杆菌B L21(DE3)中,构建了具有氨氧化功能的大肠杆菌工程菌。本论文的研究结果如下：(1)从土壤富集液中提取DNA,利用氨氧化细菌的特异引物PCR扩增amoA和hao基因序列,PCR产物分别连接克隆载体pMD18-T,转化大肠杆菌DH5a,经菌落PCR筛选,挑选目标片段的重组子测序及Blast分析,获得两种功能基因序列。结果表明amoA和hao基因分别与Nitrosomonas sp.GH22和Nitrosomonas sp. ENI-11序列同源相似性达到99%。(2)采用生物信息学对AmoA和Hao结构和功能进行分析,结果表明：AmoA氨基酸全长为276个,分子质量为31.94 kDa,属于不稳定的疏水性蛋白,该蛋白含有6个潜在跨膜螺旋区,三级结构显示空间构象不对称；Hao氨基酸全长为570个,分子质量为64.27 kDa,属于稳定的亲水性蛋白,该蛋白含有1个潜在跨膜螺旋区,三级结构显示空间构象不对称。(3)将amoA和hao基因分别连接到表达载体pQE30和pET28a,构建重组原核表达载体pQE30-amoA口pET28a-hao,分别转化大肠杆菌BL21(DE3)中,RT-PCR证实基因得到表达,然后测定AmoA和Hao粗酶液活性。以硫酸铵为底物,AmoA粗酶液的氨氮降解率为90.6%,以盐酸羟胺为底物,Hao粗酶液的羟胺降解率为86.3%,说明AmoA和Hao蛋白粗酶液具有一定的酶活性。(4)质粒pQE30-amoA和pET28a-hao,同时转化大肠杆菌BL21(DE3),经过双抗平板和菌落PCR筛选后,得到重组菌BL21 (DE3)-pQE30-amoA-pET28a-hao, RT-PCR证实两个基因均在重组菌中得到表达,扩大培养,测定此菌株的氨氮降解能力,培养60 h后氨氮的最大降解率达到92.0%,说明菌株BL21 (DE3)-pQE30-amoA-pET28a-h65tgr .1``````.ao具有一定的脱氮活性。综上所述,本研究构建了一株具有氨氧化功能的大肠杆菌工程菌,在氨氮污水处理中有一定的应用价值。
[Abstract]:Water pollution is an important environmental problem, nitrogen is one of the main pollution sources. At present, nitrification-denitrification process is used to remove nitrogen from wastewater. Ammonia-oxidizing bacteria are the main microorganisms in nitrogen removal, which can transform ammonia nitrogen into nitrite nitrogen. However, because of its slow growth rate, difficult to be cultured and difficult to isolate pure strains, it is still a restrictive factor in wastewater denitrification treatment. Therefore, the rapid growth and easy cultivation of biological denitrification strains will promote the development of biological denitrification technology. In this study, two functional genes of ammonia-oxidizing bacteria, hao gene and amoA gene, were cloned from soil enrichment solution and co-expressed in Escherichia coli BL21DE3) to construct Escherichia coli engineering bacteria with ammonia oxidation function. The results of this study were as follows: (1) DNA was extracted from soil enrichment solution. The amoA and hao gene sequence products were amplified by PCR of ammonia-oxidizing bacteria and cloned into pMD18-T, respectively, and transformed into Escherichia coli DH 5a, and screened by colony PCR. Two functional gene sequences were obtained by sequencing and Blast analysis of the target fragment. The results showed that amoA and hao genes were associated with Nitrosomonas sp.GH22 and Nitrosomonas sp. respectively. The structure and function of AmoA and Hao were analyzed by bioinformatics. The results showed that the total amino acid length of AmoA and Hao was 276, the molecular weight was 31.94 kDa, which was an unstable hydrophobic protein. The protein contains six potential transmembrane helical regions. The tertiary structure shows that there are 570 asymmetric Hao amino acids with a molecular mass of 64.27 kDa. the protein is a stable hydrophilic protein, and contains a potential transmembrane helical region. AmoA and hao genes were ligated to the expression vectors pQE30 and pET28a, respectively. The recombinant prokaryotic expression vector pET28a-haowas constructed and transformed into E. coli BL21DE3 by RT-PCR. Then the activity of AmoA and Hao crude enzyme solution was determined. The degradation rate of ammonia nitrogen was 90.6 in the crude enzyme solution of AmoA with ammonium sulfate as the substrate, and 86.3 in the crude solution of Hao with hydroxylamine hydrochloride as the substrate, indicating that the crude enzyme solution of AmoA and Hao had certain enzyme activity. PQE30-amoA and pET28a-hao. were transformed into the large intestine at the same time. The bacteria BL21 DE3 was screened by double antibody plate and colony PCR. The recombinant strain BL21 DE3- pQE30-amoA-pET28a-hao. RT-PCR confirmed that the two genes were expressed in the recombinant strain, expanded the culture, determined the ammonia-nitrogen degradation ability of the strain, and the maximum degradation rate of ammonia-nitrogen reached 92.0% after 60 h culture, indicating that the strain BL21 DE3DE3- pQE30-amoA-pET28a-h65tgr. 1 '```````````````````````````````. To sum up, a strain of Escherichia coli with ammonia oxidation function was constructed in this study, which has certain application value in ammonia nitrogen wastewater treatment.
【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X703;X172;Q78

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