本文選題：氮素污染 + 氨氧化細菌��； 參考：《陜西科技大學(xué)》2016年碩士論文

【摘要】：水污染是一個重要的環(huán)境問題,氮素是水體的主要污染源之一。目前采用硝化-反硝化工藝去除污水中的氮素,氨氧化細菌是氮素去除中起主要作用的微生物,可將氨態(tài)氮轉(zhuǎn)化為亞硝態(tài)氮。但因其生長速率緩慢,較難培養(yǎng)且不易分離到純種菌株,仍然是污水脫氮處理的一個限制性因素。因此,構(gòu)建快速生長、易培養(yǎng)的生物脫氮菌株將促進生物脫氮技術(shù)的發(fā)展。本研究從土壤富集液中克隆了氨氧化細菌的兩個功能基因(amoA基因和hao基因),并將這兩個基因共同表達于大腸桿菌B L21(DE3)中,構(gòu)建了具有氨氧化功能的大腸桿菌工程菌。本論文的研究結(jié)果如下：(1)從土壤富集液中提取DNA,利用氨氧化細菌的特異引物PCR擴增amoA和hao基因序列,PCR產(chǎn)物分別連接克隆載體pMD18-T,轉(zhuǎn)化大腸桿菌DH5a,經(jīng)菌落PCR篩選,挑選目標(biāo)片段的重組子測序及Blast分析,獲得兩種功能基因序列。結(jié)果表明amoA和hao基因分別與Nitrosomonas sp.GH22和Nitrosomonas sp. ENI-11序列同源相似性達到99%。(2)采用生物信息學(xué)對AmoA和Hao結(jié)構(gòu)和功能進行分析,結(jié)果表明：AmoA氨基酸全長為276個,分子質(zhì)量為31.94 kDa,屬于不穩(wěn)定的疏水性蛋白,該蛋白含有6個潛在跨膜螺旋區(qū),三級結(jié)構(gòu)顯示空間構(gòu)象不對稱；Hao氨基酸全長為570個,分子質(zhì)量為64.27 kDa,屬于穩(wěn)定的親水性蛋白,該蛋白含有1個潛在跨膜螺旋區(qū),三級結(jié)構(gòu)顯示空間構(gòu)象不對稱。(3)將amoA和hao基因分別連接到表達載體pQE30和pET28a,構(gòu)建重組原核表達載體pQE30-amoA口pET28a-hao,分別轉(zhuǎn)化大腸桿菌BL21(DE3)中,RT-PCR證實基因得到表達,然后測定AmoA和Hao粗酶液活性。以硫酸銨為底物,AmoA粗酶液的氨氮降解率為90.6%,以鹽酸羥胺為底物,Hao粗酶液的羥胺降解率為86.3%,說明AmoA和Hao蛋白粗酶液具有一定的酶活性。(4)質(zhì)粒pQE30-amoA和pET28a-hao,同時轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)過雙抗平板和菌落PCR篩選后,得到重組菌BL21 (DE3)-pQE30-amoA-pET28a-hao, RT-PCR證實兩個基因均在重組菌中得到表達,擴大培養(yǎng),測定此菌株的氨氮降解能力,培養(yǎng)60 h后氨氮的最大降解率達到92.0%,說明菌株BL21 (DE3)-pQE30-amoA-pET28a-h65tgr .1``````.ao具有一定的脫氮活性。綜上所述,本研究構(gòu)建了一株具有氨氧化功能的大腸桿菌工程菌,在氨氮污水處理中有一定的應(yīng)用價值。
[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.
【學(xué)位授予單位】：陜西科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X703;X172;Q78

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