本文選題：布洛芬 + 微生物降解�。� 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：布洛芬在為人類減輕疼痛的同時也帶來一定的環(huán)境污染,為了緩解此環(huán)境問題,以篩選到的3株布洛芬降解菌為研究對象,進行布洛芬的微生物降解研究,以期為布洛芬污染的微生物修復(fù)提供理論與實踐依據(jù)。將從西安污水處理廠水樣分離到的3株以布洛芬為唯一碳源的降解細(xì)菌進行菌株鑒定與生長特性研究,分別構(gòu)建基因文庫以篩選布洛芬降解基因,成功構(gòu)建3株細(xì)菌的福斯質(zhì)粒文庫后篩選得到1株可以產(chǎn)生褐色鄰苯二酚代謝物的陽性克隆菌株,再次構(gòu)建該克隆菌株的突變體文庫,篩選得到7株不再積累褐色代謝物的突變體菌株,分別對功能缺失克隆進行測序分析,初步確定布洛芬降解菌的降解相關(guān)基因。主要研究結(jié)果如下:(1)分別通過對3株布洛芬降解菌的顯微形態(tài)學(xué)觀察,生理生化特性實驗,16S rDNA鑒定及構(gòu)建系統(tǒng)發(fā)育樹對其進行種屬的鑒定,最終確定I-2為克雷伯氏菌屬(Klebsiella sp),I-4為假單胞菌屬(Pseudomonas sp),I-14為不動桿菌屬(Acinetobacter beijerinckii)。(2)分別通過生長特性實驗對3株布洛芬降解菌進行最適生長條件的探索,結(jié)果顯示3株菌的最適培養(yǎng)溫度均為30℃,I-2,I-14最適pH為7,而I-4最佳pH是6,對于布洛芬底物濃度的耐受I-2最高,為1000 mg/L,I-4次之,耐受濃度是900 mg/L,I-14最低,但也達到500 mg/L。(3)分別提取3株菌株的基因組DNA構(gòu)建了3個菌株的福斯質(zhì)粒文庫,通過對文庫的篩選在I-2福斯質(zhì)粒文庫中得到1株能夠積累鄰苯二酚代謝物的陽性克隆2H10,該陽性克隆可以利用布洛芬為唯一碳源生長,說明其含有布洛芬降解基因。(4)對含有降解基因的陽性克隆菌株2H10構(gòu)建Tn5插入突變體文庫,經(jīng)過對該突變體文庫的篩選確認(rèn)得到7株功能喪失菌株,對這些菌株進行測序分析,篩選到五個編碼基因,初步確定I-2的降解相關(guān)基因。
[Abstract]:Bloven also brought certain environmental pollution to alleviate the pain for human beings. In order to alleviate this environmental problem, 3 strains of Bloven degrading bacteria were selected as the research object, and the microbial degradation of Bloven was studied in order to provide the theory and practice basis for the microbial remediation of Bloven pollution. The water samples from the Xi'an sewage treatment plant will be provided. The isolation of 3 strains of biodegradable bacteria with ibuprofen as the only carbon source were identified and the growth characteristics were studied. The gene library was constructed to screen the ibuprofen degrading gene, and 3 strains of bacterial plasmid library were successfully constructed. 1 strains of positive clones that could produce brown catechol metabolites were screened, and the clone was constructed again. 7 mutant strains that no longer accumulated Brown metabolites were screened, and the functional deletion clones were sequenced and analyzed. The main results were as follows: (1) the micromorphological observation and physiological and biochemical characteristics of 3 strains of ibuprofen degrading bacteria were observed respectively. 16S rDNA identified and constructed phylogenetic tree for identification of its species, and finally identified I-2 as Klebsiella (Klebsiella SP), I-4 as Pseudomonas (Pseudomonas SP), I-14 as Acinetobacter (Acinetobacter beijerinckii). (2) exploration of the optimum growth conditions for 3 strains of ibuprofen degrading bacteria through growth characteristics experiments. The results showed that the optimum culture temperature of 3 strains were 30, I-2, I-14 was 7, and the best pH of I-4 was 6. The highest tolerance to ibuprofen substrate concentration was I-2, 1000 mg/L, I-4 time, 900 mg/L and I-14 lowest, but 500 mg/L. (3) also obtained 3 strains of genomic DNA to construct the FOSS plasmid library of 3 strains, respectively. By screening library, 1 positive clones that could accumulate catechol metabolites were obtained in I-2 FOS plasmid library. The positive clones could be grown with ibuprofen as the only carbon source, indicating that it contains the ibuprofen degradation gene. (4) the Tn5 insertion mutant library was constructed for the positive clon strain 2H10 containing the degrading gene, and the Tn5 inserted mutant library was constructed. The mutant library was screened and confirmed to obtain 7 strains of functional loss. The strains were sequenced and analyzed, and five encoding genes were screened to determine the related genes of I-2 degradation.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X172

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本文編號：1852668