本文關(guān)鍵詞：毒死蜱高效降解菌Cupriavidus nantongensis X1中降解基因功能分析　出處：《安徽農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 毒死蜱與TCP降解 Cupriavidus nantongensis 全基因組測序 OPH TcpA 降解路徑 單交換插入失活

【摘要】：毒死蜱是一種廣譜中等毒性的有機(jī)磷農(nóng)藥,被廣泛用作殺蟲劑、殺菌劑,除草劑,用于水果、蔬菜等經(jīng)濟(jì)作物的病蟲害防治。但是,它對哺乳動物以及其它非靶標(biāo)生物也具有毒性。其降解產(chǎn)物3,5,6-三氯-2吡啶醇(TCP),不僅比毒死蜱的半衰期更長,水溶性更高,而且會抑制其降解菌的降解活性。毒死蜱的大量和長期使用,對水體、土壤等生態(tài)環(huán)境造成了嚴(yán)重的殘留污染。本文以實驗室保存的毒死蜱及TCP的高效降解菌株X1菌株(X1菌被鑒定為嗜銅菌屬下的一個新種,被國際系統(tǒng)與進(jìn)化微生物學(xué)雜志收錄,命名為Cupriavidus nantongensis X1)作為研究對象,對其進(jìn)行了全基因組測序,得到了X1菌的基因組完成圖,確定了降解過程中的關(guān)鍵基因,并對TCP代謝過程中的重要基因進(jìn)行了基因敲除。主要研究結(jié)果如下:1.首次測定新種Cupriavidus nantongensis X1的全基因組序列。使用Illiumie Hiseq與Miseq平臺,通過組裝、優(yōu)化和補(bǔ)洞后獲得了Cupriavidus nantongensis X1基因組完成圖。X1菌基因組由2條染色體DNA序列與1條質(zhì)粒序列組成,全長7,136,420bp,共有6524個編碼基因,57個tRNAs,15個rRNAs,GC含量66.72%。序列提交Gene Bank數(shù)據(jù)庫,所獲登錄號為:CP014844,CP014845,CP014846。2.通過與GO、KEGG、COG數(shù)據(jù)庫比對分析,獲得X1菌編碼基因以及對應(yīng)的產(chǎn)物的功能注釋。并通過組裝序列、編碼基因、ncRNA信息、COG、KEGG、GO功能注釋,繪制得到了X1菌基因組圈圖。3.通過與GeneBank數(shù)據(jù)庫比對,確定毒死蜱在X1菌降解的關(guān)鍵基因--OPH基因,和毒死蜱高毒降解產(chǎn)物3,5,6-三氯-2-吡啶醇(TCP)在X1菌中降解的關(guān)鍵基因—TcpA基因,首次確定TCP降解的完整降解基因簇。結(jié)果分析顯示:OPH基因位于質(zhì)粒上,編碼有機(jī)磷水解酶,催化毒死蜱降解過程中的第一步反應(yīng),將毒死蜱水解為TCP與TCMP;降解TCP的關(guān)鍵基因TcpA基因位于1號染色體上,編碼2,4,6-三氯苯酚單加氧酶催化TCP降解過程中的氧化脫氯過程。4.分析得到毒死蜱在X1菌中的代謝路徑。毒死蜱在OPH基因作用下降解為TCP與TCMP,TCP經(jīng)過分步氧化脫氯后形成3,6-二羥基吡啶-2,5-二酮,經(jīng)過脫乙�；复蜷_吡啶環(huán),在馬來酰醋酸還原酶作用下生成β-酮己二酸,進(jìn)入三羧酸循環(huán),最終代謝成為二氧化碳和水。5.通過研究確定X1菌對氨芐青霉素,卡那霉素,氯霉素,鏈霉素,四環(huán)素,慶大霉素,紅霉素等7種抗生素的抗性譜。利用梯度平板,測定氨芐青霉素、四環(huán)素、氯霉素、鏈霉素、慶大霉素對X1菌株的MIC值分別為20.67μg/ml、12.47μg/ml、2.35μg/ml、4.02μg/ml、2.82μg/ml。X1菌株對50μg/ml卡那霉素不敏感,對100μg/ml紅霉素不敏感。6.利用同源重組對X1菌中TcpA基因進(jìn)行單交換插入失活。通過自殺質(zhì)粒PJQ200SK(慶大霉素抗性),構(gòu)建重組載體PJQ-TY-TcpA,經(jīng)過三親本接合的方式得到TcpA基因失活的突變型菌株X1-ΩTcpA。驗證了TcpA基因為TCP降解過程中的關(guān)鍵基因。
[Abstract]:Chlorpyrifos is a broad-spectrum medium toxic organophosphorus pesticide, widely used as insecticides, fungicides, herbicides, used in fruit, vegetables and other economic crops pest control. It is also toxic to mammals and other non-target organisms, and its degradation product, 35,5-trichloro-2-pyridinol, is not only longer than chlorpyrifos, but also more water-soluble than chlorpyrifos. It also inhibits the degradation activity of chlorpyrifos. A large number of chlorpyrifos are used in water for a long time. Soil and other ecological environment caused serious residual pollution. Chlorpyrifos and X1 strain X1, a highly degradable strain of TCP, were identified as a new species of Copper-eosinophilus. The whole genome was sequenced by Cupriavidus nantongensis X1, which was included in the International Journal of Systems and Evolutionary Microbiology. The complete genome map of X1 strain was obtained and the key genes in the degradation process were identified. The major results of this study are as follows: 1. For the first time, a new species of Cupriavidus nantongensis was determined. The whole genome sequence of X1. Using Illiumie Hiseq and Miseq platform. By assembling. The complete diagram of Cupriavidus nantongensis X1 genome was obtained. The genome of X1 strain consisted of two DNA sequences and one plasmid sequence. The total length of the gene was 7136Bp420bp. there were 6524 coding genes, 57 tRNAss and 15 rRNAs. The GC content was 66.72.The sequence was submitted to the Gene Bank database, and the login number was: CP014844 / CP014845C014846.2. The coding gene of X1 strain and the functional annotation of the corresponding product were obtained by comparing and analyzing the database of KEGGG. Go function annotation, draw X1 bacteria genome circle diagram. 3. By comparing with GeneBank database, determine the key gene of chlorpyrifos degradation in X1 bacteria, OPH gene. And the key gene TcpA of chlorpyrifos degradation in X1 strain. The complete degradation gene cluster of TCP degradation was identified for the first time. The results showed that the TCP gene was located on the plasmid and encoded organophosphorus hydrolase to catalyze the first step in the degradation of chlorpyrifos. Chlorpyrifos were hydrolyzed into TCP and TCMP. The key gene for TCP degradation, TcpA gene, is located on chromosome 1 and encodes 2H4. Oxidative dechlorination of chlorpyrifos in the degradation of TCP catalyzed by 6-trichlorophenol monooxygenase .4.The metabolic pathway of chlorpyrifos in X1 bacteria was analyzed. Chlorpyrifos were degraded into TCP and TCM by OPH gene. P. TCP was oxidized and dechlorinated in a step by step to form 3 ~ (6) -dihydroxypyridine-2-diketone. The pyridine ring was opened by deacetylase and 尾 -ketoadipic acid was formed by the action of maleyl acetate reductase. Enter the tricarboxylic acid cycle, the final metabolism into carbon dioxide and water .5. through the study identified X1 strains of ampicillin, kanamycin, chloramphenicol, streptomycin, tetracycline, gentamicin. The MIC values of ampicillin, tetracycline, chloramphenicol, streptomycin and gentamicin against X1 strain were 20.67 渭 g / ml by gradient plate. Strain 12.47 渭 g / ml 2.35 渭 g / ml 4.02 渭 g / ml 2.82 渭 g / ml .X1 was not sensitive to 50 渭 g / ml kanamycin. Erythromycin was insensitive to 100 渭 g / ml erythromycin. 6. Homologous recombination was used to inactivate the TcpA gene in X1 strain. The suicide plasmid PJQ200SKK (gentamicin resistance) was used. The recombinant vector PJQ-TY-TcpA was constructed. The inactivated mutant strain X1- 惟 TcpA of TcpA gene was obtained by three parent conjugation, which confirmed that the TcpA gene is the key gene in the process of TCP degradation.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X592;X172

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本文編號：1367550