毒死蜱高效降解菌Cupriavidus nantongensis X1中降解基因功能分析

发布时间：2018-01-02 04:00

本文关键词：毒死蜱高效降解菌Cupriavidus nantongensis X1中降解基因功能分析　出处：《安徽农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：毒死蜱是一种广谱中等毒性的有机磷农药,被广泛用作杀虫剂、杀菌剂,除草剂,用于水果、蔬菜等经济作物的病虫害防治。但是,它对哺乳动物以及其它非靶标生物也具有毒性。其降解产物3,5,6-三氯-2吡啶醇(TCP),不仅比毒死蜱的半衰期更长,水溶性更高,而且会抑制其降解菌的降解活性。毒死蜱的大量和长期使用,对水体、土壤等生态环境造成了严重的残留污染。本文以实验室保存的毒死蜱及TCP的高效降解菌株X1菌株(X1菌被鉴定为嗜铜菌属下的一个新种,被国际系统与进化微生物学杂志收录,命名为Cupriavidus nantongensis X1)作为研究对象,对其进行了全基因组测序,得到了X1菌的基因组完成图,确定了降解过程中的关键基因,并对TCP代谢过程中的重要基因进行了基因敲除。主要研究结果如下:1.首次测定新种Cupriavidus nantongensis X1的全基因组序列。使用Illiumie Hiseq与Miseq平台,通过组装、优化和补洞后获得了Cupriavidus nantongensis X1基因组完成图。X1菌基因组由2条染色体DNA序列与1条质粒序列组成,全长7,136,420bp,共有6524个编码基因,57个tRNAs,15个rRNAs,GC含量66.72%。序列提交Gene Bank数据库,所获登录号为:CP014844,CP014845,CP014846。2.通过与GO、KEGG、COG数据库比对分析,获得X1菌编码基因以及对应的产物的功能注释。并通过组装序列、编码基因、ncRNA信息、COG、KEGG、GO功能注释,绘制得到了X1菌基因组圈图。3.通过与GeneBank数据库比对,确定毒死蜱在X1菌降解的关键基因--OPH基因,和毒死蜱高毒降解产物3,5,6-三氯-2-吡啶醇(TCP)在X1菌中降解的关键基因—TcpA基因,首次确定TCP降解的完整降解基因簇。结果分析显示:OPH基因位于质粒上,编码有机磷水解酶,催化毒死蜱降解过程中的第一步反应,将毒死蜱水解为TCP与TCMP;降解TCP的关键基因TcpA基因位于1号染色体上,编码2,4,6-三氯苯酚单加氧酶催化TCP降解过程中的氧化脱氯过程。4.分析得到毒死蜱在X1菌中的代谢路径。毒死蜱在OPH基因作用下降解为TCP与TCMP,TCP经过分步氧化脱氯后形成3,6-二羟基吡啶-2,5-二酮,经过脱乙酰基酶打开吡啶环,在马来酰醋酸还原酶作用下生成β-酮己二酸,进入三羧酸循环,最终代谢成为二氧化碳和水。5.通过研究确定X1菌对氨苄青霉素,卡那霉素,氯霉素,链霉素,四环素,庆大霉素,红霉素等7种抗生素的抗性谱。利用梯度平板,测定氨苄青霉素、四环素、氯霉素、链霉素、庆大霉素对X1菌株的MIC值分别为20.67μg/ml、12.47μg/ml、2.35μg/ml、4.02μg/ml、2.82μg/ml。X1菌株对50μg/ml卡那霉素不敏感,对100μg/ml红霉素不敏感。6.利用同源重组对X1菌中TcpA基因进行单交换插入失活。通过自杀质粒PJQ200SK(庆大霉素抗性),构建重组载体PJQ-TY-TcpA,经过三亲本接合的方式得到TcpA基因失活的突变型菌株X1-ΩTcpA。验证了TcpA基因为TCP降解过程中的关键基因。
[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.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X592;X172

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