【作者】 姜红霞；

【导师】 闫艳春；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 化学农药在农业生产中具有重要作用,但农药的大量使用严重危害人类健康,破坏农业生态平衡;有机磷农药是化学农药中的一大类,在生产中被广泛使用,但它们属于神经毒剂,能引起人畜不同程度的中毒并造成严重的环境污染;甲基对硫磷是其中的典型代表。微生物降解在农药的生物降解中占主导地位。筛选具有降解有机磷活性的微生物,研究其降解性能,并克隆其与降解有关的基因,进而应用于农药的降解处理,具有重要的实践意义。本实验通过生理生化性质和16S rDNA同源性鉴定了实验室保存的两株甲基对硫磷降解菌X-13、X-20,结果表明:X-13 为枯草芽孢杆菌;X-20属于产碱菌属,我们将其暂编号为Alcaligenes.sp.YcX-20,16S rDNA 基因片段在GenBank 中注册号为AY628412。参与甲基对硫磷降解的酶属于磷酸酯酶,两株菌的酯酶活性都随菌液浓度的增加递增,且与菌株的生长曲线正相关。X-20 的酯酶活性高于X-13。在以甲基对硫磷为唯一碳源的液体基础培养基中黑暗培养24h,培养基混浊度增加,菌体增长,同时培养基上清中对硝基苯酚含量增加。初步预测降解菌可以将MP 降解为对硝基苯酚。以MP 为唯一碳源的基础培养基培养,X-13 在甲基对硫磷浓度小于150mg/L 时正常生长,X-20 当甲基对硫磷浓度小于500 mg/L 时正常生长;在普通培养基上两菌株分别能耐受300mg/L 和900 mg/L 的甲基对硫磷;在营养丰富的条件下,菌种能更好的利用甲基对硫磷。气相色谱法测定X-20 对甲基对硫磷的降解表明,在以50mg/L 甲基对硫磷为唯一碳源的培养基中30℃, pH7.0 培养30h 降解率50%。实验中尝试多种质粒提取方法,在两株菌中都没有提取到质粒,初步确定试验菌株可能不含有降解质粒,其降解基因可能存在于染色体上。用PCR 和鸟枪法筛选X-20 降解基因,都没有得到目的片段;推测该菌编码甲基对硫磷降解的基因属于一种新型基因。更多还原

【Abstract】 Pesticides are important to increase agricultural yield. But the pollution has harmed seriously the environment and people’s health. Organophosphorus pesticides are used widely for the control of insects all over the world; methyl parathion is a typical one of them, but they are neurotoxin to human and animal. Microbial process is found an effective way to detoxify the compounds. It is practical to isolate microorganisms that can degrade organic phosphorus, to study their characterization, and to clone the degradation genes then use them to degrade the pollution. Through chemo taxonomic characterizations and phylogenetic inference based on 16s rDNA sequence and analysis, we identified two strains of bacteria X-13 and X-20 isolated from activated sludge of Huayang Pesticide Factory in 2001. The results are: X-13 belongs to Bacillus subtilis; X-20 belongs to a member of Alcaligenes and is named as Alcaligenes.sp.YcX-20, the genus GenBank accession number of the 16s rDNA sequence is AY628412. Enzyme degraded for methyl-parathion belongs to family of phosphorus esterase. Esterase activity of the strains increases with concentration of cell dose and has derect correlation with culture time. It can grow with methyl parathion as sole carbon source and was degraded to p-nitro phenol so to reduce its toxicity for 100~200 times. In rich culture the strains can utilize methyl parathion better. The strain X-20 can tolerate high concentration methyl parathion of 500mg/L with basic medium and up to 900mg/L with ordinary medium while X-13 only 150mg/L and 300mg/L. Detected the remained methyl parathion of 50mg/L with basic culture after 30 hour, at 30℃, pH7.0 with Gas Chromatography, and found 50% was hydrolyzed. It’s possible that the two strains have no degradation plasmid and the degradation genes come from chromosome because we failed to get any plasmid from them although tried different isolated methods. The next we’ll更多还原