【作者】 周博如；

【导师】 王志英；

【作者基本信息】 东北林业大学 ， 森林保护学， 2011， 博士

【摘要】 阿特拉津(Atrazine简称AT)是一种含有苯环、结构稳定、残效期长的选择性除草剂。本研究从国内7省12个土样、1个水样中共分离出15株与AT降解有关的菌株(AT降解菌)。经生理生化分析和分子生物学鉴定,可分为3大类：第一类为节杆菌属(Arthrobacter sp.),包括4株菌,其中YS、SYM1(Y)和CS5为产脲节杆菌属(Arthrobacter ureafaciens),菌株SH为金黄色节杆菌属(A.aurescens),对AT降解率在98%以上；第二类为肺炎克雷伯氏菌属(Klebsiella pneumoniae, (Schroeter) Trevinsan),包括DY6、DY4、DY3(C)、AS、KM(B)和KM4等6个菌株,对AT降解率在56%-71%之间；第三类菌为土壤杆菌属(Agrobacterium Conn,1942,359.),包括MJ2、SYM1(W)、SYM2、CS4和CS1等5个株菌,具有还原硝酸盐及亚硝酸盐能力、可产生3-酮基乳糖及生长不需要生长素和氨基酸等特点,对AT降解率在5%以下。环境pH值、温度、金属离子和碳源对AT降解菌中产脲节杆菌菌株YS和肺炎克雷伯氏菌菌株AS的生物降解能力均有影响。在pH7.0和28℃条件下,降解100 mg·L-1AT所用时间菌株AS为168 h,菌株YS为84 h。菌株AS的最佳降解条件为pH7.0和37。C,葡萄糖可提高其降解效率；菌株YS的最佳降解条件为pH8.0和25℃,果糖、葡萄糖及乳糖可提高其降解效率,而金属离子对其降解效率有不同的影响。从AT降解菌菌株YS基因组DNA中扩增出TrzN和atzB、atzC基因,从菌株AS基因组DNA中仅扩增出TrzN基因。用可在大肠杆菌及芽孢杆菌中穿梭的质粒PHY300PLK,构建TrzN基因表达载体。将该载体导入E coli中能够表达出AT氯水解酶基因,使E coli具有AT降解能力。利用静息细胞研究环境条件对AT降解菌菌株YS生物降解能力的影响表明,Cu2+离子有明显的抑制作用,Mg2+离子有明显的促进作用；缓冲溶液有明显的抑制作用；在MgCl2溶液反应体系中最适降解温度为25℃；在BSM反应体系中,最适降解温度范围为25～37℃；有氧条件可以提高AT降解菌的生物降解能力。AT降解菌对AT污染土壤的土壤呼吸和土壤酶活性有明显影响,土壤中低含量的AT对土壤呼吸和土壤蛋白酶活性有明显的促进作用,而对土壤脲酶活性有明显的抑制作用；土壤中加入AT降解菌液对土壤CO2释放量有明显的促进作用,明显提高土壤蛋白酶活性和土壤脲酶活性。更多还原

【Abstract】 Atrazine (referred to as AT) is a selective long residual period of herbicide with a stability benzene ring. In this research,15 atrazine-degradating strains were separated from 12 soil samples and 1 water sample from the seven provinces in China. The isolated strains were divided into three categories by identification of molecular biology, physiological and biochemical:The first category including 4 strains was Arthrobacter sp., among which the YS, SYM1(Y) and CS5 were Arthrobacter ureafaciens and the SH was A.aurescens. The degradation rates of the first category strains to AT were more than 98%. The second category including DY6, DY4, DY3(C), AS, KM(B) and KM4 was Klebsiella pneumoniae. The degradation rates of the second category strains to AT were between 56% and 71%. The third category including MJ2, SYM1(W), SYM2, CS4 and CS1 was Agrobacterium sp. Those strains could restore Nitrate and Nitrite, reduce 3-keto lactose and grow without auxin and amino acid. The degradation rates of the third category strains to AT were below 5%.The environmental pH value, temperature, metal ions and carbon sources had influence on the biodegradability to AT of Arthrobacter ureafaciens YS and of Klebsiella pneumoniae AS. At the condition of pH7.0 and 28℃, the time of degradating 100mg·L-1 AT was 168 h in the strain AS and was 84 h in the strain YS. The optimum degradation conditions for the strain AS was pH7.0 and 37℃, and glucose could increase its efficiency; The optimum degradation conditions for the strain YS was pH8.0 and 25℃. Fructose, glucose and lactose could increase the efficiency of its degradation, but metal ions had different effects on the degradation efficiency.Except atzA gene, TrzN and atzB, atzC genes were amplified from Arthrobacter ureafaciens YS genomic DNA and the research showed that this set of genes were on the chromosome. Only TrzN gene was amplified from the strain of Klebsiella pneumoniae AS genomic DNA. The expression vector of TrzN gene was built using the plasmid PHY300PLK which could shuttle between Escherichia coli and Bacillus sp. After the vector was introduced, it could express the AT chlorine hydrolase gene and the E coli obtained the AT biodegradability.The research of the environmental conditions on resting cells biodegradability of the AT degrading strain YS showed:The ions of Cu2+ significantly inhibited AT-degradation, while the ions of Mg2+ significantly promoted AT- degradation; The buffer solution significantly inhibited AT- degradation; The optimum degradation temperature was 25℃in the MgCl2 reaction system but it was 25℃～37℃in the BSM reaction system. Aerobic conditions, the resting cell of the strain YS could increase biological degradation to AT.The soil respiration and soil protease was stimulated by AT, whereas the activities of soil urease were inhibited. Strain YS had a significant effect on soil respiration and soil enzyme activity of AT contaminated soil. The CO2 emission quantity was promoted and soil protease activity and urease activity were increased by bacterium.更多还原