目录
摘要 II
关键词 II
Abstract III
引言
引言 1
1 材料与方法 2
1.1 材料 2
1.1.1 重叠延伸PCR 2
1.1.2 试剂和培养基 2
1.1.3 菌株S113总DNA提取所用试剂 3
1.1.4 液质联用（HPLCMS） 3
1.1.5 污染土壤 4
1.2 测定方法 4
1.2.1 LW13sulE工程菌构建方法 4
1.2.2 LW13sulE工程菌构建验证方法 6
1.2.3 LW13sulE工程菌发酵培养方法 7
1.2.4 污染土壤修复效果验证方法 7
2 结果与分析 8
2.1 LW13sulE工程菌构建 8
2.2 LW13sulE工程菌构建验证 9
2.3 LW13sulE工程菌的污染土壤修复效果验证 10
2.3.1 玉米苗生长情况 10
2.3.2 培养10 d时玉米苗鲜重、干重和长度对比分析 11
3 讨论 13
3.1 磺酰脲类除草剂降解机理 13
3.2 菌株LW13sulE构建 13
3.3 修复效果验证 13
致谢 15
参考文献 15
利用甲烷氧化菌修复土壤除草剂污染的研究
摘 要
除草剂等难降解有机物因毒性大、易残留的原因常使作物减产甚至绝收，对生态和经济造成恶劣影响。由于功能菌株难以在土壤中定殖与大量繁殖的原因，在实际应用中生物技术的修复效率远不如预期。本实验采用的甲烷氧化菌只需要有大量甲烷的存在就可以很好在土壤中实现存活与繁殖。我们假设通过基因工程手段赋予该菌株降解目标污染物的能力，有望解决实际修复效率低下的问题。因此本研究通过把从菌株S113中获得的磺酰脲类除草剂水解酶基因sulE导入甲烷氧化菌LW13中的方法，获得具有目标污染物降解能力的工程菌株LW13sulE。再将工程菌株接种至含有不同浓度苄 *51今日免费论文网|www.jxszl.com +Q: *351916072* 
嘧磺隆的土壤中，此外在土壤中设置不接菌、接种LW13和接种S113三种处理方法作为对照，并在上述土壤中种植玉米。对比观察所种植株的长势来探究该基因工程菌对于目标污染物的降解能力。研究结果表明：接种了菌株LW13sulE的污染土壤中植株长势明显好于其他植株的长势。通过一、二级质谱鉴定确认该基因工程菌对于苄嘧磺隆的降解产物为苄嘧磺酸。综上所述，我们所做的设想在盆钵实验中具有可行性，有望将该基因工程菌的建立技术应用于污染场地的原位修复技术中。
STUDY ON USING METHANOTROPH TO REPAIR
HERBICIDE CONTAMINATED SOIL
ABSTRACT
Herbicides and other refractory organics are often toxic and tend to accumulate residues in the environment, often reducing crop yields or even failing harvests, causing bad effects on ecology and economy. Due to the difficulty of colonization and mass reproduction of functional strains in soil, the bioremediation efficiency obtained in practical applications is far less than expected. The methane oxidizing bacteria is used in this experiment only need a large amount of methane to survive and reproduce in the soil. We hypothesized that the strain was given the ability to degrade the target organic pollutants by means of genetic engineering, which is expected to solve the problem of low actual repair efficiency. Therefore, in this study, by introducing the sulfonylurea herbicide hydrolase gene sulE obtained from the strain S113 into the methane oxidizing bacteria LW13, the engineering strain LW13sulE with the target pollutant degradation ability was obtained.
Then the engineering strain LW13sulE was inoculated into soil containing different concentrations of bensulfuronmethyl. In addition, three treatment methods of noninoculation, LW13 inoculation and S113 inoculation were set in the soil as controls, and corn was planted in the above soil. Compare and observe the growth of the plant to explore the degradation ability of the genetically engineered bacteria to the target pollutants. The results of the study showed that the growth of plants in the contaminated soil inoculated with strain LW13sulE was significantly better than that obtained by other treatment methods. The firstlevel mass spectrometry and the secondlevel mass spectrometry confirmed that the degradation product of bensulfuronmethyl by this genetically engineered bacterium was bensulfuronsulfur. In summary, the ideas we have made are feasible in the pot experiment, and it is expected that the establishment of genetically engineered methane oxidizing bacteria will be further applied to insitu remediation of contaminated sites.

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