河湖湿地中重金属的不断积累对生态环境构成严重威胁。微生物是一类价格低廉、环境友好型的修复材料。然而，未经改造的微生物用于修复重金属污染存在效率低、特异性不高、环境适应能力差的缺陷。本项目将针对洞庭湖湿地的典型重金属污染，通过微生物细胞表面展示技术，将可特异性吸附、转化重金属的金属调控蛋白和甲基转移酶ArsM锚定在环境适应能力强的Pseudomonas putida细胞表面；解析展示菌的修复动力学和催化As(III)甲基化路径，分析洞庭湖湿地水环境金属离子、离子强度等的影响，探究展示菌在复杂湿地水环境中的重金属修复效率；研究重金属刺激下展示菌细胞膜离子转运酶和NADP氧化酶活、对胞内外ROS浓度的控制、胞内酶活性、非酶类抗氧化功能，阐明展示菌耐受重金属毒性的机制；探究展示菌对洞庭湖湿地种植的水稻或拟南芥耐受重金属毒性的影响。项目成果对于促进基因工程菌在湿地重金属污染修复中的应用具有重要意义。

The accumulation of heavy metals and wetland pose a long-term threat to the ecological environment. Microorganism is a kind of low cost and environment friendly repair material. However, the wild microorganisms without transformation for the remediation of heavy metal have the defect of low efficiency, no specificity and poor adaptability to the environment. This project will target at the typical heavy metal pollution in Dongting Lake wetland. The microbial cell surface display technology will be used to anchor the metal regulated proteins and arsenic(III) S-adenosylmethionine methyltransferase (arsM) which can specifically bind heavy metal and catalyse the methylation of arsenic(III) on the cell surface of Pseudomonas putida with strong adaptability to the environment. Then we will analyze remediation kinetics of heavy metal and pathway of arsenic(III) methylation by the engineered P. putida under the effect of metal ion, ions strength and pH, in case of revealing the remediation rate of heavy metal in complex wetland environment. The activity of ion invertase and NADP oxidase, physiological changes of cell membrane, ROS concentration controlled by engineered P. putida, express level of intracellular enzymes and antioxidant function of non-enzyme materials will be studied to revealing the tolerance mechanism of engineered P. putida to heavy metal toxicity. The effect of engineered P. putida on the tolerance to heavy metal toxicity of rice or Arabidopsis thaliana planted in Dongting Lake wetland will be explored through the toxicology index, growth situation and seed germination of rice or Arabidopsis thaliana. The research results of this project will have important significance for promoting the application of engineering bacteria in the remediation of heavy metal pollution in wetland.