阿特拉津AT-b3降解的分子机制与土壤污染的强化修复

Atrazine is the most widely used s-triazine herbicide for control of broadleaf weeds in resistant crops such as corn and sorghum. The widespread use of atrazine resulted in its frequent detection in both surface and ground waters beyond authorized limits, leading to potential human exposure. Toxicological studies raised major concern as atrazine was postulated to be a possible carcinogen, an endocrine disrupter and a teratogen. These facts have stimulated research on efficient bioremediation strategies for atrazine polluted environments in order to quickly prevent the dispersion of atrazine to non-atrazine application place and reduce its contamination to safe level..Microbial bioremediation is quite widely used for cleaning up contaminated soils, because it often is more efficient and less expensive than physicochemical remediation. This method is use microorganisms transform pesticide into CO2, H2O or other harmful substances, no secondary pollution. Microorganisms often respond to the input of xenobiotics into the environment by evolving mechanisms to use them as sources of nutrients and energy for their growth. Microbial degradation of atrazine and other s-triazine compounds is one of the major modes of their removal from the environment. But the degradation ability is highly affected by external environment, the degradation function of strains often decline or loss. Here we use molecular biological techniques parse the molecular mechanism of atrazine degradation, and build function enhancement strains, finally realizes the effective remediation of soil contamination..According to our earlier work, bacterial strains AT-b3 have been isolated from long term contaminated soil. AT-b3 exhibited an excellent ability to degrade atrazine. In atrazine concentration is 100 mg/L, degradation rate more than 90% in 48 h. Preliminary judgment AT-b3 is Pseudomonas bacteria. On this basis, the physical and chemical properties of AT-b3, 16S rDNA sequence homology, atrazine degradation rate and degradation products, degradation influence factors and key enzymes were determined, analysis of biological characteristics and degradation characteristics of AT-b3. Using SAGE method and qRT-PCR technology, comparison pollution and no pollution AT-b3 differentially expressed genes, screening the degradation genes, analysis of gene biology function and metabolic pathways, and reveal the metabolic network of atrazine degradation. Make degradation gene expression effectively, and strengthen remediation effect, by constructing inhibiting gene deletion strains. We use 16S rDNA gene cloning followed by restriction enzyme and sequence analysis to describe strains in soil colonization dynamics and the impact on indigenous microorganisms, environmental safety evaluation of fertilizer, and lay a foundation for further development and utilization.

微生物修复土壤农药污染，就是利用微生物将土壤中残留的有害农药转化为二氧化碳和水或其他无害物质的过程，具有安全、高效、成本低和无二次污染等优点。本项目以土壤中高残留、难降解的阿特拉津为研究对象，在筛选出降解菌AT-b3的基础上，以降解率为指标，利用分子生物技术，解析阿特拉津降解的分子机制，并构建功能强化菌株，最终实现土壤污染的高效修复。通过对AT-b3理化特性、16S rDNA序列同源性、阿特拉津降解率和降解产物、降解影响因素和关键酶等测定，分析AT-b3的生物学特性和降解特性。利用SAGE方法和qRT-PCR技术，比较污染与未污染条件下AT-b3的差异表达基因，筛选降解相关基因，分析基因生物学功能和代谢途径，揭示阿特拉津降解的代谢网络。通过构建抑制基因缺失菌株，使功能基因有效表达，强化修复效果。测定菌株的土壤定殖动态和对土著微生物的影响，评价菌株的环境安全性，为后续开发利用奠定基础。

阿特拉津为三氮苯类除草剂，长期大量使用造成了严重的环境污染，并威胁生态稳定、食品安全和人类健康。本项目依据生物修复原理，利用微生物修复技术，针对土壤阿特拉津污染治理展开研究，主要内容涵盖了阿特拉津高效降解菌的筛选和驯化；降解菌的常规和分子生物学鉴定；阿特拉津的降解特性和规律分析；降解基因筛选、基因功能和代谢途径分析；功能菌株构建及修复菌剂制备；修复功能验证和环境安全性评价等多个方面，旨在探寻一种安全高效的土壤阿特拉津污染治理方式，以缓解阿特拉津带来的环境危害。研究工作围绕研究目标，遵循研究方案和研究计划开展，主要取得了以下成果：.从污染土壤中获得阿特拉津高效降解菌，初筛时48 h对阿特拉津降解率超过90%；进行降解菌的形态学观察、生理生化特性测定、16S rDNA测序与比对，鉴定降解菌为Enterobacter sp.；测定菌体生长和不同环境条件对阿特拉津降解率的影响，明确了菌株生长与阿特拉津降解具有同步性，且在25℃-35℃、pH近中性、低盐条件下菌株降解阿特拉津效果较好，碳源对菌体生长和降解率影响不大；进一步的正交试验表明，30℃、pH 7.5、0.5%盐度为阿特拉津降解的最佳环境组合条件；通过SAGE方法筛选降解相关基因，获得368个差异表达基因，其中上调基因231个，下调基因137个；基因功能和代谢途径分析上调基因包括脲酶辅助蛋白、脲酶亚基蛋白和耐药蛋白等，下调基因有肠杆菌素合成酶、甲酸脱氢酶和α/β水解酶等，涉及了氮代谢、乙醛酸和二羧酸代谢等24个代谢途径和细胞通路；用转座子随机突变和鸟枪法获得功能强化菌株，并以硅藻土为载体制备修复菌剂；进行土壤污染修复试验时，接菌量2%、温度30℃、修复28 d，阿特拉津降解率可达83.8%；安全性测试中，菌株能有效缓解阿特拉津对敏感植物大豆的伤害，并能促进其根围微生物的数量与活性。研究成果在土壤污染治理工作中具有应用潜力。

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