土壤污染有机磷农药降解菌多样性及其分子生态效应研究

Large soil is severely contaminated due to the overuse of organophosphorous pesticides, resulting in soil problems such as the destruction of microbial community composition, its diverse alteration, and the successive decrease of land productivity. Therefore, it is imperative to restore the contaminated soil. This study mainly deals with the following three aspects: first, by development of physiological flora of different category and the method of 16S rDNA RFLP to study the alteration of microbial community composition in the soil contaminated by organophosphorous pesticides and the genetic diversity difference of soil bacteria, and to build phylogenetic tree so as to illustrate the relationship between dominant flora groups and the main flora groups in the soil contaminated by organophosphorous pesticides, and to reveal the microbial active ecological effect in the contaminated soil; second, by directive development of good microbial species in the organophosphorous pesticides of farm soil, gene cloning based on shotgun sequencing method and the analysis of 16S rDNA to separate and select diverse and new degrading bacteria for organophosphorous pesticides with the aim to discover new OPH (organophosphorus hydrolase) gene and construct the phylogenetic tree, and to disclose the relation between the diversity of degrading bacteria and its structural gene; third, by principle and method of Tetrazolium Violet to probe the profile of degrading bacteria pesticides, and to discover substance of Co-Metabolism, with a view to indicate the interaction of degrading bacteria. The studies will provide the important scientific basis for adaptation mechanism of microbial flora groups to organophosphorous pesticides, the evaluation for environmental toxicology and environmental restoration, soil microbial restoration and ecological protection.

有机磷农药大量使用使大面积土壤受到严重污染，导致了土壤微生物群落结构破坏和多样性变化，土地生产力持续下降，土壤生态修复迫在眉睫。该研究（1）通过土壤原位培养和16S rDNA RFLP方法，研究有机磷污染土壤菌群结构的变化、土壤细菌遗传多样性差异，构建系统发育树，定位降解菌系统发育地位，阐明有机磷污染土壤优势类群和主要细菌类群变化关系，揭示污染土壤微生物生态学效应；（2）通过土壤有机磷农药定向培育优良菌种和鸟枪法基因克隆及16S rDNA分析，分离、筛选多种新的有机磷降解菌种（株），发现新的降解酶基因，构建系统发育进化树，揭示降解菌多样性和结构基因的关系；（3）采用四唑紫反应原理，绘制降解菌农药降解谱，发现共代谢基质，揭示降解菌相互作用关系。这些对研究微生物群落适应有机磷农药污染机理、评价其环境毒理和污染环境的自净能力具有重大意义，为土壤生物修复和生态治理提供理论依据。

有机磷农药大量使用使大面积土壤受到严重污染，导致了土壤微生物数量、群落结构、生物活性的破坏和多样性变化，土地生产力持续下降，为此进行土壤污染有机磷降解菌多样性及其应用研究，为微生物土壤生态修复提供支持就迫在眉睫。主要进行了下列研究:①分离、鉴定、发现了3株有机磷农药降解菌并进行了系统发育定位。其中，首次发现了对农药有机磷具有降解作用的嗜麦芽假单胞菌。同时完成了对耐镉肠杆菌的分子鉴定和高效富硒菌株选育研究；②构建了有机磷农药高效降解工程菌，对甲基对硫磷、马拉硫磷和乙酰甲胺磷降解率分别达到了96.51％、94.33％和98.24％；③研究了有机磷农药污染土壤三大微生物菌群主要变化趋势，对放线菌的影响最大，对微生物菌群呼吸作用和氨化作用生物活性下降都超过了15%；④建立了降解土壤有机磷农药工程菌固定化方法，固定化的Bacillus subtilis sp.LTG1菌株对硫磷的降解率达到96.29%，甲胺磷几乎全部降解，确定了最佳投粒比为5%。⑤研究了有机磷农药污染对土壤微生物种群多样性的影响，获得了多样性指数，取得两种土壤16S rDNA文库克隆相似性系数为25.12%，揭示出农药污染造成土壤微生物结构组成的改变是影响土壤微生物种群多样性的主要原因，假单胞菌成为优势菌群。⑥建立了以四唑紫和96孔板为载体的测定农药降解谱方法，绘制各降解菌对不同农药的降解谱。揭示了不同菌种对农药的降解能力的差异性。该研究对于揭示有机磷农药的环境毒理、生物可降解性以及污染环境的修复具有重要价值，为研究微生物群落适应污染的机理、评价有机磷农药的微生物群落演替趋势及农药安全性评价具有重要意义，为土壤生物修复和生态治理提供理论和技术支持。