质粒pDOM水平转移介导土壤系统强化降解2甲4氯的功能及机制研究

To overcome the poor survival and low activity of degrading-bacteria used for bioaugmentation of persistent herbicide in soil, this study focuses on transfer and vertical mobility of pDOM plasmid in soil and its effects on persistent degradation capacity of MCPA. The insertion of gfp segment into the pDOM plasmid in donor strain Cupriavidus gilardii is performed. The relationship between transfer frequency of pDOM-gfp plasmid and species of recipient strains, selective pressure of the herbicide, temperature, humidity, nutrient content, and soil types is evaluated in soil ecosystem. Restrictive factor and mechanism of pDOM-gfp plasmid transfer are conducted. A pDOM plasmid-mediated bioaugmentation is to be performed under field condition and soil microbial community structure and function is to be assessed by BIOLOG and PCR-TGGE techniques. The available results will be useful for delineating mechanism of degradation potential formation of MCPA by soil indigenous bacteria and for developing a plasmid-mediated bioaugmentation method that could result in the persistent capacity for the degradation of persistent herbicide in soil. These results would also provide a foundation for bioremediation of pesticide soil pollution and protecting soil health.

本项目针对长残留除草剂土壤污染生物强化过程中引入的高效降解菌面临土著微生物竞争而难以存活的问题，以降解功能位于质粒pDOM的2甲4氯（MCPA）降解菌Cupriavidus gilardii为出发菌株，利用报道基因gfp标记降解质粒pDOM，研究质粒pDOM-gfp在土壤中的转移及其对 MCPA降解功能的持久性和纵深迁移性，明确质粒转移频率与受体菌种类、选择压、温湿度、养分、土壤类型的关系，探讨土壤中pDOM-gfp质粒转移的制约因子与驱动机制；并以pDOM-gfp质粒供体菌进行MCPA田间土壤污染生物强化研究，同期应用BIOLOG和PCR-TGGE评价强化过程中土壤微生物群落优势种群的结构和功能变化。研究结果可望阐明土壤微生物降解MCPA功能的形成机制，探索一条持续降解土壤长残留除草剂污染的新途径，对于控制或消减农田土壤中农药残留污染和保障土壤健康具有重要的科学意义和实际应用价值。

长残留除草剂土壤污染生物强化过程中引入的高效降解菌面临土著微生物竞争而难以存活，基于质粒水平转移的土壤除草剂污染生物强化是一种有效的修复手段。本项目以2甲4氯降解菌Cupriavidus gilardii T1为出发菌株，利用报道基因EGFP标记降解质粒pDOM，研究了质粒pDOM-EGFP在土壤中的转移及其对2甲4氯降解功能的持久性；开展质粒供体菌转移的制约因子和驱动机制研究；重点开展重复施药下质粒pDOM-EGFP供体菌对2甲4氯的降解动态、在饱和流与非饱和流土壤中质粒转移的纵深迁移性以及农田土壤中质粒供体菌强化降解2甲4氯污染效果，同期应用Illumina高通量测序评价强化过程中土壤微生物群落结构和功能变化。主要研究结果包括：1）C. gilardii T1降解2甲4氯的降解基因簇位于3号质粒(pDOM)上，构建EGFP标记的质粒供体菌C. gilardii T1（pDOM-EGFP）与出发菌株降解能力相当；2）在室内土壤4次重复施用2甲4氯，质粒pDOM-EGFP供体菌能够持续降解2甲4氯，第4次重复施用2甲4氯后10天，其降解率可达85.9%；3）质粒pDOM-EGFP供体菌在土壤中发生水平转移主要受温度和湿度影响，在饱和流与非饱和流土壤中质粒pDOM-EGFP供体菌均可向下迁移至土层30 cm处，且能有效降解2甲4氯。荧光定量PCR分析表明非饱和流土壤各土层中2甲4氯降解基因tfdA拷贝数高于饱和流土壤中；4）高通量测序结果表明质粒pDOM供体菌能在田间土壤相对丰度不占优势情况下有效降解2甲4氯，施用质粒pDOM-EGFP供体菌不仅能有效修复2甲4氯土壤污染，还对使用2甲4氯导致土壤微生物群落结构改变有正向调控作用。项目研究成果阐明了土壤微生物2甲4氯降解功能的形成机制，探索出一条持续降解土壤长残留除草剂污染的途径，相关成果已在J Agric Food Chem、Sci Total Environ等国际期刊上发表标注SCI论文7篇，另有2篇文章待发表。研究结果为控制或消减农田土壤中农药残留污染、保障土壤健康提供了科学依据。