携带人工基因簇的接合质粒介导的生物强化修复壬基酚聚氧乙烯醚污染土壤的研究

Nonylphenol polyethoxylates (NPEOs) are a class of soil pollutant with high resistance to biodegradation. However, currently available degrading bacteria do not have the complete NPEOs degradation pathway. Besides, non-native bacteria hardly accommodate to the soil environment. These reasons have hindered the development of bioremediation methods for NPEOs contaminated soils. On the basis of currently available bacteria and gene resources, this project aims at constructing a gene cluster which contains genes for complete degradation of NPEOs, followed by stimulating the NPEOs degradation in soils using conjugating plasmids containing the degradation genes: 1) illustrate the genetic mechanism behind NP degradation in Arthrobacter sp. NP and polyethoxylates chain degradation in Sphingomonas sp. Y2, then construct the gene cluster for complete degradation of NPEOs; 2) construct the broad host-range conjugating plasmid containing NPEOs degrading genes, and test the plasmid using single or mixed recipient bacteria; 3) with a combination of methods including flow cytometry and high throughput sequencing, testify the ability of conjugating plasmid to spread the genes related with NPEOs degradation in soils, as well as analyze the effect of plasmid spreading on NPEOs degradation in soils. The research will promote the understanding of molecular mechanism behind NPEOs degradation, help to bring about the efficient and complete biodegradation method for NPEOs, and help to improve the theoretic and technical system of plasmid mediated soil bioremediation.

壬基酚聚氧乙烯醚（NPEOs）是一类常见的难降解土壤污染物。然而目前已知的降解菌不具备彻底降解NPEOs的功能，而且外源降解菌难以适应土壤环境，从而限制了相应的土壤修复技术的发展。本项目在现有菌种、基因资源基础上构建可完整降解NPEOs的基因簇，采用接合型质粒携带并促进降解基因在土壤内传播，实现土壤中NPEOs有效降解：1）解析Arthrobacter sp. NP的壬基酚降解和Sphingomonas sp. Y2的乙氧基链降解基因功能，整合为完整的NPEOs降解基因簇；2）构建携带NPEOs降解基因簇的广宿主接合载体，对单一或混合受体菌测试载体功能；3）采用流式细胞检测、高通量测序等方法在土壤中测试接合载体传播降解基因簇的能力，分析载体的传播对土壤中NPEOs降解的影响。本研究旨在加深了解NPEOs降解过程分子机制，获取彻底降解NPEOs的方法，完善质粒介导土壤原位修复的理论和技术。

目前已知的微生物均不具备彻底降解壬基酚聚氧乙烯醚（NP9EO）的能力，且外源降解菌对土壤环境的低适应性，导致相应的修复技术难以成型。本研究对相关菌种的降解基因进行整合，构建并优化了可实现NP9EO完整降解的基因簇，并分析了携带该基因簇的质粒向细菌传递降解基因的能力及其介导的NP9EO污染土壤的修复水平。研究结果表明：(1) 采用BioBrick法可实现对降解基因簇中相关基因功能的解析，将确定的降解基因整合到一个基因簇中，最终我们获得了完整的NP9EO降解基因簇；(2) 通过广宿主质粒构建的pNPEmob载体具备将NP9EO降解基因传递至其他细菌的能力，且其具有传代稳定性；(3) 携带NP9EO降解基因簇的质粒可促进降解基因在土壤细菌如Streptomyces sp.、Pseudomonas sp.、Rhodococcus sp.等的传播，显著提高污染土壤的生物修复；(4) 有机质含量高的土壤有利于pNPEmob在菌种间的转移，而土壤微生物多样性越高，其抵御外界物种入侵的能力越强，在一定程度上抑制了pNPEmob的转移。本研究从分子水平对多个环节进行了技术创新，加深了对NP9EO降解过程分子机制的了解，为质粒介导的土壤中NP9EO的原位修复提供重要的理论和技术支撑。