乳酸菌Y1对土壤中毒死蜱污染修复的分子机制及安全性评价

The widespread use of chlorpyrifos in agricultural fields in China have caused the residues exceeded maximum residue limit in the surface soil, which impacts on the yield and quality of crop. Therefore, how to eliminate or reduce the residue of chlorpyrifos in soil is widely regarded as the environment and food safety problem. Previous studies found that lactic acid bacteria could degrade chlorpyrifos efficiently, but the underlying molecular mechanisms and the safety of remediation of chlorpyrifos contaminated in soil are poorly defined. In this project, the microcosmic system and molecular biology technology will be used. Lactic acid bacterium Y1 which taken as the object is focus on: (1) Degradation of chlorpyrifos in soil by lactic acid bacterium Y1; (2) The survival and colonization of lactic acid bacterium Y1 in soil and its effect on the soil microbial community; (3) RNA-Seq is planned to be used to screen and analysis the key genes of chlorpyrifos degradation by lactic acid bacterium Y1. The functions of key genes and the degradation intermediates will be identified through gene knockout and chromatographic analysis technology. The findings will elucidate the molecular mechanisms underlying the degradation of chlorpyrifos. Furthermore, the implementation of this proposal can provide necessary theoretical basis and technical assistance for microbial safety remediation of chlorpyrifos contaminated in soil and production of new microbial agents.

毒死蜱在我国农业领域应用范围广、用量大，极易残留在土壤表土层，影响农作物的产量和质量。因此，如何消除或降低土壤中毒死蜱残留已成为公认的环境安全和食品安全问题。前期工作发现，乳酸菌能够有效降解毒死蜱，但其对土壤中毒死蜱污染修复的作用机制及安全性尚不明确。本项目拟应用微宇宙系统及分子生物学手段，以乳酸菌Y1为对象，重点研究：（1）乳酸菌Y1对土壤中毒死蜱的降解作用；（2）乳酸菌Y1在土壤中的存活定殖状况及对土壤微生物群落的影响；（3）采用转录组测序（RNA-seq）筛选分析乳酸菌Y1降解毒死蜱的关键基因，通过基因敲除、色谱分析技术明确关键基因的功能及降解中间产物，阐明乳酸菌降解毒死蜱的分子机制，为毒死蜱污染土壤的微生物安全修复及新型微生物菌剂的制备提供必要的理论依据和技术参考。

本项目应用微宇宙系统，选取乳酸菌Y1对土壤中有机磷农药毒死蜱进行生物修复，研究结果表明，乳酸菌Y1对灭菌土壤和未灭菌土壤中毒死蜱的降解率为51.65 %-70.43 %，其中接种乳酸菌Y1的未灭菌土壤中降解效果最好，降解速率最快，半衰期最短，并对土壤微生物物种均一性及群落的丰富度均没有显著影响。采用高通量RNA测序（RNA-seq）结合生物信息学技术分析转录组基因表达差异特性，共筛选出可能参与毒死蜱生物降解的关键基因，如磷酸酶、脱水酶，氧化酶基因等59个；结合非靶向代谢组学及GC-MS/MS检测代谢中间产物4个，推测出乳酸菌Y1生物降解毒死蜱的代谢途径，为有机磷农药的微生物降解机制提供新的理论依据，对毒死蜱污染土壤的微生物修复及安全新型的微生物菌剂的制备具有重要的科学理论价值和实际意义。