接种功能内生细菌减低叶菜类蔬菜PAHs污染的效能及生理生化和分子机制

Polycyclic aromatic hydrocarbons (PAHs) have been commonly found with high concentrations in the soils of many regions in China. Vegetable uptake of PAHs from contaminated soils; the subsequent consumption of the PAH-contaminated vegetables lead to PAH transfer to humans via the food chain. This raises serious risks and threats to human health. In China the yield of leafy vegetables occupies one third of the total vegetable production. It has drawn a lot of attention on how to reduce the PAH contamination risks of leafy vegetables in agricultural production. Endophytic bacteria manifest positive effects on facilitating the plant development and survival in the contaminated soils. Here we coined a scientific hypothesis that the colonization of PAH-degrading endophytic bacteria (EBPAH) could effectively decontaminate PAHs accumulated during the production of leafy vegetables. Little information has been hitherto documented on the impacts of EBPAH on uptake, accumulation and metabolism of PAHs in leafy vegetables. Recently, we isolated and screened several EBPAH strains. But their performances on PAH removal in leafy vegetables are still unclear. To this end, this project seeks to investigate the plant colonization, degradation performance and mechanisms of the EBPAH strains in PAH-contaminated leafy vegetables. The colonization and distribution of EBPAH in different plant parts and sucellular fractions will be measured. The metabolities and the corresponding metabolism pathways of PAHs in leafy vegetables with inoculation of EBPAH will be elucidated using high-resolution mass spectrometry with aid of isotope-labeled compounds, and the performance of EBPAH colonization on PAH removal in leafy vegetables will be evaluated. The mechanism of the EBPAH-mediated PAH removal in leafy vegetables will be systematically clarified on the basis of the analysis of enzyme activities and the expression of PAH degradation-related genes in plants. The results from this project will provide important information in the assessment of environmental risks of PAHs and in development of management protocol to circumvent leafy vegetable from PAH contamination in agricultural production.

在我国，叶菜类蔬菜约占蔬菜总产的 1/3；污染区叶菜类蔬菜可吸收积累大量多环芳烃（PAHs），严重危害蔬菜安全和人群健康。如何有效地去除污染区叶菜类蔬菜中PAHs？国内外仍缺乏有效、可行的途径。本项目在前期筛选了具有PAHs降解功能的内生细菌（以下简称功能菌）的基础上，针对叶菜类蔬菜PAHs污染问题，尝试采用不同接种方式将功能菌定殖到蔬菜中，借助抗生素抗性标记和绿色荧光蛋白基因标记技术，揭示功能菌在蔬菜体内的定殖分布和微观定位；利用高分辨质谱结合稳定性同位素比值技术，揭示定殖功能菌的蔬菜中PAHs代谢效率和途径；分析功能菌作用下蔬菜中PAHs代谢相关酶系和降解基因的响应，阐明接种功能菌减低蔬菜PAHs污染的生理生化和分子机制。试图提出应用功能内生细菌有效规避叶菜类蔬菜PAHs污染风险的新技术及原理，为PAHs污染区叶菜类蔬菜安全生产、合理利用污染土壤资源等提供科学依据。

在我国，叶菜类蔬菜约占蔬菜总产的 1/3；污染区叶菜类蔬菜可吸收积累大量多环芳烃（PAHs），严重危害蔬菜安全和人群健康。如何有效地去除污染区叶菜类蔬菜中PAHs？国内外仍缺乏有效、可行的途径。本项目系统地研究了接种功能内生细菌减低叶菜类蔬菜PAHs污染的效能及生理生化和分子机制。①采用绿色荧光蛋白基因标记技术，借助激光共聚焦显微镜、荧光显微镜，揭示了不同接种方式下功能内生细菌在植物体内的定殖效能和微观分布。②系统地阐明了不同功能内生细菌对蔬菜中PAHs的消减规律及代谢途径。比较了浸种、浸根、喷叶等接种方式下功能菌对蔬菜中PAHs的消减效能差异。构建了由8株功能菌株配伍组成的高效降解菌群，克服单一功能菌降解广谱性较窄的问题，接种后蔬菜（上海青和小白菜）可食部位16种EPA优控PAHs含量降低>70%。首次从亚细胞水平上阐明了功能内生菌对蔬菜中PAHs的消减作用，分析了蔬菜体内PAHs的代谢产物和途径。开展了利用功能内生菌群削减蔬菜中PAHs的田间示范。③揭示了蔬菜中PAHs代谢相关酶系活性变化及其与PAHs代谢的作用关系。接种功能内生菌株Phe15可以提高空心菜不同部位及其亚细胞组分中过氧化物酶、多酚氧化酶等活性，且酶系活性与菲积累呈负相关关系。④阐明了功能内生细菌作用下植物体内PAHs降解基因响应规律及其与PAHs消减的关系。接种由6株菌组成的功能内生细菌菌群，提高了空心菜、上海青和小白菜体内PAHs降解基因丰度，蔬菜各部位降解基因丰度与功能内生细菌数量显著正相关、与PAHs含量间显著负相关。依托该项目共发表学术论文23篇，其中英文学术论文22篇。提出了应用功能内生细菌有效规避叶菜类蔬菜PAHs污染风险的新技术及原理，为PAHs污染区叶菜类蔬菜安全生产、合理利用污染土壤资源等提供了科技支撑。