奶牛瘤胃未培养尿素分解菌群的磁性纳米分离及元基因组解析

Urea is an important non-protein nitrogen source in the diet of ruminants. Nevertheless, urea is rapidly hydrolyzed by ureolytic bacteria to ammonia in the rumen of dairy cows, and such rapid ureolysis decreased urea nitrogen utilization efficiency and excessive nitrogen excretion to the environment. In our previous studies, it was revealed that the ureolytic bacteria in the rumen are of high diversity but 99% of them are yet uncultured based on pyrosequencing analysis. However, there is still lack of physiological tool to study the taxonomy and functions of those uncultured ureolytic bacteria, and to further discuss mechanisms and regulations of urea degrading. To isolate living community of the uncultured ureolytic bacteria and obtain its metagenome, this project will develop an integral analysis system combining magnetic nanoparticle mediated isolation and pyrosequencing. Through magnetic nanoparticles stability test in the rumen fluid, the magnetic nanoparticle mediated isolation will be optimized and first-time applied for uncultured ureolytic bacterial community isolation in the rumen. From the physiological evaluation of ureolytic activities of uncultured ureolytic bacteria and original rumen microbial community, this work will uncover the ecological functions of targeting bacteria in the rumen. Via taxonomy analysis of uncultured ureolytic bacterial community by pyrosequencing, the metagenome will be constructed to identify and link the functional ureolytic genes to in situ urea degrading performance. From these cutting-edge techniques, this project can reveal the structure and functions of real ureolytic bacteria community and contribute to deeper understanding on the mechanism and regulation of urea degradation in the rumen.

尿素是反刍动物重要的非蛋白氮饲料，而尿素在瘤胃中的过快分解易导致尿素利用率降低和氮素过度排放。前期研究发现，瘤胃尿素分解菌群多样性高且99%以上属于未培养细菌。而现有技术无法有效揭示尿素分解菌群的种类和原位功能，限制了对尿素分解机理和调控机制的认知。本项目针对瘤胃未培养尿素分解菌群难以分离以及功能基因认识缺乏的科学问题，提出利用磁性纳米分离和元基因组测序技术研究未培养尿素分解菌群构成和功能的科学假设。研究磁性纳米材料在瘤胃液中的稳定性，建立适合瘤胃液环境的高效磁性纳米分离技术，实现对未培养尿素分解菌群捕获和分离；研究分离的未培养尿素分解菌群功能，评价其原位生态结构与功能特征；利用高通量测序技术研究未培养尿素分解菌群构成和系统发育，解析元基因组信息，寻找与尿素代谢相关功能基因。预期将获得瘤胃未培养尿素分解菌群并阐明其构成和功能，为进一步揭示尿素分解菌群的尿素分解和调控机理提供理论依据。

尿素是反刍动物重要的非蛋白氮饲料，而尿素在瘤胃中的过快分解易导致尿素利用率降低和氮素过度排放。瘤胃尿素分解菌群多样性高且大部分属于未培养细菌，而现有技术无法有效揭示尿素分解菌群的种类和原位功能，限制了对尿素分解机理和调控机制的认知。本项目针对瘤胃未培养尿素分解菌群难以富集以及功能基因认识缺乏的科学问题，提出利用磁性纳米和元基因组测序技术研究未培养尿素分解菌群构成和功能的科学假设。通过本项目三年研究，建立了四氧化三铁磁性纳米颗粒制备方法，成功包被聚烯丙胺材料，可实现对瘤胃细菌捕获。通过对脲酶基因高通量测序和分析，发现瘤胃尿素分解菌主要来源于Methylococcaceae等。利用磁性纳米颗粒，48 h后能有效富集瘤胃尿素分解菌群。通过宏基因组测序，分析发现了优势尿素分解菌基因组及其脲酶基因簇。脲酶基因簇由3个结构基因和4个辅助基因组成，通过电镜观察发现，结构基因编码的蛋白复合物为Ure(ABC)3聚合形式，分析发现辅助蛋白对镍离子结合和传递发挥作用，有助于脲酶活性的激活。通过代谢组学分析，发现瘤胃尿素分解菌代谢尿素氮的主要去向是氨基酸合成通路。脲酶抑制剂乙酰氧肟酸抑制尿素分解，引起短期内瘤胃细菌多样性水平的升高，同时利用分子对接技术筛选获得2个高效脲酶抑制剂化合物，抑制率在40%以上。因此，本项目揭示的瘤胃未培养尿素分解菌群结构、功能和抑制机理，为进一步研究尿素分解菌群的尿素分解和调控机理提供了理论依据和技术支撑。