蛋鸡肠道上皮细胞外泌miRNA调控臭气排放的机制研究

The odor pollution seriously affects the healthy development of laying hens industry. The microbes in the cecum and feces of the laying hens are the key biological factors of odor production. Our previous study found that different strains of laying hens had different NH3 and H2S emission potentials, and there was a significant difference in cecum microbe community and the metabolic pathways related to odor production in different laying hens through 16S rRNA and metabonomics. Meanwhile, some studies have found that some miRNA secreted by host intestinal epithelium can enter the host intestinal cavity and regulate the growth and metabolism of microbes in the intestinal tract. Therefore, the host miRNAs in the intestinal contents were sequenced by using two typical strains of laying hens (low odor emission strain and high odor emission), and then select miRNAs which can regulate the growth and metabolism of odor production bacteria combined with the analysis of the metagenome, metaproteome and metabolome date. Finally, Through in vitro fermentation and in vitro culture trial, to further verify the effects of previous selected miRNAs on the odor emission and odor emission related microbes in the cecum of laying hens. The innovation and value of this research: elaborating the mechanism that miRNAs lead to the different odor emission potential by regulating the intestinal microbes indifferent laying hen strains.

蛋鸡养殖过程带来臭气污染严重影响蛋鸡养殖业健康发展，蛋鸡盲肠和粪便中的微生物是臭气产生的关键生物因子。课题组前期发现，不同品种蛋鸡具有不同的氨气和硫化氢排放潜势，并通过16SrRNA和代谢组学发现盲肠微生物群落和产臭气的相关代谢通路在不同蛋鸡品种间存在明显的差异；同时有研究发现，宿主肠上皮细胞分泌的某些miRNA可进入宿主肠腔并调控肠道中微生物的生长和代谢。因此，本研究以不同品种蛋鸡（低产臭和高产臭蛋鸡）为模式动物，对蛋鸡盲肠内容物中的宿主miRNA进行测序，并通过宏基因组、宏蛋白组和代谢组的联合分析，筛选能调控产臭菌群生长和代谢的miRNA，并通过体外发酵和体外培养试验，采用PCR等技术，进一步验证筛选的miRNA对蛋鸡盲肠产臭微生物和臭气排放的影响。主要创新与意义在于：系统解析以miRNA为纽带，经影响肠道微生物导致蛋鸡臭气排放差异的内在机制，为蛋鸡产业臭气减排提供指导。

本项目基于不同品种蛋鸡罗曼粉和海兰灰存在硫化氢（H2S）排放量差异的现象，对其中存在的机制进行探究。本项目通过对比海兰灰与罗曼粉之间H2S排放量差异、肠道菌群结构及功能差异、蛋鸡肠道上皮细胞外泌miRNA差异后筛选出与H2S产生相关并且在两品种蛋鸡间存在显著差异的菌属、miRNA和功能基因，再验证关键miRNA干预靶向宿主菌功能基因后对H2S产生的影响，以揭示不同品种蛋鸡间H2S排放量差异的潜在机制。结果显示NH3排放量在两品种蛋鸡间显著无差异，海兰灰H2S的日均排放量显著低于罗曼粉（P<0.05）。进一步对盲肠内容物扩增子测序发现海兰灰盲肠中具有较强蛋白发酵能力的菌属，并且硫酸盐还原菌的丰度显著低于罗曼粉（P<0.05），而产丁酸菌属的丰度显著高于罗曼粉（P<0.05）。通过miRNA及宏转录组测序并分析后发现在H2S代谢通路中gga-miR-222a与蛋氨酸合成酶基因Odosp_3416和BF9343_2953之间存在靶标关系。经过上述两功能基因宿主菌丰度的对比后，选择gga-miR-222a与BF9343_2953宿主菌Bacteroides fragilis NCTC9343进行验证，结果表明gga-miR-222a能被Bacteroides fragilis NCTC 9343吸收进入胞内与其核酸结合行使调控功能，并能显著提高Bacteroides fragilis NCTC 9343在对数期的丰度，上调蛋氨酸基因BF9343_2953的表达量，提高菌液中蛋氨酸含量。Bacteroides fragilis NCTC 9343与gga-miR-222a协同降低罗曼粉蛋鸡肠道H2S排放的实质是gga-miR-222a的主要作用宿主菌为Bacteroides fragilis NCTC 9343，且gga-miR-222a 只调控了 Bacteroides fragilis NCTC 9343硫代谢途径中蛋氨酸合成酶表达量增加，从而增加了“蛋氨酸-半胱氨酸”代谢通路中硫元素向蛋氨酸方向合成的量，降低了H2S代谢生成所需化合物的合成量,使H2S产量降低。本项目为后续在需要降低硫损失、硫空气污染（以H2S形式），提高蛋氨酸合成的相关领域提供生物添加剂的技术支持。

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