【作者】 王伟；

【导师】 王禹；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2025， 博士

【摘要】 反刍动物的瘤胃微生物具有独特的纤维降解能力,其代谢产物挥发性脂肪酸（VFAs）能够为宿主提供70%以上的能量供应,但此过程中伴随着大量甲烷（CH₄）温室气体产生。反刍动物瘤胃发酵是畜牧业甲烷排放的主要排放源,其中奶牛的甲烷排放量占畜牧业甲烷排放总量的30%左右,甲烷生成也会造成反刍动物饲粮摄入总能量的损耗。反刍动物的瘤胃甲烷绝大部分是由氢营养型产甲烷古菌利用氢气（H₂）和二氧化碳（CO₂）作为底物合成的,一般认为乙酸和丁酸的生成过程会释放较多的H₂和CO₂,而丙酸的生成则会消耗H₂。因此,迫切需要寻找调控瘤胃微生物及代谢产物的方法来减缓甲烷排放,提高奶牛饲料利用效率。然而,目前关于奶牛宿主遗传因素和瘤胃上皮细胞表达基因在多大程度上影响瘤胃菌群和VFAs以及通过何种方式发挥调控作用,尚不清楚。本研究收集了574头日粮、品种、性别、年龄、体重等条件高度一致的中国荷斯坦公牛群体样本,利用配对样本的宿主全基因组测序、瘤胃转录组测序、瘤胃内容物扩增子测序和代谢产物挥发性脂肪酸测定数据,将菌群丰度和VFAs浓度作为复杂性状表型,采用遗传学方法系统评估荷斯坦牛瘤胃微生物及其代谢产物的宿主遗传基础,探究宿主-微生物在瘤胃甲烷生成和VFAs利用中的互作机制,为畜牧业温室气体减排和饲料能量高效利用提供理论依据。主要研究结果如下:1.中国荷斯坦牛瘤胃微生物特征通过对中国荷斯坦牛瘤胃内容物样本分别进行了16S rRNA基因V3-V4区（细菌）和V6-V8区（古菌）扩增子测序,共鉴定到317个分类群,包括269个细菌分类群和48个古菌分类群。细菌优势菌门为拟杆菌门、厚壁菌门和变形菌门,优势菌属为普雷沃氏菌属;古菌优势菌门为广古菌门,优势菌属为甲烷短杆菌属。多样性分析表明,瘤胃细菌的物种丰富度和多样性高于古菌;属水平的菌群互作网络和检出率发现,一些菌群如Oscillospiraceae＿NK4A214＿group、Christensenellaceae＿R-7＿group、Lachnospiraceae＿NK3A20＿group、Ruminococcus和Prevotella等,具有较高的网络连接度且存在于所有个体中,可能代表了荷斯坦牛瘤胃核心菌群。微生物功能通路预测及相关性分析发现,与产甲烷古菌相关的功能通路主要包括辅酶M生物合成、F420因子合成及通过CO₂和H₂途径生成CH₄等。此外,瘤胃内不同挥发酸含量个体的菌群多样性和物种组成丰度存在差异,发现菌群α-多样性在总VFA高、低组之间存在显著差异;在高VFA组,Prevotella、Bacteroidales＿RF16＿group和Clostridia＿UCG-014等菌属丰度较高;而在低VFA组,Bacteroidales＿F082、Christensenellaceae＿R-7＿group和Papillibacter等菌属丰度较高。2.瘤胃微生物及其代谢产物的宿主遗传基础通过遗传力（h²）、表达力（e²）、肠菌力（m²）评估发现,宿主遗传因素对瘤胃菌群和VFAs的平均解释度分别为28%和23%;瘤胃基因表达对菌群和VFAs的平均解释度分别43%和61%;瘤胃菌群对VFAs的平均解释度为58%。利用全基因组关联研究（GWAS）方法,鉴定出43个遗传变异位点（SNPs）与22个微生物表型性状显著关联(p<5.71×10^-9)。例如,在牛21号染色体的遗传变异（21:422903221）与Lachnospiraceae＿ND3007＿group菌属丰度显著关联(p=1.25×10^-9)。但本研究在全基因组显著阈值下(p<5.71×10^-9),未鉴定到与瘤胃VFAs浓度表型显著关联的SNPs。相比于宿主遗传,瘤胃上皮细胞表达基因与菌群和VFAs之间的调控关系更加直接,利用全转录组关联研究（TWAS）方法,共检测到28,260个显著的基因-微生物关联对,涵盖210个分类群和4,652个基因,候选基因显著富集在代谢、免疫和病原微生物感染相关功能通路中;鉴定到4,096个基因-VFAs关联对,涉及5种VFAs表型和1,577个基因,候选基因显著富在脂肪酸合成与利用、能量与代谢调节、脂质转运与信号传导相关通路。这些TWAS显著关联基因为我们从宿主基因层面解释瘤胃上皮细胞响应菌群和VFAs变化的调控机制提供了新的机遇。此外,本研究利用全微生物组关联研究（MWAS）和孟德尔随机化（MR）分析方法,鉴定到11个与VFAs存在潜在因果关联的菌群,其中,Clostridia＿UCG-014、[Eubacterium]＿ventriosum＿group与瘤胃中总挥发酸、乙酸、丙酸和丁酸的浓度都呈正相关。3.瘤胃甲烷生成和能量利用过程中的宿主-微生物互作机制通过构建瘤胃基因-菌群-VFAs之间的多重相关性网络,发现甲烷短杆菌属的4种产甲烷古菌（Methanobrevibacter＿boviskoreani、Methanobrevibacter＿millerae、Methanobrevibacter＿thaueri和Methanobrevibacter＿gottschalkii）与乙酸生成菌（Oscillospirales＿UCG-011、Christensenellaceae＿R-7＿group和Oscillospiraceae＿NK4A214＿group等）、乙酸和乙丙比呈正相关,与丙酸生成菌（如Prevotella、Prevotellaceae＿UCG-003等）和丙酸呈负相关。宿主瘤胃上皮淀粉和糖原代谢相关基因（如AMY2B和PYGB等）与4种产甲烷古菌呈正相关,而线粒体氧化磷酸化ATP合成相关基因（如GPX3、PRDX6、COX5A、ATP5ME和ATP6V1B1等）与4种产甲烷古菌呈负相关。此外,本研究发现瘤胃主要挥发性脂肪酸（乙酸、丙酸、丁酸）与Clostridia＿UCG-014、[Eubacterium]＿ventriosum＿group、Prevotella、Bacteroidales＿RF16＿group和Ruminococcus等都呈正相关;当瘤胃菌群以高VFAs产生的丙酸型发酵为主时,宿主脂肪酸氧化相关基因（如HSD17B4、ACADVL、ACADL、CPT1A和ANGPTL4等）下调表达,而线粒体氧化磷酸化ATP合成相关基因（如NDUFA7、NDUFA2、COX6B2等）上调表达。上述宿主-微生物互作过程伴随着底物代谢氢的减少,进而降低产甲烷菌的丰度,有利于瘤胃VFAs的高效利用,减少甲烷生成和能量损失。综上所述,本研究通过整合多组学数据,利用多种关联分析方法,全面表征了中国荷斯坦牛瘤胃菌群和VFAs的宿主关联特征;通过瘤胃基因-菌群-VFAs之间的多重相关性网络,揭示出宿主-微生物互作在瘤胃甲烷生成和VFAs利用中的潜在调控机制。这些候选基因和微生物,为未来从遗传选择和微生物干预策略入手减缓奶牛甲烷排放和提高饲料能量利用效率提供了重要参考,未来有望应用于培育低甲烷排放潜力奶牛新品种,助力我国奶牛养殖业向低碳高效转型升级。更多还原

【Abstract】 The rumen microbiota of ruminants possesses unique fiber-degrading capabilities,and their metabolites,volatile fatty acids（VFAs）,can provide over 70%of the energy supply for the host.However,this process is accompanied by the generation of large amounts of methane greenhouse gas（CH₄）.Rumen fermentation in ruminants is the main source of enteric methane emissions from the livestock industry,with dairy cows accounting for approximately 30%of the total methane emissions.Methanogenesis also leads to the energy loss from the total ingested feed of ruminants.The majority of ruminal methane of ruminants is synthesized by hydrogenotrophic methanogenic archaea using hydrogen（H₂）and carbon dioxide（CO₂）as substrates.Generally,the production of acetic acid and butyric acid releases more H₂and CO₂,whereas the production of propionic acid consumes H₂.Consequently,there is an urgent need to find methods to regulate rumen microbiota and their metabolites to reduce methane emissions and improve the feed utilization efficiency of dairy cows.However,the extent to which the host genetic factors and rumen epithelial cells expressed genes of dairy cows affect rumen microbiota and VFAs,and the mechanisms underlying host-microbiome interactions in methanogenesis and VFA utilization remains unclear.In this study,we collected samples from a population of 574Chinese Holstein bulls with highly consistent diets,breeds,gender,age,and body weight.The paired samples were used to measure the host whole-genome sequencing,rumen transcriptome sequencing,rumen contents 16S rRNA gene amplicon sequencing,and VFA concentration measurements.We treated the microbiota abundance and VFA concentrations as complex trait phenotypes and systematically evaluated the host genetic basis of rumen microbiota and their metabolites in Chinese Holstein cattle using genetic approaches.We also explored the interaction mechanisms between the host and microbiota in rumen methanogenesis and VFA utilization.This research provides a theoretical foundation for mitigating greenhouse gas emissions in livestock production and enhancing feed energy efficiency.The key findings are as follows:1.Rumen microbial characteristics of Chinese Holstein cattleThrough the 16S rRNA gene amplicon sequencing targeting the V3-V4 region（for bacteria）and V6-V8 region（for archaea）in rumen content samples from Chinese Holstein cattle,we identified 317 microbial taxa,including 269 bacterial and 48 archaeal taxa.The dominant bacterial phyla were Bacteroidetes,Firmicutes and Proteobacteria,the dominant bacterial genus was Prevotella.The dominant archaeal phylum was Euryarchaeota,and the dominant genus was Methanobrevibacter.Diversity analysis indicated that the species richness and diversity of rumen bacteria were higher than those of archaea.At the genus level,the interaction networks and detection rates of the microbiota revealed that some taxa such as Oscillospiraceae＿NK4A214＿group,Christensenellaceae＿R-7＿group,Lachnospiraceae＿NK3A20＿group,Ruminococcus and Prevotella,had high network connectivity and were present in all individuals,possibly representing the core microbiota of the rumen in Holstein cattle.Microbial functional pathway prediction and correlation analysis revealed that the functional pathways related to methanogenic archaea mainly include coenzyme M biosynthesis,factor F420 biosynthesis,and methanogenesis from CO₂and H₂ pathway,etc.Additionally,there were differences in microbiota diversity and species composition abundance among individuals with different rumen volatile fatty acids concentration.We found that theα-diversity of the microbiota was significantly different between the high and low total VFA groups.In the high-VFA group,taxa such as Prevotella,Bacteroidales＿RF16＿group,and Clostridia＿UCG-014 were more abundant.Conversely,the low-VFA group showed higher abundances of Bacteroidales＿F082,Christensenellaceae＿R-7＿group,and Papillibacter.2.Host genetic basis of rumen microbiota and metabolitesThrough evaluations of heritability（h²）,expressability（e²）,and microbiability（m²）,we found that host genetic factors explained an average of 28%and 23%of the variance in microbiota abundance and volatile fatty acids concentration,respectively.Rumen gene expression accounted for 43%and 61%of the variance in microbiota and VFAs,while rumen microbiota explained 58%of the variance in VFAs.Using genome-wide association study（GWAS）approach,we identified 43 genetic variants（SNPs）significantly associated with 22 microbial phenotypic traits(p<5.71×10^-9).For instance,a genetic variant on bovine chromosome 21（21:422903221）was significantly associated with the abundance of Lachnospiraceae＿ND3007＿group genus(p=1.25×10^-9).However,at the genome-wide significance threshold(p<5.71×10^-9),no SNPs were identified to be significantly associated with VFAs concentration phenotypes in this study.Compared with host genetics,the regulatory relationship between rumen epithelial cell expressed genes and microbiota and VFAs is more direct.In contrast,using transcriptome-wide association study（TWAS）method,we revealed 28,260 significant gene-microbe associations,covering 210 taxa and4,652 genes,and candidate genes were significantly enriched in metabolic,immune,and pathogenic microbe infection-related functional pathways.We also identified 4,096gene-VFA associations involving 5 VFA phenotypes and 1,577 genes,and and candidate genes were significantly enriched in fatty acid synthesis and utilization,energy and metabolism regulation,and lipid transport and signal transduction-related pathways.These TWAS significantly associated genes provide new opportunities for us to explain the regulatory mechanism of rumen epithelium cells response to microbiota and VFA changes at the host gene level.Additionally,in this study,using the microbiome-wide association study（MWAS）and Mendelian randomization（MR）approaches,we identified potentially causal relationships between 11 microbiota and 8 VFAs,among which Clostridia＿UCG-014 and[Eubacterium]＿ventriosum＿group were positively correlated with the concentrations of total VFA,acetic acid,propionic acid,and butyric acid in the rumen.3.Host-microbial interaction mechanisms during rumen methanogenesis and energy utilizationThrough construction a multiple correlation network among rumen genes,microbiota,and VFAs,we observed that the four methanogenic archaea（M.boviskoreani,M.millerae,M.thaueri,and M.gottschalkii）of Methanobrevibacter genus were positively correlated withacetate-producingbacteria（e.g.,Oscillospirales＿UCG-011,Christensenellaceae＿R-7＿group,and Oscillospiraceae＿NK4A214＿group）,acetate acid,and A/P ratio.They were negatively correlated with propionate-producing bacteria（e.g.,Prevotella and Prevotellaceae＿UCG-003）and propionic acid.The host rumen epithelial starch and glycogen metabolism-related genes（e.g.,AMY2B and PYGB）were positively correlated with the four methanogenic archaea,while the mitochondrial oxidative phosphorylation ATP synthesis-related genes（e.g.,GPX3,PRDX6,COX5A,ATP5ME,and ATP6V1B1）were negatively correlated with the four methanogenic archaea.In addition,we found that the main rumen volatile fatty acids（acetic acid,propionic acid,and butyric acid）were positively correlated with Clostridia＿UCG-014,[Eubacterium]＿ventriosum＿group,Prevotella,Bacteroidales＿RF16＿group,and Ruminococcus,etc.When the rumen microbiota is dominated by propionic acid-type fermentation with high VFAs production,the host fatty acid oxidation-related genes（e.g.,HSD17B4,ACADVL,ACADL,CPT1A,and ANGPTL4）were down-regulated,while the mitochondrial oxidative phosphorylation ATP synthesis-related genes（e.g.,NDUFA7,NDUFA2,and COX6B2）were up-regulated.The above host-microbe interaction processes were accompanied by a reduction in substrate metabolic hydrogen,thereby decreasing the abundance of methanogens.This mechanism facilitates efficient utilization of ruminal VFAs,mitigating methanogenesis and energy loss.In summary,this study comprehensively characterized the host-associated features of rumen microbiota and VFAs in Chinese Holstein cattle by integrating multi-omics data and employing various association analysis methods.Through the multiple correlations network among rumen genes,microbiota,and VFAs,we uncovered the potential regulatory mechanisms underlying host-microbiome interactions in ruminal methanogenesis and VFA utilization.These candidate host genes and microbial taxa provide critical insights for future strategies aimed at mitigating methane emissions and enhancing feed energy utilization efficiency in ruminants through genetic selection and microbial intervention.These findings offer a scientific foundation to cultivate a new breed of dairy cows with low methane emission potential and contribute to the transformation of China’s dairy cattle farming towards low-carbon and high-efficient practices.更多还原