动物营养学报    2020, Vol. 32 Issue (1): 234-243    PDF    
环湖牦牛瘤胃微生物区系特征及性别之间的差异
韩学平1,2,3,4 , 刘宏金1,2,3 , 胡林勇1,2,3 , 徐世晓1,2,3 , 艾德强4 , 付弘贇4 , 陈永伟4 , 赵建忠5     
1. 中国科学院西北高原生物研究所, 西宁 810008;
2. 中国科学院高原生物适应和进化重点实验室, 西宁 810008;
3. 中国科学院大学, 北京 100049;
4. 青海省畜牧总站, 西宁 810001;
5. 青海省农牧业工程项目咨询中心, 西宁 810001
摘要: 本试验旨在研究环湖牦牛瘤胃微生物区系特征及性别之间的差异。随机挑选4~5岁环湖牦牛健康公牛5头[平均体重为(228.0±19.4)kg]和母牛5头[平均体重为(198.0±8.4)kg],采用16S rDNA扩增子高通量测序的方法研究公、母牦牛瘤胃微生物区系特征和性别之间的差异。结果表明:2个性别间瘤胃微生物在门水平上组成相似,其中拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrobia)和厚壁菌门(Firmicutes)是优势菌门,样品中平均相对丰度分别为51.74%、15.00%和14.34%。在属水平上,普氏菌属_1(Prevotella_1)、理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)和普雷沃氏菌科_UCG-001(Prevotellaceae_UCG-001)是优势菌属,平均相对丰度分别为16.11%、7.74%和3.43%。Alpha多样性指数中,公牛的Sobs指数为2 689,极显著高于母牛(P < 0.01),其余指数无显著性别差异(P>0.05)。主坐标分析(PCoA)和相似性分析(Anosim)结果说明2个性别之间瘤胃微生物区系存在明显差异。在属水平上,公牦牛Rikenellaceae_RC9_gut_group的相对丰度显著高于母牦牛(P < 0.05),母牦牛Prevotella_1、毛螺菌科AC2044群(Lachnospiraceae_AC2044_group)、SaccharofermentansAnaerovoraxElusimicrobium的相对丰度显著高于公牦牛(P < 0.05)。瘤胃微生物TAX4FUN预测功能比较发现,公、母牦牛瘤胃微生物功能在糖类代谢、氨基酸代谢、脂肪代谢及疾病相关等功能存在差异。从上述分析来看,性别对牦牛瘤胃微生物区系的组成有显著影响,尤其是在属水平上差异较大。
关键词: 牦牛    16S rDNA    瘤胃    微生物区系    性别    TAX4FUN    
Rumen Microbiota Characteristics and Its Difference between Sex of Huanhu Yaks
HAN Xueping1,2,3,4 , LIU Hongjin1,2,3 , HU Linyong1,2,3 , XU Shixiao1,2,3 , AI Deqiang4 , FU Hongyun4 , CHEN Yongwei4 , ZHAO Jianzhong5     
1. Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China;
2. Key Laboratory of Adaptation and Evolution of Plateau Biota, Chinese Academy of Sciences, Xining 810008, China;
3. University of Chinese Academy of Science, Beijing 100049, China;
4. Technology Extension Service of Animal Husbandry of Qinghai, Xining 810001, China;
5. Consultation Center of Agricultural and Animal Husbandry Engineering Project of Qinghai, Xining 810001, China
Abstract: This experiment was conducted to study the rumen microbiota characteristics and its difference between sex of Huanhu yaks. Five healthy male yaks[average body weight was (228.0±19.4) kg] and five healthy female yaks[average body weight was (198.0±8.4) kg] during 4 to 5 years were selected, and 16S rDNA amplicon high-throughput sequencing technology was applied to study the rumen microbiota characteristics and its difference between sex of yaks. The results showed that yaks of two sex had similar rumen microbiota composition at the phylum level, and Bacteroidetes, Verrucomicrobia and Firmicutes were predominant phyla with relative abundances of 51.74%, 15.00% and 14.34%, respectively. Prevotella_1, Rikenellaceae_RC9_gut_group and Prevotellaceae_UCG-001 were predominated at the genus level with relative abundance of 16.11%, 7.74% and 3.43%, respectively. Among alpha diversity indexes, the Sobs index of male yaks was 2 689, and significantly higher than that of female yaks (P < 0.01), while other indexes were no significant differences between sex (P>0.05). The results of principle coordination analysis (PCoA) and analysis of similarity (Anosim) showed that there were obvious difference in microbiota between two sex. At the generic level, the relative abundance of Rikenellaceae_RC9_gut_group of male yaks was significantly higher than that of female yaks (P < 0.05), and the relative abundance of Prevotella_1, Lachnospiraceae_AC2044 _group, Saccharofermentans, Anaerovorax and Elusimicrobium of female yaks were significantly higher those of male yaks (P < 0.05). Meanwhile, the TAX4FUN predicted function comparison found that there were difference on sugar metabolism, amino acid metabolism, fat metabolism and disease-related functions of rumen microbial function of male and female yaks. In short, sex has significant effect on rumen microbiota composition of yaks, especially at genus level.
Key words: yaks    16S rDNA    rumen    microbiota community    sex    TAX4FUN    

反刍动物瘤胃内聚集着大量的微生物,这些微生物主要是细菌、真菌和原生动物,它们对反刍动物消化起着重要作用[1]。这些微生物将植物性饲料发酵形成挥发性脂肪酸(volatile fatty acids,VFAs)和微生物蛋白(microbial proteins,MPs),为反刍动物生长和生产提供所需营养[2]。研究不同家畜品种瘤胃微生物特征及其影响因素,对于人们通过调节各种因素提高家养动物生产性能具有重要作用。过去几十年间,基于传统培养方法,部分瘤胃微生物优势菌被分离和鉴定出来[3-5]。但是,这种方法能分离出来的微生物仅占瘤胃微生物的一小部分[6]。基于16S rRNA/rDNA的分子生物学技术的出现为研究人员提供了研究微生物多样性、系统进化等的新方法[7-8]。近年来,研究人员对绵羊、山羊、奶牛等家养动物和鹿、麋鹿等非家养动物的瘤胃微生物区系特征进行了大量研究[9-11],这些研究使人们对不同动物瘤胃微生物区系特征有了更深入的认识。同时,宿主、饲粮、饲养方式、年龄等环境因素对瘤胃微生物区系的影响也开展了诸多研究[12-14]。而牦牛作为青藏高原地区特有的家养反刍动物,其瘤胃微生物区系特征及性别对瘤胃微生物区系影响的研究对于阐述牦牛对高原地区高寒、低氧自然环境适应能力具有重要意义。Miyoshi等[15]研究报道,年龄和性别对大鼠肠道微生物区系有显著影响。最近的几项研究结果表明,青春期后女性肠道微生物多样性明显高于男性;人类和啮齿类动物肠道微生物与性别有关[16-18]。这些研究证明了性别可影响肠道微生物区系特征,但研究对象为人和其他单胃动物,目前,性别对反刍动物瘤胃微生物组成的影响尚未见报道。

牦牛作为青藏高原特有的家养反刍动物,对高寒、低氧的高原环境有很强的适应能力。牦牛瘤胃微生物区系特征的研究及公、母牦牛瘤胃微生物区系差异的研究对于丰富现有肠道微生物研究和阐述牦牛独特适应性具有重要意义。因此,本试验采用16S rRNA V3~V4区扩增子高通量测序技术,对环湖牦牛成年公、母牛瘤胃微生物区系特征和性别对微生物区系的影响进行研究,旨在阐明环湖牦牛瘤胃微生物区系特征及其在性别之间的差异。

1 材料与方法 1.1 试验动物与样品采集

2018年11月,随机挑选在青海省海晏县境内全放牧饲养的4~5岁环湖牦牛健康公牛5头[平均体重为(228.0±19.4) kg]和母牛5头[平均体重为(198.0±8.4) kg],送到青海夏华肉食品有限公司(位于海晏县)屠宰车间,经停食24 h、停水2 h后屠宰,屠宰后立即采集经4层纱布过滤的瘤胃液样品2 mL于具塞的消毒管中,迅速置于液氮中保存,带回实验室用于DNA提取和后续分析。

1.2 DNA提取与测序

根据E.Z.N.A stool DNA Kit(Omega Bio-Tek公司,美国)使用说明提取样品微生物DNA。微生物16S rRNA V3~V4区通用引物为341F(5′-CCTACGGGNGGCWGCAG-3′)和806R(5′-GGACTACHVGGGTATCTAAT-3′),PCR反应体系为:5 μL的10×KOD Buffer,5 μL的2.5 mmol/L dNTPs,1.5 μL引物(5 μmol/L),1 μL的KOD聚合酶和100 ng板DNA。扩增条件为:95 ℃预变性2 min,随后98 ℃变性10 s,62 ℃退火30 s,68 ℃延伸30 s,共27个循环,最后68 ℃延伸10 min。对扩增产物切胶回收,用QuantiFluorTM荧光计进行定量。后送广州基迪奥生物科技有限公司根据Illumina说明构建文库,在Hiseq2500的PE250模式上机测序。

1.3 数据生物信息学处理

去除测序结果reads中N碱基比例大于10%的序列和低质量序列,用Flash 1.2.11[19]进行reads拼接,Qiime 1.9.1[20]进行tags质控过滤,在与Gold database (r20110519)数据库进行对比(UCHIME algorithm)去嵌合体处理,根据barcode确定对应样品,去除barcode和引物序列。利用Mothur v1.39.1软件进行去冗余处理(https://www.mothur.org/),计算tags丰度。按照相似度大于97%用Uparse (usearch v9.2.645)[21]将序列聚类为分类操作单元(operational taxonomic unit,OTU),使用RDP classifier 2.2[22]与SILVA分类学数据库进行物种注释[23],得到OTU的分类学信息。用Tax4FUN[24]进行KEGG微生物群落功能预测。

1.4 数据统计与分析方法

数据采用Excel 2016进行整理,组间Alpha多样性采用OriginPro 2017软件配对样本t检验方法进行检验[25],菌群丰度差异显著性检验采用STAMP软件Welch’s t检验方法进行检验[26]P < 0.05表示差异显著。应用Jaccard算法在OTU水平上对所有样品进行主坐标分析(principle coordination analysis,PCoA)[27]、相似性分析(analysis of similarity,Anosim)和Beta多样性分析[28]

2 结果与分析 2.1 测序结果

经测序,10份瘤胃液样品中共得到原始序列2 332 256条,经质控和去嵌合体等处理后,有效序列为1 913 877条,平均每个样品191 387.7条。香浓(Shannon)稀释曲线呈现缓和趋势,说明测序深度趋于饱和(图 1-A),序列覆盖度(goods coverage)为99.6%。有效序列在按相似度大于97%的方法聚类为2 878个OTU,其中2 340个OTU为2个性别共享(图 1-B)。

F1~F5分别为公牛的5个样品,M1~M5分别为母牛的5个样品。 F1 to F5 were 5 samples of female yaks, and M1 to M5 were 5 samples of male yaks, respectively. 图 1 样品稀释曲线(A)和OTU维恩图(B) Fig. 1 Rarefaction curve of sample (A) and OTUs Venn diagram (B)
2.2 物种注释

基于SILVA分类学数据库和RDP分类工具,样品有效序列被分类到从门到种不同分类水平上。界水平上,微生物的相对丰度高于99.90%。所有样品共检测到10个门,2个性别门水平组成相似,其中拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrobia)和厚壁菌门(Firmicutes)是优势菌门,样品中平均相对丰度分别为51.74%、15.00%和14.34%(图 2-A)。在属水平上,普氏菌属_1(Prevotella_1)、理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)和普雷沃氏菌科_UCG-001(Prevotellaceae_UCG-001)是优势菌属,平均相对丰度分别为16.11%、7.74%和3.43%,公、母牛中未分类的序列比例分别为50.9%和44.3%(图 2-B)。

Bacteroidetes:拟杆菌门;Verrucomicrobia:疣微菌门;Firmicutes:厚壁菌门;Lentisphaerae:黏胶球形菌门;Fibrobacteres:纤维杆菌门;Proteobacteria:变形菌门;Spirochaetae:螺旋菌门;Cyanobacteria:蓝细菌门;Tenericutes:软壁菌门;Elusimicrobia:迷踪菌门;Other:其他;Unclassified:未分类序列;Prevotella_1:普氏菌属_1;Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;Prevotellaceae_UCG-001:普雷沃氏菌科_UCG-001;Prevotellaceae_UCG-003:普雷沃氏菌科_UCG-003;Fibrobacter:纤维杆菌属;Erysipelotrichaceae_UCG-004:韦荣球菌科_UCG-004;Ruminococcaceae_UCG-010:疣微菌科_UCG-010;Christensenellaceae_R-7_group:梭菌目R-7群;Treponema_2:螺旋体科_2。 图 2 环湖牦牛瘤胃门水平(A)和属水平(B)细菌组成 Fig. 2 Bacterial composition at phylum level (A) and genus level (B) in rumen of Huanhu yaks
2.3 Alpha多样性指数

表 1可见,公牛的Sobs指数为2 689,极显著高于母牛(P < 0.01),其余指数无显著性别差异(P>0.05)。

表 1 公、母牦牛瘤胃微生物Alpha多样性指数 Table 1 Alpha diversity indexes of rumen microbiota of male and female yaks
2.4 Beta多样性分析

应用Jaccard算法在OTU水平上对所有样品进行PCoA(图 3-A),可以看出2个性别的样品明显聚为2类,说明2个性别之间瘤胃微生物区系存在明显差异。Anosim结果也表明牦牛瘤胃微生物组成存在显著性别差异(P < 0.05)(图 3-B)。

图 3 公、母牦牛瘤胃微生物Beta多样性PCoA(A)和Anosim(B) Fig. 3 Beta diversity of PCoA (A) and Anosim (B) of rumen microbiota of male and female yaks
2.5 性别间差异物种分析

用STAMP v2.1.3的Welch’s t检验在门水平和属水平上进行性别间物种差异分析,由图 4可见,门水平上牦牛性别之间无显著差异(P>0.05);属水平上,公牦牛Rikenellaceae_RC9_gut_group的相对丰度显著高于母牦牛(P < 0.05),母牦牛Prevotella_1、毛螺菌科AC2044群(Lachnospiraceae_AC2044_group)、SaccharofermentansAnaerovoraxElusimicrobium的相对丰度显著高于公牦牛(P < 0.05)。

Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;Prevotella_1:普氏菌属_1;Lachnospiraceae_AC2044_group:毛螺菌科AC2044群。 图 4 公、母牦牛之间差异物种分析 Fig. 4 Analysis of differential species between male and female yaks
2.6 功能预测

用OriginPro2017工具对牦牛瘤胃微生物TAX4FUN预测功能进行性别之间差异Welch’s t检验,由表 2可见,在相对丰度大于0.1%的预测功能中,母牛缬氨酸、亮氨酸和异亮氨酸生物合成(phenylalanine, tyrosine and tryptophan biosynthesis)、果糖和甘露糖代谢(fructose and mannose metabolism)、戊糖和葡萄糖醛酸相互转化(pentose and glucuronate interconversions)、缬氨酸、亮氨酸和异亮氨酸生物合成(valine, leucine and isoleucine biosynthesis)、泛酸盐和辅酶A生物合成(pantothenate and CoA biosynthesis)和硫中继系统(sulfur relay system)等预测功能的相对丰度显著高于公牛(P < 0.05),公牛过氧化物酶体增殖物激活受体信号通路(PPAR signaling pathway)、叶酸碳池(one carbon pool by folate)、军团菌病(legionellosis)、脂肪酸代谢(lipoic acid metabolism)、植物病原菌互作(plant-pathogen interaction)、萜类骨架生物合成(terpenoid backbone biosynthesis)、赖氨酸生物合成(lysine biosynthesis)、细胞周期-茎杆菌(cell cycle-caulobacter)、甘油磷脂代谢(glycerophospholipid metabolism)和脂多糖生物合成(lipopolysaccharide biosynthesis)等预测功能的相对丰度显著高于母牛(P < 0.05)。这些功能主要涉及萜类化合物和聚酮类化合物的代谢,辅因子和维生素的代谢,脂类代谢,传染性疾病,糖的生物合成和代谢,折叠、排序与降解,环境适应,内分泌系统,细胞生长与死亡,碳水化合物代谢和氨基酸代谢等。

表 2 环湖牦牛性别之间相对丰度有显著差异的预测功能 Table 2 Predicted functions with different relative abundance between sex of Huanhu yaks
3 讨论 3.1 环湖牦牛瘤胃微生物区系特征

诸多学者围绕宿主、饲粮、管理方式[10, 29-30]等因素对瘤胃微生物的影响进行了研究。但鲜有报道对青藏高原牦牛瘤胃微生物组成及其在2个性别之间的差异进行研究。本研究结果中Bacteroidetes、Verrucomicrobia和Firmicutes是环湖牦牛瘤胃中的优势菌门,在门水平上2个性别之间没有显著差异,Prevotella_1是优势菌属。曹连宾等[31]应用构建16S rRNA克隆文库的方法研究了放牧牦牛和舍饲牦牛瘤胃微生物的多样性,结果表明牦牛瘤胃微生物多样性高,Firmicutes和Bacteroidetes是2种养殖方式下牦牛瘤胃微生物共同的优势菌门。Xue等[32]对舍饲牦牛和放牧牦牛瘤胃微生物区系特征的研究结果表明,Firmicutes、Bacteroidetes和变形菌门(Proteobacteria)是牦牛的优势菌门,普氏菌属(Prevotella)是优势菌属,该结论与本研究结论基本一致,本文研究对象是青海省环湖牦牛,而报道中的牦牛为四川省牦牛,两者遗传或饲养条件差异是引起差别的可能原因。此外,本文采用16S rDNA扩增子高通量测序的方法进行研究,而上述2个报道采用的均是构建克隆文库的方法,这也是产生差异的可能原因。Shen等[33]研究表明,随着非纤维碳水化合物含量在饲粮中的增加,Verrucomicrobia的丰度极显著增加。Fujio-Vejar等[34]研究结果表明,在青年智利人肠道中Bacteroidetes、Verrucomicrobia和Firmicutes丰度最高,相对丰度分别为43.6%、41.6%和8.5%,Verrucomicrobia是PVC(Planctomycetes-Verrucomicrobia-Chlamydiae)超级门的成员,该门包括系统发育相关且具有一些不常见特征的细菌,如复杂的动态膜内系统,这些特征使该门微生物更像是原核生物。Derrien等[35]研究结果表明,嗜黏蛋白阿卡曼氏菌(Akkermansia muciniphila)是在人类肠道中发现的该门主要成员,该菌是严格厌氧的革兰氏阴性菌,是一类高度专门化的利用黏蛋白作为氮源和碳源的微生物。嗜黏蛋白阿卡曼氏菌在使用抗生素后和使用二甲双胍治疗治疗病人体内丰度更高[36]。前期研究表明,Prevotella是反刍动物瘤胃中最优势的菌属[37-39]。De Filippo等[40]研究表明,在饮食中植物性纤维含量高的非洲儿童肠道中Prevotella丰度较高,而在饮食中动物性蛋白质、脂肪较高的欧洲儿童肠道中拟杆菌属(Bacteroides)丰度较高。

3.2 性别对牦牛瘤胃微生物组成的影响

本研究中性别之间有差别的属Anaerovorax、Lachnospiraceae_AC2044_group、Saccharofermentans属于梭菌目(Clostridiales),Prevotella_1属于拟杆菌目(Bacteroidales),Prevotella_1是Bacteroidales中丰度最高的菌属。Söllinger等[41]研究结果表明,Clostridiales是Firmicutes的重要组成菌目,其在瘤胃植物性纤维降解过程中发挥重要作用。Bacteroidales能降解纤维素,并含有能降解植物多糖能力的基因[42]。最近的几项关于人类肠道微生物研究结果表明,女性肠道中微生物多样性高于男性,性别影响肠道微生物的组成[17-18, 43]。而Miyoshi等[15]研究结果表明,人肠道微生物组成不存在性别差异。De La Cuesta-Zuluaga等[44]认为人肠道微生物受性激素调节,因为激素水平不同和女性提前到达青春期,所以女性肠道微生物多样性较高,而男性到了中年之后肠道微生物多样性水平才能提高。而本研究中牦牛年龄均已达到成年,且公牛肠道微生物多样性高于母牛,其原因还需深入研究。

3.3 性别对牦牛瘤胃微生物功能的影响

性别之间存在差异的TAX4FUN预测功能主要为萜类化合物和聚酮类化合物的代谢,辅因子和维生素的代谢,脂类代谢,传染性疾病,糖的生物合成和代谢,折叠、排序与降解,环境适应,内分泌系统,细胞生长与死亡,碳水化合物代谢和氨基酸代谢等,其中差异主要为碳水化合物代谢、氨基酸代谢等与代谢相关的功能。可以看出2个性别的牦牛瘤胃微生物功能在糖类代谢、氨基酸代谢、脂肪代谢及疾病相关等功能存在显著差异。

本研究首次采用高通量测序结束对青藏高原牦牛瘤胃微生物特征及性别对微生物组成的影响进行了研究,不仅丰富了动物胃肠道微生物研究的内容,也为今后青藏高原反刍家畜瘤胃微生物的深入研究和调控提供了借鉴。但因本研究没有开展不同年龄和性别牦牛瘤胃微生物组成的研究,也没有测定不同性别个体的激素水平,难以全面阐释差异产生的原因,因此今后应深入研究年龄、性别及激素水平对青藏高原反刍家畜瘤胃微生物组成的影响。

4 结论

① Bacteroidetes、Verrucomicrobia和Firmicutes是环湖牦牛瘤胃中的优势菌门,Prevotella_1是优势菌属。

② 环湖牦牛2个性别之间瘤胃微生物组成、多样性和功能存在差异,尤其是植物性纤维降解相关微生物丰度差异显著,这些差异物种可能为2个性别的牦牛适应高海拔地区气候条件发挥了不同作用。

③ 环湖牦牛瘤胃微生物糖类、脂肪、氨基酸代谢及疾病相关功能的丰度也存在差异。

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