基于16S rRNA测序技术对不同年龄放牧公牦牛肠道细菌区系的比较分析

doi:10.16431/j.cnki.1671-7236.2021.10.012

摘要/Abstract

摘要： 试验旨在比较分析不同年龄放牧公牦牛肠道细菌区系特征，为牦牛肠道微生物功能调控规律提供理论基础。采集青海省玉树州歇武镇0.5（Ac组）、1.5（Bc组）、2.5（Cc组）、3.5岁（Dc组）放牧公牦牛直肠粪便，采用16S rRNA测序技术对不同年龄牦牛肠道细菌区系进行测序分析。使用Qiime软件进行Alpha多样性和Beta多样性分析，用R软件绘制PCoA图，使用Anosim函数进行组间差异显著性检验，利用Mothur方法与SILVA132的SSUrRNA数据库进行物种注释分析，并利用Tax4Fun对肠道细菌进行功能预测。结果表明，年龄对肠道细菌区系操作分类单元（OTUs）数目和Simpson指数有显著影响（P<0.05），Simpson指数在Cc组最低；Beta多样性分析结果显示，Cc分别与Ac、Dc组之间、Ac与Dc组之间有显著差异（P<0.05）。在门水平上，各组厚壁菌门所占丰度最高，拟杆菌门居于第二。Bc和Cc组厚壁菌门相对丰度显著高于Ac和Dc组（P<0.05），疣微菌门相对丰度在各年龄组之间有显著差异（P<0.05），且Cc组最高；在属水平上，随着年龄的增加，拟普雷沃菌属丰度逐渐增加（P<0.05），一些非优势菌属罗姆布茨菌属、颤杆菌属、艾克曼菌属和Mailhella在组间存在显著差异（P<0.05）；功能预测结果表明，牦牛肠道细菌区系的代谢功能显示出最高的相对丰度，在各组中相对丰度均超过44%。Cc组氨基酸代谢、信号转导和细胞运动功能基因表达丰度显著高于Ac和Dc组（P<0.05），Ac、Bc和Dc组之间功能基因表达丰度无显著差异（P>0.05）。综上，随着年龄增长，放牧公牦牛肠道细菌区系多样性发生变化；2.5岁牦牛肠道细菌区系的变化提示此阶段牦牛的蛋白质消化代谢和免疫能力较高，为育肥饲养提供了良好的基础。

关键词: 16S rRNA测序; 放牧牦牛; 肠道细菌区系; 功能预测

Abstract: The gut bacterial composition of grazing male yaks at different ages were compared and analyzed to provide a basis for the regulation of bacterial function. Rectal fecal samples were collected from 0.5-year-old (Ac group), 1.5-year-old (Bc group), 2.5-year-old (Cc group) and 3.5-year-old (Dc group) grazing male yaks in Xiewu town, Yushu prefecture, Qinghai province. 16S rRNA sequencing technology was used to analyze the bacterial composition of yaks at different ages. Alpha diversity and Beta diversity were calculated by Qiime software. PCoA diagram was drawn by R software. Anosim analysis was used to test the difference between groups. Mothur method and the SSUrRNA database of SILVA132 were used for species annotation analysis, and Tax4Fun was used for functional prediction of gut bacterial. The results showed that the increase of age had a significant effect on the number of OTUs and Simpson index (P<0.05), and simpson index of gut bacterial composition was the lowest in Cc group. The results of Beta diversity analysis showed that there were significant differences between Cc and Ac, Dc groups, and between Ac and Dc groups (P<0.05). At the phylum level, the relative abundance of Firmicutes in each group was the highest, and that of Bacteroidetes was the second. The relative abundance of Firmicutes in Bc and Cc groups were significantly higher than that in Ac and Dc groups (P<0.05). The relative abundance of Verrucomicrobia was significantly different among different age groups and the highest in Cc group (P<0.05). At the genus level, with the increase of age, the relative abundance of Alloprevotella increased gradually (P<0.05). There were significant differences in some non-dominant Romboutsia, Oscillibacter, Akkermansia and Mailhella between groups (P<0.05). In function prediction, the metabolic function of yaks gut bacterial showed the highest relative abundance, which was more than 44% in each group. The expression abundance of functional genes of amino acid metabolism, signal transduction and cell motility in Cc group were significantly higher than that in Ac and Dc groups (P<0.05). There was no significant difference in the expression abundance of functional genes among Ac, Bc and Dc groups (P>0.05). In summary, the gut bacterial composition of grazing male yaks changed in diversity with the increase of age. The changes of gut bacterial composition of 2.5-year-old yaks indicated that the protein digestion, metabolism and immunity of yaks were high at this stage, which provided a good foundation for fattening.

Key words: 16S rRNA sequence; grazing yak; gut bacterial composition; function prediction

中图分类号:

S823.8⁺5

郭文杰, 刘书杰, 崔占鸿, 孙璐. 基于16S rRNA测序技术对不同年龄放牧公牦牛肠道细菌区系的比较分析[J]. 中国畜牧兽医, 2021, 48(10): 3614-3624.

GUO Wenjie, LIU Shujie, CUI Zhanhong, SUI Lu. Comparative Analysis of Gut Bacterial Composition of Grazing Male Yaks at Different Ages Based on 16S rRNA Sequencing Technology[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3614-3624.

参考文献

[1] 马国兰.浅谈青海的牦牛资源现状及保护措施[J]. 中国牛业科学, 2014, 40(3):89-91. MA G L.Resource status and protections of yaks in Qinghai[J]. China Cattle Science, 2014, 40(3):89-91.(in Chinese)
[2] O'HARA E, NEVES A L A, SONG Y, et al.The role of the gut microbiome in cattle production and health:Driver or passenger?[J]. Annual Review of Animal Biosciences, 2020, 8:199-220.
[3] FUJIMURA K E, SLUSHER N A, CABANA M D, et al.Role of the gut microbiota in defining human health[J]. Expert Review of Anti-Infective Therapy, 2010, 8(4):435-454.
[4] 左愈臻, 高世杰, 邵建华, 等.成年牦牛小肠结构及黏膜免疫相关细胞数量变化研究[J]. 畜牧兽医学报, 2011, 42(12):1776-1781. ZUO Y Z, GAO S J, SHAO J H, et al.Study on the structure and quantitative changes of mucosal immunity-associated cells in small intestine of the adult yak[J]. Acta Veterinaria et Zootechnica Sinica, 2011, 42(12):1776-1781.(in Chinese)
[5] 苟娜娜.青藏高原牦牛和黄牛机体激素、免疫球蛋白和肠道微生物的季节性变化及相关性[D].兰州:兰州大学, 2020. GOU N N.Seasonal change and correlation of body hormone, immunoglobulin and intestinal microbes in yak and cattle on the Tibetan plateau[D].Lanzhou:Lanzhou University, 2020.(in Chinese)
[6] ZHANG L H, JIANG X, LI A Y, et al.Characterization of the microbial community structure in intestinal segments of yak(Bos grunniens)[J]. Anaerobe, 2020, 61:102115.
[7] DING X Z, LIANG C N, GUO X, et al.Physiological insight into the high-altitude adaptations in domesticated yaks(Bos grunniens) along the Qinghai-Tibetan plateau altitudinal gradient[J]. Livestock Science, 2014, 162:233-239.
[8] HUANG X D, TAN H Y, LONG R, et al.Comparison of methanogen diversity of yak (Bos grunniens) and cattle (Bos taurus) from the Qinghai-Tibetan plateau, China[J]. BMC Microbiology, 2012, 12:237.
[9] SHAPIRA M.Gut microbiotas and host evolution:Scaling up symbiosis[J]. Trends in Ecology & Evolution, 2016, 31(7):539-549.
[10] 聂远洋, 邓岳, 刘戎梅, 等.不同年龄麦洼牦牛肠道菌群的分离鉴定及其群落结构的变化[J]. 中国测试, 2016, 42(12):53-59. NIE Y Y, DENG Y, LIU R M, et al.Isolation, identification and community structure changes of intestinal flora in Maiwa yaks at different ages[J]. China Measurement & Test, 2016, 42(12):53-59.(in Chinese)
[11] 付洋洋.年龄和性别对牦牛舍饲育肥效果和肉品质的影响研究[D].成都:西南民族大学, 2018. FU Y Y.Effects of age and sex on fattening and meat quality of house-feeding yaks[D].Chengdu:Southwest University of Nationalities, 2018.(in Chinese)
[12] CAPORASO J G, LAUBER C L, WALTERS W A, et al.Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample[J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(Suppl 1):4516-4522.
[13] YOUSSEF N, SHEIK C S, KRUMHOLZ L R, et al.Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys[J]. Applied and Environmental Microbiology, 2009, 75(16):5227-5236.
[14] 李东萍, 郭明璋, 许文涛.16S rRNA测序技术在肠道微生物中的应用研究进展[J]. 生物技术通报, 2015, 31(2):71-77. LI D P, GUO M Z, XU W T.Advances and applications on methodology of 16S rRNA sequencing in gut microbiota analysis[J]. Biotechnology Bulletin, 2015, 31(2):71-77.(in Chinese)
[15] 刘驰, 李家宝, 芮俊鹏, 等.16S rRNA基因在微生物生态学中的应用[J]. 生态学报, 2015, 35(9):2769-2788. LIU C, LI J B, RUI J P, et al.Application of 16S rRNA gene in microbial ecology[J]. Acta Ecologic Sinica, 2015, 35(9):2769-2788.(in Chinese)
[16] 中华人民共和国国家质量监督检验检疫总局.饲料中水分的测定GB/T 6435-2014[S].北京:中国标准出版社, 2014. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of moisture in feedstuff GB/T 6435-2014[S].Beijing:China Standard Press, 2014.(in Chinese)
[17] 中华人民共和国国家质量监督检验检疫总局.饲料中粗蛋白的测定凯氏定氮法GB/T6432-2018[S].北京:中国标准出版社, 2018. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of crude protein in feedstuff:Kjeldahl method GB/T 6432-2018[S].Beijing:China Standard Press, 2018.(in Chinese)
[18] 中华人民共和国国家质量监督检验检疫总局.饲料中粗脂肪的测定GB/T 6433-2006[S].北京:中国标准出版社, 2006. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of crude fat in feedstuff GB/T 6433-2006[S].Beijing:China Standard Press, 2006.(in Chinese)
[19] 中华人民共和国国家质量监督检验检疫总局.饲料中粗灰分的测定GB/T 6438-2007[S].北京:中国标准出版社, 2007. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of crude ash in feedstuff GB/T 6438-2007[S].Beijing:China Standard Press, 2007.(in Chinese)
[20] 中华人民共和国农业部.饲料中酸性洗涤纤维的测定NY/T 1459-2007[S].北京:中国标准出版社, 2007. AGRICULTURAL MINISTRY OF THE PEOPLE'S REPUBLIC OF CHINA.Determination of acid detergent fiber in feedstuff NY/T 1459-2007[S].Beijing:China Standard Press, 2007.(in Chinese)
[21] 中华人民共和国国家质量监督检验检疫总局.饲料中中性洗涤纤维(NDF)的测定GB/T 20806-2006[S].北京:中国标准出版社, 2006. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of neutral detergent fiber in feedstuff GB/T 20806-2006[S].Beijing:China Standard Press, 2006.(in Chinese)
[22] 中华人民共和国国家质量监督检验检疫总局.食品安全国家标准婴幼儿食品和乳品中乳糖、蔗糖的测定GB 5413.5-2010[S].北京:中国标准出版社, 2010. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.National food safety standard:Determination of lactose and sucrose in foods, milk and milk products for infants and young children GB 5413.5-2010[S].Beijing:China Standard Press, 2010.(in Chinese)
[23] 中华人民共和国国家质量监督检验检疫总局.食品安全国家标准乳和乳制品中非脂乳固体的测定GB 5413.39-2010[S].北京:中国标准出版社, 2010. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.National food safety standard:Determination of nonfat total milk solids in milk and milk products GB 5413.39-2010[S].Beijing:China Standard Press, 2010.(in Chinese)
[24] 中华人民共和国国家质量监督检验检疫总局.饲料中钙、铜、铁、镁、锰、钾、钠和锌含量的测定原子吸收光谱法GB/T 13885-2017[S].北京:中国标准出版社, 2017. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of the contents of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc in feeds:Method using atomic absorption spectrometry GB/T 13885-2017[S].Beijing:China Standard Press, 2017.(in Chinese)
[25] 中华人民共和国国家质量监督检验检疫总局.饲料中总磷的测定分光光度法GB/T 6437-2018[S].北京:中国标准出版社, 2017. GENERAL ADMINISTRATION OF QUALITY SUPERVISION, INSPECTION AND QUARANTINE OF THE PEOPLE'S PEPUBLIC OF CHINA.Determination of phosphorus in feedstuff:Spectrophotometry GB/T 6437-2018[S].Beijing:China Standard Press, 2018.(in Chinese)
[26] 王慧慧, 李寅虎, 邱创钊, 等.1067例不同年龄及地域人群肠道菌群特征[J]. 中国微生态学杂志, 2019, 31(9):1000-1004. WANG H H, LI Y H, QIU C Z, et al.The characterization of human gut microbiota across ages and continents based on 1067 fecal samples[J]. Chinese Journal of Microecology, 2019, 31(9):1000-1004.(in Chinese)
[27] 王润盈.大型食草动物粪便环境对小鼠肠道菌群演替的促进[D].大连:大连医科大学, 2020. WANG R Y.Enhancement of gut microbial succession in mice in environment contaminated with herbivore feces[D].Dalian:Dalian Medical University, 2020.(in Chinese)
[28] 昝林森.牛生产学[M].第2版.北京:中国农业出版社, 2007. ZAN L S.Cattle Production[M].2rd Edition.Beijing:China Agriculture Press, 2007.(in Chinese)
[29] CHEN Y M, WEI L, CHIU Y S, et al.Lactobacillus plantarum TWK10 supplementation improves exercise performance and increases muscle mass in mice[J]. Nutrients, 2016, 8(4):205.
[30] HAN J H, KIM I S, JUNG S H, et al.The effects of propionate and valerate on insulin responsiveness for glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes via G protein-coupled receptor 41[J]. PLoS One, 2014, 9(4):e95268.
[31] SCOTT K P, GRATZ S W, SHERIDAN P O, et al.The influence of diet on the gut microbiota[J]. Pharmacological Research, 2013, 69(1):52-60.
[32] WU G D, CHEN J, HOFFMANN C, et al.Linking long-term dietary patterns with gut microbial enterotypes[J]. Science, 2011, 334(6052):105-108.
[33] MALMUTHUGE N, LI M, GOONEWARDENE L A, et al.Effect of calf starter feeding on gut microbial diversity and expression of genes involved in host immune responses and tight junctions in dairy calves during weaning transition[J]. Journal of Dairy Science, 2013, 96(5):3189-3200.
[34] 刘传发, 张良志, 付海波, 等.野牦牛和家牦牛粪便菌群与短链脂肪酸关系的研究[J]. 兽类学报, 2019, 39(1):1-7. LIU C F, ZHANG L Z, FU H B, et al.Relationship research between fecal microbes and short chain fatty acid between wild yak and domestic yak[J]. Acta Theriologica Sinica, 2019, 39(1):1-7.(in Chinese)
[35] NIE Y, ZHOU Z, GUAN J, et al.Dynamic changes of yak(Bos grunniens) gut microbiota during growth revealed by polymerase chain reaction-denaturing gradient gel electrophoresis and metagenomics[J]. Asian-Australasian Journal of Animal Sciences, 2017, 30(7):957-966.
[36] 高凤.奶牛肠道微生物群落结构与多样性研究[D].邯郸:河北工程大学, 2017. GAO F.The study on intestinal microbial community structure and diversity of dairy cows[D].Handan:Hebei University of Engineering, 2017.(in Chinese)
[37] 陈宝剑, 吴永绍, 覃兆鲜, 等.猪不同发育阶段肠道微生物菌群特征分析[J]. 中国畜牧杂志, 2021, 57(1):101-108. CHEN B J, WU Y S, QIN Z X, et al.Characteristic analysis of intestinal microflora in different developmental stages of pigs[J]. Chinese Journal of Animal Science, 2021, 57(1):101-108.(in Chinese)
[38] 李晓斌, 赵国栋, 刘振, 等.3~6月龄伊犁马肠道微生物群落多样性的研究[J]. 动物营养学报, 2017, 29(5):1535-1544. LI X B, ZHAO G D, LIU Z, et al.A study on intestinal microbota diversity of 3- to 6-month-old Yili horses[J]. Chinese Journal of Animal Nutrition, 2017, 29(5):1535-1544.(in Chinese)
[39] 马晓聪, 熊兴江, 莫毅, 等.基于16S rDNA测序技术的自发性高血大鼠肠道菌群结构变化及中药干预初探[J]. 中华中医药学刊, 2020, 38(8):71-74. MA X C, XIONG X J, MO Y, et al.The changes of intestinal flora structure in spontaneously hypertensive rats based on 16S rDNA sequencing technology and the intervention of traditional Chinese medicine[J]. Chinese Archives of Traditional Chinese Medicine, 2020, 38(8):71-74.(in Chinese)
[40] TUN H M, BRAR M S, KHIN N, et al.Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing[J]. Journal of Microbiological Methods, 2012, 88(3):369-376.
[41] LOUIS P, FLINT H J.Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine[J]. FEMS Microbiology Letters, 2009, 294(1):1-8.
[42] 彭典, 胡卓伟, 张晓伟.肠道益生菌A.muciniphila抗代谢性疾病的治疗学展望[J]. 药学学报, 2019, 54(5):768-777. PENG D, HU Z W, ZHANG X W.Therapeutic perspectives of intestinal probiotics A. muciniphila in metabolic disorders[J]. Acta Pharmaceutica Sinica, 2019, 54(5):768-777.(in Chinese)
[43] PLOVIER H, EVERARD A, DRUART C, et al.A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice[J]. Nature Medicine, 2017;23(1):107-113.
[44] OTTMAN N.Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila[D].Wageningen:Wageningen University, 2015.
[45] RAABIS S, LI W, CERSOSIMO L.Effects and immune responses of probiotic treatment in ruminants[J]. Veterinary Immunology Immunopathology, 2019, 208:58-66.
[46] 郝晨阳, 程艳芬, 徐丽婧, 等.广叶绣球菌多糖对免疫低下小鼠肠道免疫功能的调节作用[J]. 菌物学报, 2020, 39(7):1380-1390. HAO C Y, CHENG Y F, XU L J, et al.Regulatory effects of Sparassis latifolia polysaccharide on intestinal immune function in immunosuppressed mice[J]. Mycosystema, 2020, 39(7):1380-1390.(in Chinese)
[47] WALTER J, HERTEL C, TANNOCK G W, at al.Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis[J]. Applied Environmental Microbiology, 2001, 67(6):2578-2585.
[48] 聂召龙, 刘书杰, 崔占鸿, 等.新疆巴州幼年与成年牦牛瘤胃细菌区系多样性分析[J]. 核农学报, 2019, 33(11):2147-2157. NIE Z L, LIU S J, CUI Z H, et al.Analysis of rumen bacterial flora diversity in young and adult Bazhou yaks in Xinjiang[J]. Journal of Nuclear Agricultural Sciences, 2019, 33(11):2147-2157.(in Chinese)
[49] QIN R, WANG J, CHAO C, et al.RS5 produced more butyric acid through regulating the microbial community of human gut microbiota[J]. Journal of Agricultural and Food Chemistry, 2021, 69(10):3209-3218.
[50] HAGI T, BELZER C.The interaction of Akkermansia muciniphila with host-derived substances, bacteria and diets[J]. Applied Microbiology and Biotechnology, 2021, 105(12):4833-4841.