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

Comparative Analysis of Gut Bacterial Composition of Grazing Male Yaksat Different Ages Based on 16S rRNA Sequencing Technology

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。

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.