基于宏基因组测序技术研究正常与腹泻牦牛犊牛肠道菌群特征

Study on Characterization of Intestinal Flora in Normal and Diarrhea Yak Calves Based on Metagenomic Sequencing Technology

本试验旨在探究正常与腹泻牦牛犊牛肠道菌群组成特征,为牦牛犊牛培育中腹泻的早期预防提供重要参考。试验选取2月龄牦牛犊牛(公)6头,其中正常牦牛犊牛3头,腹泻牦牛犊牛3头,采用直肠取样法采集所有犊牛粪便样品,提取高质量的细菌总DNA,利用宏基因组测序技术分析正常与腹泻牦牛犊牛肠道微生物群落结构及功能基因组成。结果表明:腹泻牦牛犊牛粪便微生物Shannon指数、Simpson指数、Chao1指数和ACE指数低于正常牦牛犊牛;在门水平上,相比正常牦牛犊牛,腹泻牦牛犊牛的粪便微生物中变形菌门(Proteobacteria)的相对丰度显著升高(P<0.05),软壁菌门(Tenericutes)、衣原体门(Chlamydiae)的相对丰度显著降低(P<0.05);在KEGG分析中,正常与腹泻牦牛犊牛在代谢通路上没有显著差异(P>0.05);在EggNOG水平功能分析中,腹泻牦牛犊牛的基因在细胞外结构、细胞运动性、转录以及复制、重组和修复上显著富集(P<0.05),正常牦牛犊牛的基因在翻译、核糖体结构与生物发生、细胞壁/膜/包膜生物发生上显著富集(P<0.05);在CAZy水平功能分析中,正常和腹泻牦牛犊牛肠道微生物基因均主要在糖苷水解酶、糖基转移酶基因家族中有显著差异(P<0.05);在CARD的抗性基因分析中,腹泻牦牛犊牛携带抗性基因较多的主要菌门有4个,依次是厚壁菌门(Firmicutes)、Proteobacteria、拟杆菌门(Bacteroidetes)、梭杆菌门(Fusobacteria),正常牦牛犊牛携带抗性基因较多的主要菌门有6个,其中占比较高的有3个,依次是Firmicutes、Bacteroidetes、Proteobacteria,同时发现正常和腹泻牦牛犊牛中Proteobacteria的占比与其携带抗性基因的占比呈负相关;在LEfSe分析中,腹泻牦牛犊牛的抗性基因多于正常牦牛犊牛,且多为多重耐药基因。综上可知,发生腹泻的牦牛犊牛肠道菌群的多样性降低,会引起变形菌门相对丰度的增加和厚壁菌门相对丰度的减少;同时,腹泻牦牛犊牛携带的抗性基因增加,且多数基因属于多重耐药基因。

The purpose of this experiment was to explore the composition characteristics of intestinal flora of normal and diarrhea yak calves,to provide an important reference for the early prevention of diarrhea in yak calf cultivation.Six 2-month-old yak calves(male)were selected for the experiment,including 3 normal yak calves and 3 diarrhea yak calves.Fecal samples from all calves were collected using rectal sampling method,and the high-quality total bacterial DNA was extracted to analyze the gut microbial community structure and functional gene composition of normal and diarrhea yak calves by metagenomic sequencing technology.The results showed that the fecal microbial Shannon index,Simpson index,Chao1 index and ACE index of diarrhea yak calves were lower than those of normal yak calves.At phylum level,compared with the normal yak calves,the relative abundance of Proteobacteria in fecal microorganisms of the diarrhea yak calves was significantly increased(P<0.05),and the relative abundances of Chlamydomonasa and Chlamydia were significantly decreased(P<0.05).In the KEGG analysis,there were no significant differences in metabolic pathways between the normal and diarrhea yak calves(P>0.05).In the EggNOG level function analysis,the genes of the diarrhea yak calves were significantly enriched in extracellular structure,cell movement,transcription,replication,recombination,and repair(P<0.05),and the genes of the normal yak calves were significantly enriched in translation,ribosome structure and biogenesis,and cell wall/membrane/envelope biogenesis(P<0.05).In the CAZy level function analysis,the intestinal microbial genes of the normal and diarrhea yak calves had significant differences mainly in the glycoside hydrolase and glycosyltransferase gene families(P<0.05).In the CARD resistance gene analysis,there were 4 main phyla carrying more resistance genes of the diarrhea yak calves,namely Firmicutes,Proteobacteria,Bacteroidetes and Fusobacterium;there were 6 main phyla carrying resistance genes of the normal yak calves,there were 3 phyla of them had higher proportion,namely Firmicutes,Bacteroidetes and Proteobacteria,and it was found that the proportion of Proteobacteria of the normal and the diarrhea yak calves was negatively correlated with the proportion of resistance genes.In the LEfSe analysis,the resistance genes of the diarrhea yak calves were more than those of the normal yak calves,and most of them were multi-drug resistant genes.In conclusion,the diversity of intestinal flora in yak calves with diarrhea is decreased,which causes the increase of Proteobacteria relative abundance and the decrease of Firmicutes relative abundance.At the same time,a variety of resistance genes carried by diarrhea yak calves is increased,and most genes are multi-drug resistant genes.