SPF级雄性SD大鼠回肠与结肠菌群结构比较分析

doi:10.12300/j.issn.1674-5817.2022.103

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (1): 53-60.DOI: 10.12300/j.issn.1674-5817.2022.103

所属专题：实验动物资源开发与利用

• 实验动物资源开发与利用 • 上一篇下一篇

SPF级雄性SD大鼠回肠与结肠菌群结构比较分析

许琴¹(), 倪艳¹^,²(), 是文辉¹, 李建瑛¹, 刘江伟¹, 赵红琼², 徐新明³()()

^1.新疆军区总医院, 新疆特殊环境医学重点实验室, 乌鲁木齐 830000
^2.新疆农业大学动物医学学院, 乌鲁木齐 830052
^3.新疆畜牧科学院生物技术研究所, 乌鲁木齐 830000

收稿日期:2022-07-07 修回日期:2022-09-16 出版日期:2023-02-25 发布日期:2023-03-09
通讯作者: 徐新明（1973—），男，副研究员，研究方向：动物遗传育种，动物科学生物技术研究。E-mail：xuxinmingcool@163.com。ORCID： 000-0002-7247-2774
作者简介:许琴（1974—），女，博士，研究方向：人类疾病动物模型制作及特殊环境医学病理生理学。E-mail： xuqin740831@163.com；
倪艳（1998—），女，硕士研究生，研究方向：基础兽医学。E-mail： 2460548072@qq.com
基金资助:
全军实验动物专项科研课题“高原缺氧寒冷应激环境功能性肠病动物模型制作及干预”(SYDW〔2018〕10)

Analysis on Ileum and Colon Microflora of SPF Male SD Rats based on High-throughput Sequencing

Qin XU¹(), Yan NI¹^,²(), Wenhui SHI¹, Jianying LI¹, Jiangwei LIU¹, Hongqiong ZHAO², Xinming XU³()()

^1.General Hospital of Xinjiang Military Districts, Key Laboratory of Special Environmental Medicines of Xinjiang, Urumqi 830000, China
^2.Veterinary Medicine College of Xinjiang Agricultural University, Urumqi 830052, China
^3.Biotechnology Research Institute of Xinjiang Animal Husbandry Sciences Academy, Urumqi 830000, China

Received:2022-07-07 Revised:2022-09-16 Published:2023-02-25 Online:2023-03-09
Contact: XU Xinming (ORCID: 000-0002-7247-2774), E-mail: xuxinmingcool@163.com

摘要/Abstract

摘要：

目的使用高通量测序技术分析SPF级雄性SD大鼠回肠与结肠菌群结构与丰度。方法收集30只SPF级雄性SD大鼠的回肠及结肠腔内容物，提取内容物中细菌总DNA并进行PCR扩增。使用Ilumina NovaSeq测序平台对样本中细菌16S rRNA的V3-V4区进行测序。然后基于有效数据在门、属水平上分析肠道菌群的物种结构和丰度，采用QIIME软件（扩增子）分析工具分析回肠和结肠菌群的多样性及差异，同时应用Tax4Fun程序预测回肠及结肠菌群的优势基因富集通路。结果在门水平上，雄性SD大鼠回肠内微生物优势菌群主要是厚壁菌门（Firmicutes）和放线菌门（Actinobacteria），占比超过98%；结肠内微生物优势菌群主要是厚壁菌门（Firmicutes）和拟杆菌门（Bacteroidetes），占比超过95%。在属水平上，回肠以乳酸杆菌属（Lactobacillus）和另枝菌属（Alistipes）为主要优势菌群，结肠以乳酸杆菌属（Lactobacillus）及罗姆布茨菌属（Romboutsia）为优势菌群。在菌群多样性方面，结肠菌群的α多样性包括丰富度指数（Chao1指数）及多样性指数（香农指数Shannon index）都显著高于回肠（P＜0.01），其菌群物种的组成结构差异性比回肠小；回肠菌群结构差异性显著的主要是厚壁菌门（Firmicutes）、罗姆布茨菌属（Romboutsia）和消化链球菌科（Peptostreptococcaceae），而结肠菌群结构差异性显著的是拟杆菌门（Bacteroidetes）、拟杆菌纲（Bacteroidia）和拟杆菌目（Bacteroidales）。在菌群功能方面，回肠菌群优势基因显著富集于类脂物代谢、多酮代谢、膜运输、生物降解等通路，而结肠菌群优势基因显著富集于甘聚糖生物合成代谢、能量代谢、辅助因子和维生素及其他产物的生物合成等通路。结论 SPF级雄性SD大鼠回肠与结肠菌群在结构与丰度上存在显著差异，结肠菌群的丰富度及多样性高于回肠。

关键词: 肠道菌群, 菌群丰度, 回肠, 结肠, SD大鼠

Abstract:

Objective To analyze the microflora structure and abundance of ileum and colon of SPF male SD rats by high-throughput sequencing technique. Methods The ileal and colonic lumen contents of 30 SPF male SD rats were collected, the total bacterial DNA in the contents was extracted and amplified by PCR. The V3-V4 region of bacterial 16S rRNA in the samples was sequenced using Illumina NovaSeq sequencing platform. Then the species structure and abundance of the intestinal flora were analyzed at the phylum and genus level based on validated data. The diversity and differences between ileal and colonic flora were analyzed using the QIIME software (amplicon) analysis tool. The Tax4Fun program was applied to predict the dominant gene enrichment pathways of ileal and colonic flora. Results At the phylum level, the dominant microflora in the ileum of male SD rats were Firmicutes and Actinobacteria, accounting for more than 98%. The dominant colonic microbial community were mainly Firmicutes and Bacteroidetes, accounting for more than 95%. At the genus level, Lactobacillus and Alistipes were the predominant bacteria in the ileum. The colon was dominated by Lactobacillus and Romboutsia. In terms of flora diversity, the alpha diversity of colonic flora including richness index (Chao1 index) and diversity index (Shannon index) were significantly higher than those of ileum (P < 0.01), and the structural variability of the composition of its flora species was smaller than that of ileum; the ileal flora with significant structural variability were mainly of the phylum Firmicutes, Romboutsia, Peptostreptococcaceae, while the colonic flora had significant structural differences in Bacteroidales. In terms of flora function, the ileal flora dominant genes were significantly enriched in lipid-like metabolism, polyketide metabolism, membrane transport, biodegradation and other pathways, while the colonic flora dominant genes were significantly enriched in glycan biosynthesis metabolism, energy metabolism, biosynthesis of cofactor and vitamins and other products, and other pathways. Conclusion There are significant differences in structure and abundance between the ileal and colonic flora of SPF male SD rats, and the abundance and diversity of colonic flora are higher than those of ileal flora.

Key words: Intestinal microflora, Bacteria abundance, Ileum, Colon, SD rats

中图分类号:

Q95-33

许琴, 倪艳, 是文辉, 李建瑛, 刘江伟, 赵红琼, 徐新明. SPF级雄性SD大鼠回肠与结肠菌群结构比较分析[J]. 实验动物与比较医学, 2023, 43(1): 53-60.

Qin XU, Yan NI, Wenhui SHI, Jianying LI, Jiangwei LIU, Hongqiong ZHAO, Xinming XU. Analysis on Ileum and Colon Microflora of SPF Male SD Rats based on High-throughput Sequencing[J]. Laboratory Animal and Comparative Medicine, 2023, 43(1): 53-60.

图/表 4

图1 大鼠回肠、结肠菌群基于OTUs的门、属水平分类注：A，韦恩图，图中粉色圆圈代表结肠（ZJ）独有OTU个数，蓝色圆圈代表回肠（ZH）独有OTU个数，重叠部分代表回肠、结肠共有的OTUs个数；B，门水平的物种相对丰度柱形图；C，属水平物种系统发生关系图。

Figure 1 Classification of phylum and genus of ileum and colon microflora in rats based on operation taxonomic unitsNote：A， Venn graph， in which， the pink circle represents the number of operation taxonomic units （OTU） unique to colon （ZJ）， the blue circle represents the number of OTUs unique to ileum （ZH）， and the overlap part represents the number of OTUs shared by ileum and colon； B， Histogram of relative species abundance at phylum level； C， Phylogenetic graph of genus level species.

表1 大鼠回肠与结肠菌群的α多样性指数统计

Table1 Alpha diversity indices statistics of microflora in ileum and colon of rats

样品名称

Sample name

观察数

Observed number

OTU数

OTU number

香农指数

Shannon index

辛普森指数

Simpson index

Chao1指数

Chao1 index

测序深度

Goods_Coverage

图2 大鼠回肠、结肠菌群多样性分析注：A，基于Weighted Unifrac距离的主坐标分析（PCoA），横坐标表示一个主成分（PC1），纵坐标表示另一个主成分（PC2），百分比表示主成分对样本差异的贡献值。B，线性判别分析（LDA）值分布图。ZJ，结肠；ZH，回肠。

Figure 2 Diversity analysis of microflora in ileum and colon of ratsNote： A， the principal co-ordinates analysis （PCoA） based on the weighted unifrac distance. The abscissa represents one principal component （PC1）， the ordinate represents another principal component （PC2）， and the percentage represents the contribution value of principal component to sample difference. B， the linear discriminant analysis （LDA） value distribution. ZJ， colon； ZH， ileum.

图3 大鼠回肠、结肠菌群功能预测图注：A，Tax4Fun功能注释相对丰度柱形图；B，基于Tax4Fun的功能注释聚类热图。

Figure 3 Functional prediction charts of microflora in ileum and colon of ratsNote：A， Tax4Fun relative abundance histogram of function annotation； B， clustering heat map of function annotation based on Tax4Fun.

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SPF级雄性SD大鼠回肠与结肠菌群结构比较分析

Analysis on Ileum and Colon Microflora of SPF Male SD Rats based on High-throughput Sequencing

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