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

doi:10.12300/j.issn.1674-5817.2022.103

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 4

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.

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

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.

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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