In Experiment 1, the salinity tolerance of saline-tolerant strains has been effectively demonstrated.
Our research team in this experiment we compared the gene expression of six strains of bacteria in normal saline environment and high saline environment to analyze the differential expression genes of bacterial saline tolerance, and analyze the significant enrichment pathway of differential genes, which lays the foundation for differential gene mining from the perspective of gene annotation function. The conditions of transcriptomics and proteomics experiments were determined by experiment 1 (pH=7.0, 10 g/L NaCl and pH=10.0, 60 g/L NaCl).
Differential analysis of gene expression
Genes with significant differences in gene expression levels under different conditions of salinity are called differentially expressed genes (DEGs). Using |log2(FoldChange)|>0 and padj＜0.05 (p-value corrected for multiple hypothesis testing) as the screening criteria for differentially expressed genes, the number of differentially expressed genes in the six strains obtained from the identification was compared, and analyzed in proportion to the number of all the expressed genes (see Table 3.1).
Table 3.1 Statistical table of the number of differentially expressed genes
It can be seen that the six strains of bacteria had 1043, 1567, 654, 1963, 1560 and 1892 differentially expressed genes. Meanwhile, volcano plots were used to analyze the up- and down-regulation of gene expression and the significance level of six strains of salinity-tolerant bacteria in samples from the high salinity group (Group G) and normal salinity group (Group Z) (Figure 3.5).
Fig3.5 Volcano map of DEGs(a:B.casei G20,b:B.haynesii P19,c:M.luteus R17,d:H. bluephagenesis TD01,e:H.campaniensis LS21,f:E.cloacaeRS35)
The volcano diagram visualizes the distribution of DEGs in group Z and group G of the six strains, from which it can be seen that there are 541, 315, 651, 977, 785 and 972 up-regulated differentially expressed genes, and 502, 339, 916, 986, 775 and 920 down-regulated differentially expressed genes in each of the six strains, and there are several DEGs in each of the six strains with large and significant differences in the multiplicity of the change in the difference DEGs
Differential gene GO functional enrichment analysis
In order to determine which biological functions are significantly associated with the differential genes, we performed GO (Gene Ontology) functional significance enrichment analysis, and screened the GO functional entries with significant enrichment of differential genes using the threshold value of padj < 0.05 (p < 0.05 for R17 strain).
The results of enrichment of DEGs in group Z and group G of six strains are shown in Table 3.2.
Table 3.2 GO enrichment entry statistic stable
Differential gene KEGG functional enrichment analysis
In order to identify the most important biochemical metabolic pathways and signaling pathways in which the differential genes are involved, our team conducted KEGG Pathway enrichment analysis by using KEGG (Kyoto Encyclopedia of Genes and Genomes), a comprehensive database that integrates genomic, chemical, and systemic functional information. The KEGG metabolic pathways that were significantly enriched for up- and down-regulated differentially differentiated genes were screened using p < 0.05 as the threshold for significant enrichment, and the number of enrichments is summarized in Table 3.3.
Table 3.3 KEGG enrichment entry statistic stable
Based on the above enrichment results, we plotted the up- and down-regulated.
Fig 3.12 Differential gene KEGG enrichment bubble chart of B.casei G20(a:up-regulated DEGs,b:down-regulatedDEGs)
Fig 3.13 Differential gene KEGG enrichmentbubble chart of B.haynesii P19(a:up-regulated DEGs,b:down-regulatedDEGs)
Fig 3.14 Differential gene KEGG enrichment bubble chart of M.luteus R17(a:up-regulated DEGs,b:down-regulatedDEGs)
Fig 3.15 Differential gene KEGG enrichment bubble chart of H. bluephagenesis TD01 (a. up-regulated DEGs, b: down-regulated DEGs)
Fig 3.16 Differential gene KEGG enrichment bubble chart of H. campaniensis LS21 (a: up-regulated DEGs, b: down-regulated DEGs)
Fig 3.17 Differential gene KEGG enrichment bubble chart of E. cloacae RS35 (a: up-regulated DEGs, b: down-regulated DEGs)
Overall, we found that the six differentially expressed genes were enriched in ribosomes, oxidative phosphorylation, secondary metabolite biosynthesis, nucleic acid synthesis and metabolism, salt metabolism, pyruvate metabolism, carbon metabolism, amino acid synthesis, metabolism and degradation, microbial metabolism and metabolism of 2-oxocarboxylic acid in different environments, suggesting that the regulation of the above metabolic pathways plays an important role in the response to salinity stress in the saline tolerant bacteria. This suggests that the regulation and modification of the above metabolic pathways play an important role in the response of salinity-tolerant bacteria to saline and alkaline stress.
