Study On Gene Expressin Pattern And Regulatory Network Of ?-toxin-induced Bombyx Mori

The silkworm has a long history of breeding in China and is an important part of husbandry.Although modern farming has drastically reduced the cost of farming,various diseases still cause huge economic losses to the sericulture.Bacillus bombyseptieus?Bb?is a common pathogen of the silkworm,which can infect the silkworm and cause bacterial sepsis.In the process of infection,Bb secretes parasporal crystal toxins,damages the silkworm tissues and promotes invasion and infection.The a-toxin is a major bacterial toxin secreted from Bb and is lethal to silkworms.In this study,transcriptome sequencing RNA-seq was used in combination with bioinformatics analysis to analyze gene expression patterns and regulatory networks in the midgut,blood,fatbody and BmE cell line of silkworm before and after a-toxin treatment,in order to elucidate the immune response of silkworm to Bb toxin.1.Analysis of transcriptome data analysisIn order to study the immune response of silkworms induced by a-toxin,a-toxin was used to treat larvae and BmE cells.The midgut,blood,fatbody and BmE cells of silkworms were collected.Three biological replicates were collected and then total RNA was extracted.A strand-specific total RNA transcriptome sequencing library was constructed and transcriptome sequencing was performed.A total of 48 samples were used for sequencing and 157 Gb raw data were obtained,with an average of 30M reads per sample.After data filtering and sequence correction,132 Gb high quality data was obtained,with an average of 23M reads per sample.By calculating the medTIN value of each sample,the integrity of the original sequence was analyzed and the results showed no degradation.Then the quality-controlled reads were aligned to the reference genome in the silkDB database,and the mapping rate of each sample reached about 80%.In summary,the data of all samples meet the quality control requirements and can be used for comparative analysis of genome-wide gene expression patterns.Based on the alignment with the reference genome,the expression level of genes was calculated.The results showed that the distribution of expression in different tissues was similar,that is,most of the genes were expressed at high levels around 32 TMM,and the overall level of gene expression in fatbody,midgut and blood after toxin treatment showed an upward trend.PCA analysis showed that the percentage variance of PC1-3 was:45.06%,26.92%,and 18.07%,respectively,and different tissues were separated from each other and there was no obvious correlation between them,indicating that the gene expression patterns of different tissue samples were significantly different.2.Analysis of differentially expressed genesBased on the negative binomial distribution model,the differential expression probabilities of all genes were calculated,and genes with a change fold over 2 and an FDR value less than 0.05 were defined as differential expressed genes?DEGs?.A total of 952 genes were identified.The number of DEGs in the midgut,fatbody,blood,and BmE cells is 238,555,295,and 32,respectively,and the number of up-regulated genes is 113,344,159,and 16,respectively,and the number of down-regulated genes is 125,211,136,and 16,respectively.The distribution of DEGs in various tissues showed that there were 11 up-regulated genes in the midgut,blood,and fatbody.Changes in expression of 31 genes in different tissues are inconsistent.The above results indicate that the treatment with a-toxins leads to changes in the expression levels of many genes,and there are significant differences in the genes responding to toxin in different tissues.Functional enrichment analysis was performed on DEGs and 89 different GO classes were enriched,including 36 biological processes,46 molecular functions,and 7 cell components.The biological pathways for enrichment of DEGs are mainly:trehalose synthesis pathway,citric acid transport pathway,and catabolic process of peptidoglycan;molecular functions are mainly citric acid transmembrane transport activity,UDP synthesis activity,metalloproteinase activity,and oxygen transport activity;cellular components are mainly extracellular regions,muscle protein complexes,and mitochondrial inner membrane transport protein complexes.The result of enrichment analysis of DEGs in different tissues differed significantly.Up-regulated genes in the midgut were mainly enriched in the tyrosine kinase signaling pathway,down-regulated genes in the midgut were mainly enriched in the chitin metabolic pathway,and up-regulated genes in fatbody were mainly enriched in inositol triphosphate activity.In summary,a-toxins affect many signaling pathways in the silkworms,and there are significant differences in the response patterns of a-toxins in different tissues.3.Analysis of differential expressed lncRNAsTo analyze the immune responses of lncRNAs to a-toxins,the quality-controlled data were aligned to the BmncRNA database including 6281 silkworm IncRNAs.By calculating the level of expression,a total of 1135 lncRNAs has the reads count greater than 5 in each sample,and the expression level was concentrated at 16 TMM,and the overall expression level was relatively low compared to the coding gene.LncRNAs with a fold change greater than 2 and an FDR value less than 0.05 were defined as differential expressed IncRNAs.A total of 353 differentially expressed IncRNAs were identified.The number of differentially expressed IncRNAs in the midgut,blood,fatbody,and BmE cells is 103,85,193 and 19,respectively,and the number of up-regulated IncRNAs is 58,42,87,and 11,respectively,and the number of down-regulated IncRNAs is 45,43,106,and 8,respectively.The expression level of differentially expressed IncRNAs is concentrated at 32 TMM and show a decreasing trend overall in treated samples.By calculating the Jensen-Shannon score,the tissue-specificity of differentially expressed IncRNAs versus the coding genes is determined.The results show that IncRNAs have a higher tissue specificity than the coding genes.4.Co-expression network analysisTo construct a co-expression network,an analysis was performed using expression levels of differentially expressed IncRNAs and DEGs,and two major co-expression modules were identified.One of the modules contained 118 nodes,including 113 genes and 5 IncRNAs.The other module contains 87 nodes,including 83 genes and 4 IncRNAs.The clique analysis results show that the two modules contain a total of 139 cliques?82 and 57,respectively?,with a minimum size of 2 and a maximum of 40.Among them,9 IncRNAs form 51 cliques,and 5 IncRNAs can form multiple cliques,the most being bmlnct₁109 which can form 32 clusters.The largest clique in the module was extracted,resulting in 2 cliques.The results show that one of the cliques had an IncRNA numbered AK382731 while the other clique contain only genes.It suggests that the DEGs may be regulated by lncRNAs,or may perform biological functions together with IncRNAs.The results of enrichment analysis show that the genes in one of the modules are involved in the chitin pathway,and the other module is not enriched in specific biological pathways,but the proteins encoded by the genes in the module associated with the detoxification process of organs in the silkworm,including serine protease,cytochrome P450 and so on.Co-expression network analysis revealed a potential correlation between IncRNAs and the coding gene,providing a reference for functional studies of IncRNAs in the immune response in the silkworm.