Deep Insight into the Transcriptome of the Single Silk Gland of Bombyx mori

Abstract

The silk gland synthesizes and secretes a large amount of protein and stores liquid silk protein at an extremely high concentration. Interestingly, silk proteins and serine protease inhibitors are orderly arranged in the silk gland lumen and cocoon shells. Silk fiber formation and the spinning mechanism have not been fully elucidated. Therefore, we conducted a comparative transcriptome analysis of seven segments of the single silk gland to characterize internal changes in the silk gland during the 5th instar of mature larvae. In total, 3121 differentially expressed genes were identified in the seven segments. Genes highly expressed in the middle silk gland (MSG) were mainly involved in unsaturated fatty acid biosynthesis, fatty acid metabolism, apoptosis-fly, and lysosome pathways, whereas genes highly expressed in the posterior silk gland (PSG) were mainly involved in ribosome, proteasome, citrate cycle, and glycolysis/gluconeogenesis pathways. Thus, the MSG and PSG differ greatly in energy source use and function. Further, 773 gradually upregulated genes (from PSG to MSG) were involved in energy metabolism, silk protein synthesis, and secretion, suggesting that these genes play an important role in silk fiber formation. Our findings provide insights into the mechanism of silk protein synthesis and transport and silk fiber formation.

Keywords: Bombyx mori; gradually upregulated genes; single silk gland; transcriptome.

Figure 1

Overview of samples and transcriptome data. (A) Silk gland at full maturation of 5th instar. The single silk gland was divided into MSG-A (anterior segment of the middle silk gland), MSG-M (middle segment of the middle silk gland), MSG-P (posterior segment of the middle silk gland), PSG-1, PSG-2, PSG-3, and PSG-4. Red boxes indicate where the samples were collected. (B) Pearson correlation analysis of samples. The number is the correlation coefficient between each two samples. (C) Heatmap of silk protein-related genes, including silk protein genes, transcription factors, and protease inhibitors.

Figure 2

Cluster analysis of all differentially expressed genes (DEGs) between single silk gland samples. (A) Hierarchical clustering of DEGs. Red, high expression level; Green, low expression level. DEGs, differentially expressed genes. (B) Coexpression analysis of DEGs. Red, high correlation; yellow, low correlation.

Figure 3

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene ontology (GO) classification of high expression genes in the MSG and PSG. (A,C) KEGG pathway enrichment analysis of high expression genes in the MSG (A) and PSG (C), respectively. “*” (qvalue < 0.05) represents a significantly enriched pathway. MSG, middle silk gland; PSG, posterior silk gland. (B,D) GO scatterplot constructed with REVIGO for high expression genes in the MSG (B) and PSG (D), respectively. Colors represent the p-value according to the legend. Bubble size reflects the frequency of each GO term.

Figure 4

Cluster and KEGG enrichment analysis of MSG-A highly expressed genes. (A) Hierarchical clustering of high expression genes in the MSG-A. (B) KEGG pathway enrichment analysis of high expression genes in the MSG-A. “*” (qvalue < 0.05) represents a significantly enriched pathway.

Figure 5

Functional analysis of gradually upregulated genes. (A,B) GO scatterplot constructed with REVIGO for gradually upregulated genes related to biological process (A) and molecular function (B). (C) KEGG pathway enrichment analysis of gradually upregulated genes in cluster 2 and cluster 8. “*” (qvalue < 0.05) represents a significantly enriched pathway. (D) Detection of the peripheral pH of the silk gland by ion-selective microelectrode (ISM).

Figure 6

Quantitative PCR verification of partial gradually upregulated genes. “*” stands for p-value < 0.05, “**” stands for p-value < 0.01, and “***” stands for p-value < 0.001.