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. 2015 Feb 6;16(1):60.
doi: 10.1186/s12864-015-1287-9.

Comparative analysis of the silk gland transcriptomes between the domestic and wild silkworms

Shou-Min Fang  1   2 , Bi-Li Hu  3 , Qiu-Zhong Zhou  4 , Quan-You Yu  5 , Ze Zhang  6
Affiliations

Comparative analysis of the silk gland transcriptomes between the domestic and wild silkworms

Shou-Min Fang et al. BMC Genomics. .

Abstract

Background: Bombyx mori was domesticated from the Chinese wild silkworm, Bombyx mandarina. Wild and domestic silkworms are good models in which to investigate genes related to silk protein synthesis that may be differentially expressed in silk glands, because their silk productions are very different. Here we used the mRNA deep sequencing (RNA-seq) approach to identify the differentially expressed genes (DEGs) in the transcriptomes of the median/posterior silk glands of two domestic and two wild silkworms.

Results: The results indicated that about 58% of the total genes were expressed (reads per kilo bases per million reads (RPKM) ≥ 1) in each silkworm. Comparisons of the domestic and wild silkworm transcriptomes revealed 32 DEGs, of which 16 were up-regulated in the domestic silkworms compared with in the wild silkworms, and the other 16 were up-regulated in the wild silkworms compared with in the domestic silkworms. Quantitative real-time polymerase chain reaction (qPCR) was performed for 15 randomly selected DEGs in domestic versus wild silkworms. The qPCR results were mostly consistent with the expression levels determined from the RNA-seq data. Based on a Gene Ontology (GO) enrichment analysis and manual annotation, five of the up-regulated DEGs in the wild silkworms were predicted to be involved in immune response, and seven of the up-regulated DEGs were related to the GO term "oxidoreductase activity", which is associated with antioxidant systems. In the domestic silkworms, the up-regulated DEGs were related mainly to tissue development, secretion of proteins and metabolism.

Conclusions: The up-regulated DEGs in the two domestic silkworms may be involved mainly in the highly efficient biosynthesis and secretion of silk proteins, while the up-regulated DEGs in the two wild silkworms may play more important roles in tolerance to pathogens and environment adaptation. Our results provide a foundation for understanding the molecular mechanisms of the silk production difference between domestic and wild silkworms.

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Figures

Figure 1
Figure 1
Anatomy structures of intact silk glands from the domestic and wild silkworms. The short red lines indicate the boundary regions among the anterior silk gland (ASG), median silk gland (MSG), and posterior silk gland (PSG).
Figure 2
Figure 2
Boxplot of the log transformed RPKM expression values across four silkworms. RPKM: Reads per kilo bases per million reads. The solid horizontal line represents the median, and the box encompasses the lower and upper quartiles.
Figure 3
Figure 3
Hierarchical clustering and Venn diagram of the differentially expressed genes in the silk glands. (A) Hierarchical clustering of the differentially expressed genes, using the RNA-seq data derived from the silk glands of four silkworms based on log10 RPKM values. The blue bands indicate low gene expression quantity; the red bands indicate high gene expression quantity. (B) Venn diagram showing the overlaps between the differentially expressed genes (DEGs) in the domestic and wild silkworms. (C) Venn diagram of the DEGs in the domestic D_CH vs D_CY and and in the wild W_AKBH vs W_AKSQ.
Figure 4
Figure 4
Expression patterns of differentially expressed genes in domestic versus wild silkworms. (A) Expression levels of the differentially expressed genes (DEGs) in four silkworm strains. (B) Tissue expression patterns of the DEGs in different larvae tissues based on microarray data [11]. Twenty-one of the 32 DEGs in domestic versus wild silkworms had probes in the microarray data (Table 3). Hierarchical clustering was performed using Cluster software (http://genome-www.stanford.edu/clustering/) with the average linkage method. F: female; M: male. A/MSG: anterior/median silk gland; PSG: posterior silk gland.
Figure 5
Figure 5
Scatterplot of enriched KEGG pathways for differentially expressed genes between any two silkworms. Rich factor is the ratio of the differentially expressed gene number to the total gene number in a certain pathway. The characters a, b, c, d, e, and f correspond to the comparisons D_CH vs D_CY, W_AKSQ vs W_AKBH, W_AKBH vs D_CH, W_AKBH vs D_CY, W_AKSQ vs D_CH, and W_AKSQ vs D_CY, respectively. The size and color of the dots represent the gene number and the range of the FDR value, respectively. The grey dot indicates a pathway that was not significantly enriched (the FDR value is 0.08).
Figure 6
Figure 6
Quantitative real-time PCR validation of the differentially expressed genes. The relative expression of a candidate gene was normalized against RpL3. For the up-regulated DEGs in the domestic silkworms, the fold-change of each gene was calculated by dividing the relative expression level in the W_AKSQ. For the up-regulated DEGs in the wild silkworms, the fold-change of each gene was calculated by dividing the relative expression level in the D_CH. The data are the average ± standard error of three independent replicated qPCR experiments. An absolute value of fold-change ≥ 2 and one-way analysis of variance analysis (P-value < 0.005) were used to estimate the significance of gene expression changes. Significant differential expressions of genes between any two silkworms were marked by a star.
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