Comparative transcriptomes analysis of the wing disc between two silkworm strains with different size of wings

Abstract

Wings of Bombyx mori (B. mori) develop from the primordium, and different B. mori strains have different wing types. In order to identify the key factors influencing B. mori wing development, we chose strains P50 and U11, which are typical for normal wing and minute wing phenotypes, respectively. We dissected the wing disc on the 1st-day of wandering stage (P50D1 and U11D1), 2nd-day of wandering stage (P50D2 and U11D2), and 3rd-day of wandering stage (P50D3 and U11D3). Subsequently, RNA-sequencing (RNA-Seq) was performed on both strains in order to construct their gene expression profiles. P50 exhibited 628 genes differentially expressed to U11, 324 up-regulated genes, and 304 down-regulated genes. Five enriched gene ontology (GO) terms were identified by GO enrichment analysis based on these differentially expressed genes (DEGs). KEGG enrichment analysis results showed that the DEGs were enriched in five pathways; of these, we identified three pathways related to the development of wings. The three pathways include amino sugar and nucleotide sugar metabolism pathway, proteasome signaling pathway, and the Hippo signaling pathway. The representative genes in the enrichment pathways were further verified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The RNA-Seq and qRT-PCR results were largely consistent with each other. Our results also revealed that the significantly different genes obtained in our study might be involved in the development of the size of B. mori wings. In addition, several KEGG enriched pathways might be involved in the regulation of the pathways of wing formation. These results provide a basis for further research of wing development in B. mori.

Fig 1. The wings of pupa and adults of P50 and U11.

(A) The pupa of P50 male (left) and female (right). (B) The pupa of U11 male (left) and male (right). (C) The wings of P50. (D) The wings of U11.

Fig 2. Statistics of transcript numbers and distribution at P50 and U11.

Fig 3. Volcano plot of DEGs.

The red points indicate genes up-regulated in P50, green points indicate the genes down-regulated in P50, and blue points indicate genes for which the log2 fold change was less than 1 (and are therefore not differentially expressed between P50 and U11).

Fig 4. GO classification of the differentially expressed genes.

(A) These genes were annotated as biological processes. (B) These genes were annotated as cellular components. (C) These genes were annotated as molecular functions. The up-regulated genes are marked with a pink bar and down regulated genes are marked with a blue bar.

Fig 5. Quantitative real-time PCR validation for genes.

Blue bars indicate gene expression in P50 and orange bars indicate gene expression in U11. ‘*’ indicates significant difference (0.01<P-value ≤0.05); ‘**’ indicates the most significant difference (P-value<0.01). A-E are related to proteasome enriched genes, F and G are involved in the Hippo signaling pathway, H-J are related to amino sugar and nucleotide sugar metabolism. D1: 1^st-day of wandering stage; D2; 2^nd-day of wandering stage; D3: 3^rd-day of wandering stage.