Comparative mRNA and LncRNA Analysis of the Molecular Mechanisms Associated With Low Silk Production in Bombyx mori

Abstract

Naked pupa sericin and Naked pupa are two mutant strains of Bombyx mori with extremely low or no fibroin production compared to the Qiufeng and Baiyu strains, both of which exhibit very high silk fibroin production. However, the molecular mechanisms by which long non-coding RNAs regulate fibroin synthesis need further study. In this study, we performed high-throughput RNA-seq to investigate lncRNA and mRNA expression profiles in the posterior silk gland of Qiufeng, Baiyu, Nd-s^D, and Nd silkworms at the third day of the 5th instar. Our efforts yielded 26,767 novel lncRNAs and 6,009 novel mRNAs, the expression levels of silk protein genes and silk gland transcription factors were decreased in Qiufeng vs. Nd-s^D and Qiufeng vs. Nd, while those of many genes related to autophagy, apoptosis, RNA degradation, ubiquitin-mediated proteolysis and heat shock proteins were increased. Moreover, the expression of a large number of genes responsible for protein synthesis and secretion was significantly decreased in Nd. GO and KEGG analysis results showed that nucleotide excision repair, mRNA surveillance pathways, amino acid degradation, protein digestion and absorption, ER-associated degradation and proteasome pathways were significantly enriched for the Qiufeng vs. Nd-s^D and Qiufeng vs. Nd comparisons. In conclusion, our findings contribute to the lncRNA and mRNA database of Bombyx mori, and the identified differentially expressed mRNAs and lncRNAs help to reveal the molecular mechanisms of low silk production in Nd-s^D and Nd, providing new insights for improvement of silk yield and elucidation of silk mechanical properties.

Keywords: Bombyx mori; RNA-seq; lncRNA; posterior silk gland; silk production.

Figure 1

Comparison of Qiufeng, Baiyu, Nd-s^D and Nd. The dissected silk glands of silkworms at I5D3. The white long scale bars mark the PSG (A). The pupa of Qiufeng, Baiyu, Nd-s^D and Nd from left to right (B). The cocoon of Qiufeng, Baiyu, Nd-s^D and Nd from left to right (C). Comparison of the weight of the pupa (D) and cocoon shell (E) of Qiufeng, Baiyu, Nd-s^D and Nd. Sixteen pupae and cocoons were collected for each species including eight males and eight females, and Student's t-test was performed to analyze their pupa and cocoon shell weight differentials. **P < 0.01. Scale bars, 1 cm in (A–C).

Figure 2

Predicted lncRNAs and mRNAs of the PSG in Bombyx mori as filtered with CPC, txCdsPredict, and CNCI and compared with the Pfam database. Venn diagram of the lncRNAs (A). Venn diagram of the mRNAs (B).

Figure 3

Scatter plot of mRNA and lncRNA expression levels. mRNA gene expression levels in Qiufeng vs. Baiyu (A), Qiufeng vs. Nd-s^D (B) and Qiufeng vs. Nd (C). LncRNA gene expression levels in Qiufeng vs. Baiyu (D), Qiufeng vs. Nd-s^D (E) and Qiufeng vs. Nd (F). The red points represent upregulated DELs, the blue points represent downregulated DELs, and the gray points represent genes that were not significantly different between strains.

Figure 4

Hierarchical clustering of mRNAs (A) and lncRNAs (B) in Qiufeng, Baiyu, Nd-s^D and Nd.

Figure 5

Top 20 KEGG pathways of the DEGs in Qiufeng vs. Baiyu (A), Qiufeng vs. Nd-s^D (B) and Qiufeng vs. Nd (C). “Rich factor” indicates the ratio of the number of DEGs enriched in the pathway to the total number of genes annotated in the pathway.

Figure 6

Top 20 KEGG pathways for the target genes of the DELs in Qiufeng vs. Baiyu (A), Qiufeng vs. Nd-s^D (B) and Qiufeng vs. Nd (C). “Rich factor” indicates the ratio of the number of DEGs enriched in the pathway to the number of genes annotated in the pathway.

Figure 7

Pathway enrichment analysis of the differentially expressed mRNAs The enrichment results related to protein processing in the ER in Qiufeng vs. Baiyu (A), Qiufeng vs. Nd-s^D (B) and Qiufeng vs. Nd (C) and the enrichment results related to the proteasome in Qiufeng vs. Baiyu (D), Qiufeng vs. Nd-s^D (E) and Qiufeng vs. Nd (F) are shown.