New insight into the mechanism underlying the silk gland biological process by knocking out fibroin heavy chain in the silkworm

Abstract

Background: Exploring whether and how mutation of silk protein contributes to subsequent re-allocation of nitrogen, and impacts on the timing of silk gland degradation, is important to understand silk gland biology. Rapid development and wide application of genome editing approach in the silkworm provide us an opportunity to address these issues.

Results: Using CRISPR/Cas9 system, we successfully performed genome editing of Bmfib-H. The loss-of-function mutations caused naked pupa and thin cocoon mutant phenotypes. Compared with the wild type, the posterior silk gland of mutant showed obviously degraded into fragments in advance of programmed cell death of silk gland cells. Comparative transcriptomic analyses of silk gland at the fourth day of the fifth instar larval stage(L5D4)identified 1456 differential expressed genes (DEGs) between posterior silk gland (PSG) and mid silk gland (MSG) and 1388 DEGs between the mutant and the wild type. Hierarchical clustering of all the DEGs indicated a remarkable down-regulated and an up-regulated gene clade in the mutant silk glands, respectively. Down-regulated genes were overrepresented in the pathways involved in cancer, DNA replication and cell proliferation. Intriguingly, up-regulated DEGs are significantly enriched in the proteasome. By further comparison on the transcriptome of MSG and PSG between the wild type and the mutant, we consistently observed that up-regulated DEGs in the mutant PSG were enriched in protein degrading activity and proteasome. Meantime, we observed a series of up-regulated genes involved in autophagy. Since these protein degradation processes would be normally occur after the spinning time, the results suggesting that these progresses were activated remarkably ahead of schedule in the mutant.

Conclusions: Accumulation of abnormal fib-H protein might arouse the activation of proteasomes as well as autophagy process, to promote the rapid degradation of such abnormal proteins and the silk gland cells. Our study therefore proposes a subsequent process of protein and partial cellular degradation caused by mutation of silk protein, which might be helpful for understanding its impact of the silk gland biological process, and further exploration the re-allocation of nitrogen in the silkworm.

Keywords: Autophagy; Bmfib-H; Bombyx mori; CRISPR/Cas9; Proteasome; Transcriptome.

Fig. 1

Cas9/sgRNA mediated gene editing of fib-H of the silkworm. a Schematic diagram of the sgRNA-targeting sites. The two boxes indicate the two exons of Bmfib-H, and the black line indicates the gene locus. The sgRNA targeting sites S is located on the sense strand of exon-2. F′ and R’ were used to anneal the upstream and downstream regions of the targeting site. The sgRNA targeting sequence is in black, and the protospacer adjacent motif (PAM) sequence is in red. b The different phenotypes of Cas9/sgRNA induced mutations. The left is the normal cocoon, the middle side is the thin-layered cocoon and the right is the naked pupae. c Various types of insertion mutations screened from homozygous mutant silkworms (G3). H1, 19 bp insertion; H2, 13 bp insertion, and H3, 20 bp insertion. Deletions are indicated by hyphens and insertions are shown in green lowercase letters. The PAM sequence is in red. d Comparison of predicted amino acid sequences of H3 and wild type amino acid sequence. The missing amino acids are replaced with dashes. The numbers on the right indicate the amino acid residue positions of the proteins. Premature stop codons are shown in blue letters and black point. e Silk glands of the wild type (WT) and the mutant from the 2nd day to 4th day of 5th larvae stage. All the scale bars represent 1 cm

Fig. 2

Expression patterns of the differentially expressed genes in the MSG and PSG of the wild type and the mutant. a Venn diagram of the differential expressed genes (DEGs) between the mid silk gland and posterior silk gland (b) Venn diagram of the DEGs between the wild type and the mutant in the same tissue. MSG, middle silk gland; PSG, posterior silk gland. c Heatmap and Hierarchical clustering of the differentially expressed genes. d KEGG enrichment pathways for genes that are nearly uniformly down-regulated in both the MSG and PSG of the mutant. e KEGG enrichment pathways for genes that are generally obviously upregulated in both the MSG and PSG of the mutant. Corrected p-value: p-value in hypergeometric test after correction. All pathways had Corrected p-value < 0.05

Fig. 3

Scheme of proteasome pathway. The up-regulated transcripts in the Bmfib-H gene knock-out PSG are indicated by a red frame

Fig. 4

Expression levels of up-regulated DEGs related to protein degradation. a FPKM of the autophagy-related genes (ATG). b FPKM of the two important genes involved in autophagy and proteasome pathway. Ubiquitin-conjugating enzyme E2L functions in targeting a protein for degradation in the proteasome complex. Acid phosphatase is an indirect autophagic marker. *, p < 0.05; **, p < 0.01; ***, p < 0.001