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

Yong Cui,Lei Chen,Hui Xiang,Yongjian Lin,Wen Wang,Qili Feng,Yanan Zhu

doi:10.1186/s12864-018-4602-4

Yong Cui, Lei Chen + Show 5 more

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https://doi.org/10.1186/s12864-018-4602-4

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Abstract

BackgroundExploring 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.ResultsUsing 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.ConclusionsAccumulation 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.

Highlights

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
Silk fibroin which consists of the fibroin heavy chain (Fib-H), fibroin light chain (FibL) and the 25-kD polypeptide proteins, at a molar ratio of 6:6:1 [3], is synthesized in the posterior silk gland (PSG), accumulates and coats with sericin when it goes through the middle silk gland (MSG), and is secreted via the anterior silk gland (ASG) [4]
It is sensible to propose that these extremely abundant transcripts were further translated. These extremely abundant transcripts were further translated to the nonsense proteins with 284aa in length, resulting in accumulation of the abnormal proteins, which could accounts for the activation of synthesis for more proteasomes, as we have observed. Supporting to this inference, we found that the three important heat shock proteins in the Degradation via proteasome requires mark of unneeded protein firstly, by another kind of protein, i.e. ubiquitin, a small regulatory protein found in most tissues of eukaryotic organisms

Summary

Introduction

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. The domestic silkworm (Bombyx mori) is a significant economic insect for synthesizing and spinning cocoon, which is an important material for textiles and industrial applications and biomaterials and cosmetics [1, 2]. The character of cocoon of silkworm is a typical complex trait and the expression of silk protein gene is strictly subject to time and space constraints. The silk glands grow sharply and synthesize silk proteins dramatically [6]. Once the cocoon is formed, the silk glands degenerate during the larva-pupa transition [7]. Autophagy and cathepsins play essential roles in this process and autophagy precedes apoptosis [8, 9]

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