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

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.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4602-4) contains supplementary material, which is available to authorized users.

Mass spider silk production through targeted gene replacement in Bombyx mori

“…Disruption of FibH expression significantly reduced silk production, and the FibH −/− animals only formed very thin cocoon shells ( Fig. 4 A ), similar to previous reports ( 32 , 33 ). In comparison, both MaSp1 +/− and MaSp1 +/+ animals spun silk and formed elliptical cocoon shells ( Fig.…”

CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells

“…These cellular stress responses were also observed in FibH knock-out silkworms using CRISPR/Cas9 system [47]. The loss-offunction mutation caused an accumulation of abnormal FibH protein and aroused the activation of proteasomes as well as autophagy process in the silk gland cells [47], which was consistent with our results for the autophagy assay and suggested that accumulation of secretion-deficient (abnormal or mutant) FibH protein in the silk gland cells might lead to more activation of autophagy process to promote the rapid degradation of such abnormal proteins and in cells. Thus, we suspected that the cellular stress responses might be caused by the secretion-deficient fibroin protein that is encoded by the CRISPR-activated FibH gene in BmE cells, further raising the possibility that secretion-deficient FibH was responsible for the PSG cell atrophy in fibroin-deficient B. mori mutants.…”

“…Therefore, the stressed cells initiate adaptive responses aiming at reducing damage to the cells, such as a decrease in global protein translation and an increase in proteins of the proteolytic system [38,[44][45][46]. These cellular stress responses were also observed in FibH knock-out silkworms using CRISPR/Cas9 system [47]. The loss-offunction mutation caused an accumulation of abnormal FibH protein and aroused the activation of proteasomes as well as autophagy process in the silk gland cells [47], which was consistent with our results for the autophagy assay and suggested that accumulation of secretion-deficient (abnormal or mutant) FibH protein in the silk gland cells might lead to more activation of autophagy process to promote the rapid degradation of such abnormal proteins and in cells.…”

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

“…Ubiquitin is a small and highly conserved polypeptide of 76 amino acids reported in different organisms that is involved in proteins degradation [ 43 ] and (in mammals) in the posttranslational modifications of plasma membrane proteins and voltage-gated sodium channels (Na v ) in a process called ubiquitylation [ 44 ]. Although the role of this protein in spider venoms is not clear (besides the role in silk glands and proteins degradation [ 43 , 45 ]), it may be involved in the Nav activation after envenomation, since P. verdolaga ubiquitin’s similarity and a conserved domain at the α-subunit C-terminal make Na v potential targets for the ubiquitins. The activation of these channels may play an important role during envenomation, facilitating the action of toxins and affecting Na v , which plays a synergic role, the later being previously reported in P. verdolaga [ 10 ].…”