Developmental and nuclear proteomic signatures characterize the temporal regulation of fibroin synthesis during the last molting-feeding transition of silkworm.

Silkworm fibroins are natural proteinaceous macromolecules and provide core mechanical properties to silk fibers. The synthesis process of fibroins is posterior silk gland (PSG)-exclusive and appears active at the feeding stage and inactive at the molting stage. However, the molecular mechanisms controlling it remain elusive. Here, the silk gland's physiological and nuclear proteomic features were used to characterize changes in its structure and development from molting to feeding stages. The temporal expression profile and immunofluorescence analyses revealed a synchronous transcriptional on-off mode of fibroin genes. Next, the comparative nuclear proteome of the PSG during the last molting-feeding transition identified 798 differentially abundant proteins (DAPs), including 42 transcription factors and 15 epigenetic factors. Protein-protein interaction network analysis showed a "CTCF-FOX-HOX-SOX" association with activated expressions at the molting stage, suggesting a relatively complex and multifactorial regulation of the PSG at the molting stage. In addition, FAIRE-seq verification indicated "closed" and "open" conformations of fibroin gene promoters at the molting and feeding stages, respectively. Such proteome combined with chromatin accessibility analysis revealed the detailed signature of protein factors involved in the temporal regulation of fibroin synthesis and provided insights into silk gland development as well as silk production in silkworms.

Overexpression of bond-forming active protein for efficient production of silk with structural changes and properties enhanced in silkworm.

Silkworm silk exhibits excellent mechanical properties, biocompatibility, and has potential applications in the biomedical sector. This study focused on enhancing the mechanical properties of Bombyx mori silk by overexpressing three bond-forming active proteins (BFAPs): AFP, HSP, and CRP in the silk glands of silkworms. Rheological tests confirmed increased viscoelasticity in the liquid fibroin stock solution of transgenic silkworms, and dynamic mechanical thermal analysis (DMTA) indicated that all three BFAPs participated in the interactions between fibroin molecular networks in transgenic silk. The mechanical property assay indicated that all three BFAPs improved the mechanical characteristics of transgenic silk, with AFP and HSP having the most significant effects. A synchrotron radiation Fourier transform infrared spectroscopy assay showed that all three BFAPs increased the ß-sheet content of transgenic silk. Synchrotron radiation wide-angle X-ray diffraction assay showed that all three BFAPs changed the crystallinity, crystal size, and orientation factor of the silk. AFP and HSP significantly improved the mechanical attributes of transgenic silk through increased crystallinity, refined crystal size, and a slight decrease in orientation. This study opens new possibilities for modifying silk and other fiber materials.