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.

Abnormal overexpression of SoxD enhances melanin synthesis in the Ursa mutant of Bombyx mori.

The pigment and structural color of insects play crucial roles in body protection, ecological adaptation, and signal communication. Epidermal melanization is a common and main coloring pattern, which results in broad phenotypic diversity. Melanin is one of the compounds contributing to dark brown-black pigmentation, which is synthesized from dopamine and tyrosine by the melanin metabolism pathway. The Ursa mutant of the silkworm Bombyx mori is a body-color mutant characterized by excessive melanin pigmentation in the larval epidermis. However, the exact gene responsible for this phenotype remains unclear. Here, we performed positional cloning of the gene responsible for Ursa, which was mapped to an 83-kb region on chromosome 14. The genomic region contains a protein-coding gene encoding a transcription factor, which was designated BmSoxD. The mutation site was determined by analysis of nucleotide sequences of the genomic region corresponding to BmSoxD, which identified a 449-bp transposable sequence similar to that of the B. mori transposon Helitron inserted into the sixth intron. BmSoxD was dramatically overexpressed in the epidermis of Ursa at the end of the molting stage compared with that of wild-type B. mori. Overexpression of BmSoxD led to upregulation of genes involved in the melanin metabolism pathway, whereas knocking down BmSoxD via small interfering RNAs blocked melanin pigment production in the larval epidermis. These data indicate that the mutation in BmSoxD is responsible for the Ursa mutant phenotype. We propose that the transposable sequence insertion causes abnormal overexpression of BmSoxD at the molting stage in the Ursa mutant, resulting in excessive melanin synthesis and its accumulation in epidermal cells.

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

The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.

Effects of Osiris9a on Silk Properties in Bombyx mori Determined by Transgenic Overexpression.

Osiris is an insect-specific gene family with multiple biological roles in development, phenotypic polymorphism, and protection. In the silkworm, we have previously identified twenty-five Osiris genes with high evolutionary conservation and remarkable synteny among several insects. Bombxy mori Osiris9a (BmOsi9a) is expressed only in the silk gland, particularly in the middle silk gland (MSG). However, the biological function of BmOsi9a is still unknown. In this study, we overexpressed BmOsi9a in the silk gland by germline transgene expression. BmOsi9a was overexpressed not only in the MSG but also in the posterior silk gland (PSG). Interestingly, BmOsi9a could be secreted into the lumen in the MSG but not in the PSG. In the silk fiber, overexpressed BmOsi9a interacted with Sericin1 in the MSG, as confirmed by a co-immunoprecipitation assay. The overexpression of BmOsi9a altered the secondary structure and crystallinity of the silk fiber, thereby changing the mechanical properties. These results provide insight into the mechanisms underlying silk proteins secretion and silk fiber formation.

Structural and Mechanical Properties of Silk from Different Instars of Bombyx mori.

Silkworm silk has excellent mechanical properties, biocompatibility, and promising applications in the biomedical sector. Silkworms spin silk at the beginning and end of each of their five instar stages, as well as spinning mature silk after the fifth instar. We evaluated the mechanical properties and structure of 10 kinds of silk fibers from different stages. A tensile test showed that instar beginning silk, instar end silk, and mature silk possess distinct properties. Attenuated total reflectance Fourier-transform infrared spectroscopy and X-ray diffraction results showed that the excellent mechanical properties of instar end silk are attributed to higher ß-sheet content and suitable crystallinity. Liquid chromatography-tandem mass spectrometry showed that P25 protein content in IV-E silk is 2.9× higher than that of cocoon silk. This study can offer guidelines for further biomimetic investigations into the design and manufacture of artificial silk protein fibers with novel function.

Proteomic Analysis of Larval Integument in a Dominant Obese Translucent (Obs) Silkworm Mutant.

The dominant obese translucent (Obs) mutant of the silkworm (Bombyx mori) results in a short and stout larval body, translucent phenotype, and abnormal pigmentation in the integument. The Obs mutant also displays deficiency in ecdysis and metamorphosis. In the present study, to gain an understanding of multiple Obs phenotypes, we investigated the phenotypes of Obs and performed a comparative analysis of the larval integument proteomes of Obs and normal silkworms. The phenotypic analysis revealed that the Obs larvae were indeed short and fat, and that chitin and uric acid content were lower but melanin content was higher in the Obs mutant. Proteomic analysis revealed that 244 proteins were significantly differentially expressed between Obs and normal silkworms, some of which were involved in uric acid metabolism and melanin pigmentation. Twenty-six proteins were annotated as cuticular proteins, including RR motif-rich cuticular proteins (CPR), glycine-rich cuticular protein (CPG), hypothetical cuticular protein (CPH), cuticular protein analogous to peritrophins (CPAPs), and the chitin_bind_3 motif proteins, and accounted for over 84% of the abundance of the total significantly differentially expressed proteins. Moreover, 22 of the 26 cuticular proteins were downregulated in the Obs mutant. Comparative proteomic analysis suggested that the multiple phenotypes of the Obs mutant might be related to changes in the expression of proteins that participate in cuticular formation, uric acid metabolism, and melanin pigmentation. These results could lay a basis for further identification of the gene responsible for the Obs mutant. The data have been deposited to ProteomeXchange with identifier PXD010998.

A Simple Method for the Cross-Section Area Determination of Single Profiled Fibers and Its Application.

One of the critical prerequisites for accurately measuring the mechanical properties of profiled fibers is the precise determination of their cross-sectional areas (CSAs). In this study, a new method is established for determining a single profiled fibers' CSA based on the frozen section method and digital photo, pixel-ratio method (FS-DP). FS-DP is used to obtain a transverse section of a fiber, by acquiring an image of the cross section using optical microscopy or scanning electron microscopy, and then calculating the CSA using Photoshop. Using FS-DP, it was found that the shape of a fiber of silk changes little in a range of 50 µm, but varies considerably over a range of 1 m, while the CSA of cocoon silk (900 m) first increases and then decreases. Mechanical property tests showed that the elongation, strength, elastic modulus, and toughness values of the cocoon silk are consistent with those reported previously. Additionally, FS-DP was also used to observe other profiled fibers. The application tests indicated that FS-DP can be used to quickly and accurately obtain the CSA of a single profiled fiber, and that it is suitable for the large-scale determination and analysis of the mechanical properties of profiled fibers.

Osiris9a is a major component of silk fiber in lepidopteran insects.

In a previous high-throughput proteomics study, it was found that the silkworm cocoon contains hundreds of complex proteins, many of which have unknown functions, in addition to fibroins, sericins, and some protease inhibitors. Osiris was one of the proteins with no known function. In this study, we identified the Osiris gene family members and constructed a phylogenetic tree based on the sequences from different species. Our results indicate that the Osiris9 gene subfamily contains six members; it is specifically expressed in lepidopteran insects and has evolved by gene duplication. An Osiris gene family member from Bombyx mori was designated as BmOsiris9a (BmOsi9a) on the basis of its homology to Drosophila melanogaster Osiris9. The expression pattern of BmOsi9a showed that it was highly expressed only in the middle silk gland of silkworm larvae, similar to Sericin1 (Ser1). BmOsi9a was visualized as two bands in western blot analysis, implying that it probably undergoes post-translational modifications. Immunohistochemistry analysis revealed that BmOsi9a was synthesized and secreted into the lumen of the middle silk gland, and was localized in the sericin layer in the silk fiber. BmOsi9a was found in the silk fibers of not only three Bombycidae species, viz. B. mori, B. mandarina, and B. huttoni, but also in the fibers collected from Saturniidae species, including Antheraea assama, Antheraea mylitta, and Samia cynthia. Although the exact biological function of Osi9a in the silk fibers is unknown, our results are important because they demonstrate that Osi9a is a common structural component of silk fiber and is expressed widely among the silk-producing Bombycidae and Saturniidae insects. Our results should help in understanding the role of Osi9a in silk fibers.

Genome-wide identification and analysis of JHBP-domain family members in the silkworm Bombyx mori.

Juvenile hormone (JH) regulates the insect growth and development. JH appears in the hemolymph bound by a specific glycoprotein, juvenile hormone-binding protein (JHBP), which serves as a carrier to release the hormone to target tissues and cells. However, JHBP family candidates, expression patterns, and functional implications are still unclear. In this study, we identified 41 genes-containing conserved JHBP domains distributed across eight chromosomes of the silkworm Bombyx mori. A phylogenetic tree showed that the silkworm JHBP (BmJHBP) genes could be classified into two major branches and four subfamilies. Microarray data revealed that BmJHBP genes exhibit various expression patterns and are expressed in different tissues, periods, and sexes. The expression of BmJHBP genes was generally higher in the head, integument, midgut, fat body, testis, and ovary than in the anterior of the silk gland (ASG), median of the silk gland (MSG), posterior of the silk gland (PSG), hemocyte, and Malpighian tubule. BmJHBPd2, in particular, was investigated by Western Blotting, and immunofluorescent assay and was found to be highly expressed in the PSG cytoplasm on day 3 of the fifth instar, coinciding with silk production. Taken together, our findings will be useful in improving understanding the complexity of the JHBP family, and will lay the foundation of explaining functional characterization for further research.

Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application.

Natural silk fiber spun by the silkworm Bombyx mori is widely used not only for textile materials, but also for biofunctional materials. In the present study, we genetically engineered an advanced silk material, named hSFSV, using a transgenic silkworm, in which the recombinant human acidic fibroblast growth factor (hFGF1) protein was specifically synthesized in the middle silk gland and secreted into the sericin layer to surround the silk fiber using our previously optimized sericin1 expression system. The content of the recombinant hFGF1 in the hSFSV silk was estimated to be approximate 0.07% of the cocoon shell weight. The mechanical properties of hSFSV raw silk fiber were enhanced slightly compared to those of the wild-type raw silk fiber, probably due to the presence of the recombinant of hFGF1 in the sericin layer. Remarkably, the hSFSV raw silk significantly stimulated the cell growth and proliferation of NIH/3T3 mouse embryonic fibroblast cells, suggesting that the mitogenic activity of recombinant hFGF1 was well maintained and functioned in the sericin layer of hSFSV raw silk. These results show that the genetically engineered raw silk hSFSV could be used directly as a fine biomedical material for mass application. In addition, the strategy whereby functional recombinant proteins are expressed in the sericin layer of silk might be used to create more genetically engineered silks with various biofunctions and applications.

Basic helix-loop-helix transcription factor Bmsage is involved in regulation of fibroin H-chain gene via interaction with SGF1 in Bombyx mori.

Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix-loop-helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells.