Gland Of Silkworm Research Articles

Fe-reinforced silkworm silk with superstrong mechanical properties for mass production

Silk is a versatile material known for its outstanding properties, including good mechanical performance, luster, and biocompatibility in various fields. However, the growing demand for silk in diverse applications necessitates further improvements in its performance. Traditional methods for improving silk performance face challenges such as low strength, toughness, and limited mass production. Here, we report a novel approach that combines genetic engineering and ion reinforcement to produce silk fibers with exceptional strength and stiffness. By overexpressing iron ion-related protein genes in the anterior silk gland of silkworms, this study successfully increased the iron ion content in the resulting silk. Furthermore, when applied to the large-scale production of hybrid silkworm varieties, the mechanical properties of silk were significantly enhanced, thus facilitating mass production. The resulting silk exhibited remarkable mechanical properties, including a high fracture strength (790.3 MPa), fracture energy (123.2 MJm−3), stretchability (∼21.41 % strain) and Young’s modulus (15.41 GPa). Comprehensive analysis involving dynamic simulation, synchrotron radiation infrared spectroscopy, X-ray diffraction, fluorescence spectroscopy, and circular dichroism analysis confirmed the specific interaction between iron ions and tyrosine within the silk protein. This interaction promoted the formation of increased the β-sheet and crystal contents in the silk fibers, contributing to their improved mechanical properties. Importantly, this concept can be extended to other metal ions, offering a promising method for enhancing silk structure and mechanical properties without changing the silk protein sequence.

Mutation in the Bombyx mori BmGMC2 gene impacts silk production and silk protein synthesis

Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2, differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers.

Physiological and transcriptomic responses of silkworms to graphene oxide exposure

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis–Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

Bombyx moriSuppressor of Hairless is involved in the regulation of the silkworm cell cycle and endoreplication of the silk glands

The Notch signaling pathway is important in cell cycle regulation and cell proliferation. The transcriptional repressor Suppressor of Hairless [Su(H)] is a molecular switch for downstream target genes of the Notch signaling pathway but the regulatory mechanism of the Su(H) gene in the cell cycle is unclear. We determined the function of the Notch signaling pathway and Bombyx mori Su(H) [BmSu(H)] in the regulation of the silkworm cell cycle. Inhibition of Notch signaling promoted the replication of DNA in silkworm gland cells and expression of the BmSu(H) gene was significantly reduced. Overexpression of the BmSu(H) gene inhibited DNA replication and cell proliferation of silkworm cells, whereas knockout of the BmSu(H) gene promoted DNA replication and cell proliferation. Knockout of the BmSu(H) in silkworms improved the efficiency of silk gland cell endoreplication and increased important economic traits. We demonstrated that BmSu(H) protein can directly bind to the promoters of BmCyclinA, BmCyclinE and BmCDK1 genes, inhibiting or promoting their transcription at the cell and individual level. This study identified molecular targets for genetic improvement of the silkworm and also provided insights into the regulatory mechanism of the cell cycle.

Identification and function of the Pax gene Bmgsb in the silk gland of Bombyx mori.

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development.

Identification and characterization of extrachromosomal circular DNA in the silk gland of Bombyx mori.

The silk gland cells of silkworm are special cells which only replicate DNA in the nucleus without cell division throughout the larval stage. The extrachromosomal circular DNAs (eccDNAs) have not yet been reported in the silk gland of silkworms. Herein, we have explored the characterization of eccDNAs in the posterior silk gland of silkworms. A total of 35346 eccDNAs were identified with sizes ranging from 30 to 13569549bp. Motif analysis revealed that dual direct repeats are flanking the 5' and 3' breaking points of eccDNA. The sequences exceeding 1kb length in eccDNAs present palindromic sequence characteristics flanking the 5' and 3' breaking points of the eccDNA. These motifs might support possible models for eccDNA generation. Genomic annotation of the eccDNA population revealed that most eccDNAs (58.6%) were derived from intergenic regions, whereas full or partial genes were carried by 41.4% of eccDNAs. It was found that silk protein genes fib-H, fib-L, and P25, as well as the transcription factors SGF and sage, which play an important regulatory role in silk protein synthesis, could be carried by eccDNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the genes carried by eccDNAs were mainly associated with the development and metabolism-related signaling pathways. Moreover, it was found that eccDNAfib-L could promote the transcription of fib-L gene. Overall, the results of the present study not only provide a novel perspective on the mechanism of silk gland development and silk protein synthesis but also complement previously reported genome-scale eccDNA data supporting that eccDNAs are common in eukaryotes.

Dynamic Changes and Characterization of the Metal Ions in the Silk Glands and Silk Fibers of Silkworm

Metal ions are involved in the conformational transition of silk fibroin and influence the structure and mechanical properties of silk fibers. However, the dynamic characteristics of metal ions during the formation of silk fibers remain unclear. In this study, we found that the silk glands of silkworms contain various metal elements, with varying levels of the metal elements in different zones of the glands and higher levels in the anterior silk glands. Additionally, the content of various metallic elements in the silk glands varied greatly before and after spinning, similar to their content in different cocoon layers, thus, indicating that the anterior silk glands maintain a certain metal ion environment for the transport and conformational transformation of the silk proteins. Most of the metallic elements located in fibroin were confirmed using degumming experiments. For the first time, a scanning electron microscope energy spectrometry system was used to characterize the metal elements in the cross-section of silk and cocoons. These findings have deepened our understanding of the relationship between the overall metal ion environment and silk fiber formation and help us further conceptualize the utilization of metal ions as targets to improve the mechanical properties of the silk fibers.

The tissue-specific expression of silkworm cuticle protein gene ASSCP2 is mediated by the Sox-2 transcription factor

The silk gland of silkworm is a unique organ in which silk proteins are synthesized, secreted, and transformed into fibers. The anterior silk gland (ASG) is located at the end of the silk gland, and is thought to be involved in silk fibrosis. In our previous study, a cuticle protein, ASSCP2, was identified. This protein is specifically and highly expressed in the ASG. In this work, the transcriptional regulation mechanism of ASSCP2 gene was studied by a transgenic route. The ASSCP2 promoter was analyzed, truncated sequentially, and used to initiate the expression of EGFP gene in silkworm larvae. After egg injection, seven transgenic silkworm lines were isolated. Molecular analysis revealed that the green fluorescent signal could not be detected when the promoter was truncated to −257 bp, suggesting that the −357 to −257 sequence is the key region responsible for the transcriptional regulation of the ASSCP2 gene. Furthermore, an ASG specific transcription factor Sox-2 was identified. EMSA assays showed that Sox-2 binds with the −357 to −257 sequence, and thus regulates the tissue-specific expression of ASSCP2. This study on the transcriptional regulation of ASSCP2 gene provides theoretical and experimental basis for further studies of the regulatory mechanism of tissue-specific genes.

Genetically engineered Blue silkworm capable of synthesizing natural blue pigment

Synthetic biology is an eco-friendly and sustainable approach for the production of compounds, particularly used when the production processes involve toxic reagents. In this study, we used the silk gland of silkworm to produce indigoidine, a valuable natural blue pigment that cannot be synthesized naturally in animals. We genetically engineered these silkworms by integrating the indigoidine synthetase (idgS) gene from S. lavendulae and the PPTase (Sfp) gene from B. subtilis into the silkworm genome. In the resulting Blue silkworm, indigoidine was detected at a high level in the posterior silk gland (PSG), spanning all developmental stages from larvae to adults, without affecting silkworm growth or development. This synthesized indigoidine was secreted from the silk gland and subsequently stored in the fat body, with only a small fraction being excreted by the Malpighian tubule. Metabolomic analysis revealed that Blue silkworm efficiently synthesized indigoidine by upregulating l-glutamine, the precursor of indigoidine, and succinate, which is related to energy metabolism in the PSG. This study represents the first synthesis of indigoidine in an animal and therefore opens a new avenue for the biosynthesis of natural blue pigments and other valuable small molecules.

GASTROPROTECTIVE EFFECT OF CRUDE SERICIN POWDER, AGAINST ASPIRIN INDUCED GASTRIC MUCOSAL INJURIES IN RATS

Crude Sericin Powder, derived from the silk gland of silkworm (Bombyx mori) has many biomedical applications due to its antioxidant properties. In this present study , to evaluate the gastro-protective consequences of hydroxyl amino acid rich sericin solution on aspirin induced gastric mucosal injuries in albino rat models were tested following Institutional Ethical committee rules and regulations .In our experiments, 36 male Holtzman strain adult rats were considered and divided into Control group, Crude sericin treated group, Crude Sericin Pre-treated control group, Crude Sericin Pre-treated Aspirin treated group, Omeprazole treated control group and Omeprazole Pre-treated experiment group. AST, ALT and ALP, SOD, CAT, LPO, and GSH were measured of all groups. Crude Sericin Pre-treated group shows increase in UI, GSH, ALT ALP and AST activities, whereas it shows decrease in CAT and SOD activities.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

Fiber Formation and Mechanical Properties of Bombyx mori Silk Are Regulated by Vacuolar-Type ATPase.

The mechanism of silk fiber formation in silkworms, Bombyx mori, is of particular scientific interest because it is closely related to the mechanical properties of silk fibers. However, there are still substantial knowledge gaps in understanding the details of this mechanism. Studies have found a pH gradient in the silk gland of silkworms. A vacuolar-type ATPase (V-ATPase) is thought to be involved in establishing this pH gradient. Although it is reported that the pH gradient plays a role in silk fibrillogenesis, the direct relationship between V-ATPase and silk mechanical properties is unclear. Thus, this study aims to clarify this relationship. We found that V-ATPase is highly and stably expressed in the anterior silk gland (ASG) and maintains the pH gradient and the fine structure of ASG. Inhibition of V-ATPase activity increased the β-sheet content and crystallinity of silk fibers. Tensile testing showed that the mechanical properties of silk fibers improved after inhibiting V-ATPase activity. All the data suggest that V-ATPase is a key factor in regulating silk fibrillogenesis and is related to the final mechanical properties of the silk fibers. V-ATPase is a potential target for silk mechanical property improvement.

High mechanical property silk produced by transgenic silkworms expressing the spidroins PySp1 and ASG1

Spider silk is one of the best natural fibers with excellent mechanical properties; however, due to the visual awareness, biting behavior and territory consciousness of spiders, we cannot obtain spider silk by large-scale breeding. Silkworms have a spinning system similar to that of spiders, and the use of transgenic technology in Bombyx mori, which is an ideal reactor for producing spider silk, is routine. In this study, the piggyBac transposon technique was used to achieve specific expression of two putative spider silk genes in the posterior silk glands of silkworms: aggregate spider glue 1 (ASG1) of Trichonephila clavipes (approximately 1.2 kb) and two repetitive units of pyriform spidroin 1 (PySp1) of Argiope argentata (approximately 1.4 kb). Then, two reconstituted spider silk-producing strains, the AG and PA strains, were obtained. Finally, the toughness of the silk fiber was increased by up to 91.5% and the maximum stress was enhanced by 36.9% in PA, and the respective properties in AG were increased by 21.0% and 34.2%. In summary, these two spider genes significantly enhanced the mechanical properties of silk fiber, which can provide a basis for spidroin silk production.

Methacrylated Silk Fibroin Hydrogels: pH as a Tool to Control Functionality.

The last decade has witnessed significant progress in the development of photosensitive polymers for in situ polymerization and 3D printing applications. Light-mediated sol-gel transitions have immense potential for tissue engineering applications as cell-laden materials can be crosslinked within minutes under mild environmental conditions. Silk fibroin (SF) is extensively explored in regenerative medicine applications due to its ease of modification and exceptional mechanical properties along with cytocompatibility. To efficiently design SF materials, the in vivo assembly of SF proteins must be considered. During SF biosynthesis, changes in pH, water content, and metal ion concentrations throughout the silkworm gland divisions drive the transition from liquid silk to its fiber form. Herein, we study the effect of the glycidyl-methacrylate-modified SF (SilkMA) solution pH on the properties and secondary structure of SilkMA hydrogels by testing formulations prepared at pH 5, 7, and 8. Our results demonstrate an influence of the prepolymer solution pH on the hydrogel rheological properties, compressive modulus, optical transmittance, and network swellability. The hydrogel pH did not affect the in vitro viability and morphology of human dermal fibroblasts. This work demonstrates the utility of the solution pH to tailor the SilkMA conformational structure development toward utility and function and shows the need to strictly control the pH to reduce batch-to-batch variability and ensure reproducibility.

Effects of transgenic silk materials that incorporate FGF-7 protein microcrystals on the proliferation and differentiation of human keratinocytes.

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor‐7 (FGF‐7) fused with the polyhedron‐encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF‐7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF‐7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF‐7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF‐7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF‐7‐polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.

Fiber Length of Eri Silk Cocoons and Influence of Staple Cutting during Spinning Preparatory Process

ABSTRACT The range of single fiber length in both white and brick red eri cocoons is very high due to inactivity of Filipi’s gland of silkworm along with open structure of cocoons’ shells. This wide range can be reduced by staple cutting process after cocoons’ opening. The average fiber length is improved with more value of staple cut length. Due to staple cutting, short fiber generation occurred and descending trend was noticed with increase of staple cut length. Hauteur value is enhanced with increase of staple cut length and significant difference exists between 100 mm and 200 mm staple. Similarly, higher proportion of longer fiber percentage was revealed with increase of staple length. Since there is restriction of long fiber up to 200 mm for setting of rollers in drafting zone of speed frame and ring frame in case of long staple spinning, length can be set between 100 mm and 150 mm. Due to difference in measurement principle, the average fiber length assessed by BISFA oil plate method and Hauteur and Barbe evaluated by Almeter is not similar. So, expressions have been derived for prediction of Hauteur as well as Barbe from fiber length measured by BISFA oil plate method.

Induction of ER stress, antioxidant and detoxification response by sublethal doses of chlorantraniliprole in the silk gland of silkworm, Bombyx mori

Sublethal doses of chlorantraniliprole (CAP) disrupt spinning disorder in the silkworm Bombyx mori (B. mori) and cause reduced cocoon production. In the present study, we investigated the effects of trace amounts of CAP on morphology and gene expression of the B. mori silk gland, found the posterior silk gland cells were possessed of disintegrated Endoplasmic reticulum (ER), unevenly distributed chromatin after exposure to CAP (0.01 mg/L). Gene expression analysis revealed that IRE1 and ATF6 ER stress-signaling pathways were inhibited, the PERK/CncC pathway was activated. Digital gene expression (DGE) analysis showed that detoxification-related genes, antioxidant genes and genes involved in ER protein processing pathway were expressed differentially in CAP-treated silkworm larvae. Notably, the transcript levels of the detoxification-related genes (CYP4M5, CYP6AB4, GSTD3 and GSTS1) and the antioxidant genes (CAT, TPX and SOD) were significantly increased, and the expression of ER protein processing-related genes (Sec61β, Sec61γ, Sec23α and ERGIC-53) was significantly decreased after CAP exposure. The results showed that sublethal doses of CAP exposure caused ER stress, oxidative damage to the silk gland and the perturbation of protein processing in ER, thereby probably leading to abnormal growth of the silk gland and triggering the spinning failure in silkworm.

Facile synthesis of 3D silk fibroin scaffolds with tunable properties for regenerative medicine

Silk fibroin (SF) porous scaffolds provide mechanical support and biochemical signals to encourage cell attachment and modify biological performance. The available methods for the preparation of SF scaffolds are still required. The crosslinkers used are likely to impact the biocompatibility. Herein, water-insoluble SF scaffolds were prepared by physical methods. The phosphate solution promoted SF molecules aggregate from SF/heparin mixed solution. Then SF scaffolds were prepared in centrifuge tubes under different centrifugal speed. The phosphate was leached from the scaffolds, leaving porous structure. The centrifugal force produced shear-induced silk crystallinity to tune the mechanical performance like the natural silkworm gland. The relationship between performance and second structure of the scaffolds have been revealed by X-ray Diffraction (XRD) and deconvoluting Fourier Transform Infrared spectroscopy (FTIR). Due to changes in the β-sheet content, pore structure, mechanical strength, and drug-loaded behavior was adjustable. The scaffolds performed excellent on the Activated Partial Thromboplastin Time (APTT) value, and it can keep sustainable released for 7 days. The scaffolds prepared in mild environment showed tunable stiffness, good anticoagulation, and improved cell compatibility, suggesting its potential application in regenerative medicine.

Genetic Manipulation of MicroRNAs in the Silk Gland of Silkworm, Bombyx Mori

BackgroundMicroRNAs (miRNAs) are a class of non-coding RNAs with important post-transcriptional regulatory functions. To reveal the function of miRNAs in vivo, the critical step is to change their expression levels in the tissues or organs. In this work, we explored the application of several important genetic techniques in altering the expression of silk gland-specific miR-274 of silkworm (Bombyx mori).ResultsInjection of synthesized microRNA mimics and antagomirs exerted no effect on the expression of miR-274 in the silk gland, miR-274 sponge specifically absorbed miR-274 and down-regulated its expression, transgenic overexpression of miR-274 precursor significantly up-regulated miR-274, and finally tissue-specific CRISPR/Cas9 system achieved deletion of miR-274.ConclusionsA practical technical system was established for studying the functions of miRNAs in silk gland of Bombyx mori. Our research provides methodological support for the functional study of miRNAs and other noncoding RNAs in the silk gland and more organs in other species.

A silkworm based silk gland bioreactor for high-efficiency production of recombinant human lactoferrin with antibacterial and anti-inflammatory activities

BackgroundSilk glands are used by silkworms to spin silk fibers for making their cocoons. These have recently been regarded as bioreactor hosts for the cost-effective production of other valuable exogenous proteins and have drawn wide attention.ResultsIn this study, we established a transgenic silkworm strain which synthesizes the recombinant human lactoferrin (rhLF) in the silk gland and spins them into the cocoon by our previously constructed silk gland based bioreactor system. The yield of the rhLF with the highest expression level was estimated to be 12.07 mg/g cocoon shell weight produced by the transgenic silkworm strain 34. Utilizing a simple purification protocol, 9.24 mg of the rhLF with recovery of 76.55% and purity of 95.45% on average could be purified from 1 g of the cocoons. The purified rhLF was detected with a secondary structure similar with the commercially purchased human lactoferrin. Eight types of N-glycans which dominated by the GlcNAc (4) Man (3) (61.15%) and the GlcNAc (3) Man (3) (17.98%) were identified at the three typical N-glycosylation sites of the rhLF. Biological activities assays showed the significant evidence that the purified rhLF could relief the lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells and exhibit potent antibacterial bioactivities against the Escherichia coli (E. coli) and Bacillus subtilis.ConclusionsThese results show that the middle silk gland of silkworm can be an efficient bioreactor for the mass production of rhLF and the potential application in anti-inflammation and antibacterial.