Gland Of Silkworm Research Articles

Comparative Proteome Analysis Reveals that Cuticular Proteins Analogous to Peritrophin-Motif Proteins are Involved in the Regeneration of Chitin Layer in the Silk Gland of Bombyx mori at the Molting Stage.

The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography-tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR-motif chitin-binding proteins is identified during day 2 of the fourth instar (IV-2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)-motif chitin-binding proteins is higher during the fourth molt (IV-M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP-motif proteins. Furthermore, the expression of CPAP3-G, the most abundant CPAP-motif cuticular protein in the silk gland during the IV-M stage, is investigated using western blot and immunofluorescence analyses; CPAP3-G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP-motif chitin-binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO2 NPs treated and control groups, respectively. Compared with the control, TiO2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO2 NPs promoted silk protein synthesis.

Role of BmDredd during Apoptosis of Silk Gland in Silkworm, Bombyx mori.

Silk glands (SGs) undergo massive apoptosis driven degeneration during the larval-pupal transformation. To better understand this event on molecular level, we investigated the expression of apoptosis-related genes across the developmental transition period that spans day 4 in the fifth instar Bombyx mori larvae to day 2 pupae. Increases in the expression of BmDredd (an initiator caspase homolog) closely followed the highest BmEcR expression and resembled the expression trend of BmIcE. Simultaneously, we found that BmDredd expression was significantly higher in SG compared to other tissues at 18 h post-spinning, but reduced following injection of the apoptosis inhibitor (Z-DEVD-fmk). Furthermore, BmDredd expression correlated with changes of caspase3-like activities in SG and RNAi-mediated knockdown of BmDredd delayed SG apoptosis. Moreover, caspase3-like activity was increased in SG by overexpression of BmDredd. Taken together, the results suggest that BmDredd plays a critical role in SG apoptosis.

Fabrication of nanofibrous silkworm gland three-dimensional scaffold containing micro/nanoscale pores and study of its effects on adipose tissue-derived stem cell growth

An ideal tissue engineering scaffold requires a structure similar to that of the natural extracellular matrix. The structure of a scaffold is the most important parameter for determining the behavior and functions of cells. Therefore, the importance of designing a suitable scaffold has been greatly emphasized. Electrospinning has been extensively used to fabricate scaffolds for tissue engineering. In this study, we developed a nanofibrous scaffold made from silkworm gland (SG), which contains the silk protein fibroin and other proteins that have functions including antigenotoxicity and the promotion of cell growth. To investigate the physical and biological functions of SG nanofibrous (SGN) scaffold, we fabricated silk fibroin nanofibrous (SFN) scaffold as a control lacking the SG functional proteins. Field emission scanning electron microscope images revealed that the SGN three-dimensional scaffold had a homogenous pore distribution with a high porosity and interconnected pore walls. In biological tests, the SGN scaffold supported excellent cell viability and had a stronger antioxidant property compared to the SFN scaffold. In addition, the SGN scaffold efficiently supported the growth of adipose tissue-derived stem cells and their osteogenic and fibrocartilage differentiation. Therefore, this novel SGN three-dimensional scaffold shows high promise for use in tissue engineering.

20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.

Structure, evolution, and expression of antimicrobial silk proteins, seroins in Lepidoptera

The silks of silkworm and waxworm contain abundant antimicrobial proteins, including protease inhibitors and seroins. Protease inhibitors have antifungal activities, whereas seroins have antiviral and antibacterial activities. In order to obtain insights into the structure, evolution, and expression of seroins, we performed an extensive survey based on the available genome, transcriptome, and expressed sequence tags datasets. Sixty-four seroins were identified in 32 lepidopteran species. The phylogenetic and structural analyses revealed that seroins can be classified into five subfamilies: seroin 1, seroin 2, seroin 3, seroin 2 + 1, and seroin 3 + 3. It is interesting that seroin 2 + 1 contains two tandem seroin domains, seroin 2 and seroin 1, whereas seroin 3 + 3 has two tandem seroin 3 domains. Each seroin domain contains a proline-rich N-terminal motif and a conserved C-terminal motif. The transcriptome and EST data indicated that seroin 1 and seroin 2 genes were expressed in the silk gland but seroin 3 genes were not. Semi-quantitative RT–PCR and western blot analyses suggested that seroin 1 and seroin 2 were constantly accumulated in the silk gland of silkworm during the fifth instar, and then secreted into cocoon silk during spinning. Immunofluorescence analyses indicated that seroin 1 was secreted into the fibroin and sericin layers, whereas seroin 2 protein was only secreted into the sericin layer. However, the antimicrobial activity of seroin 2 was more effective than that of seroin 1. The presence of seroin 1 in the fibroin layer suggested that this protein not only acts as an antimicrobial protein, but might also play a role in the assembly and secretion of fibroins. Seroin 3, which was first identified here, might be related to pheromone synthesis or recognition, as it was highly expressed in male antennae and in the pheromone gland.

Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori

Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.

Changes in 30K protein synthesis during delayed degeneration of the silk gland by a caspase-dependent pathway in a Bombyx (silkworm) mutant.

The silk gland in silkworm (Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval-pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval-pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals.

Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons.

With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

Transcriptomic Analysis of the Anterior Silk Gland in the Domestic Silkworm (Bombyx mori) - Insight into the Mechanism of Silk Formation and Spinning.

Silk proteins are synthesized in the middle and posterior silk glands of silkworms, then transit into the anterior of the silk gland, where the silk fibers are produced, stored and processed. The mechanism of formation and spinning of the silk fibers has not been fully elucidated, and transcriptome analyses specific to the anterior silk gland have not been reported. In the present study, we explored gene expression profiles in five regions of silk gland samples using the RNA-Seq method. As a result, there were 959,979,570 raw reads obtained, of which 583,068,172 reads were mapped to the silkworm genome. A total of 7419 genes were found to be expressed in terms of reads per kilobase of exon model per million mapped reads ≥ 5 in at least one sample. The gene numbers and expression levels of the expressed genes differed between these regions. The differentially expressed genes were analyzed, and 282 genes were detected as up-regulated in the anterior silk gland, compared with the other parts. Functions of these genes were addressed using the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases, and seven key pathways were enriched. It suggested that the ion transportation, energy metabolism, protease inhibitors and cuticle proteins played essential roles in the process of silk formation and spinning in the anterior silk gland. In addition, 210 genes were found differently expressed between males and females, which should help to elucidate the mechanism of the quality difference in silk fibers from male and female silkworms.

Reinforced and Ultraviolet Resistant Silks from Silkworms Fed with Titanium Dioxide Nanoparticles

As the perfect combination of strength and luster, silkworm silks have been widely used in many fields but still need improvements. This paper demonstrates an in vivo uptake of titanium dioxide (TiO2) nanoparticles by silkworms, leading to the direct production of intrinsically modified silk. The nanoparticles can be easily incorporated into the silk gland of silkworm by using this method due to the interactions between TiO2 and silk fibroin molecules. Infrared spectra indicate that TiO2 nanoparticles confine the conformation transition of silk fibroin from random coil/α-helix to β-sheet. Results of synchrotron radiation wide-angle X-ray diffraction and small-angle X-ray scattering suggest that modified silks have lower crystallinity, higher mesophase content, and higher Herman’s orientation functions of crystalline region and mesophase region than control group. The breaking strength and elongation at break of the modified silk can be improved up to 548 ± 33 MPa and 16.7 ± 0.8%, respectively, by adding 1...

Spider silk gut: development and characterization of a novel strong spider silk fiber.

Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering.

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.

Naturally functionalized silk as useful material for photonic applications

Silk is a natural fibre obtained from the Bombyx mori silkworm cocoons that can be used in a wide range of fields thanks to its inherent multifunctionality.Post-production steps are necessary to impart colour to the fibres to employ the material for optics and photonic applications, such as in fluorescence-based optofluidic devices in lab-on-a-chip realization.Here we present an intrinsically greener dyeing approach for fabricating naturally functionalized silk, where highly-fluorescent organic dyes with lasing properties are in vivo up-taken by silkworms once introduced in the artificial diet. A detailed photoluminescence spectroscopy investigation is implemented to test whether the dyes are effectively incorporated within the silk proteins, in correlation with the silkworm gland positions where proteins extraction is held. Light amplification characteristics are demonstrated in silk extracted from glands of silkworm fed with artificial diet doped with Rhodamine B dye.

MicroRNA of the fifth-instar posterior silk gland of silkworm identified by Solexa sequencing.

No special studies have been focused on the microRNA (miRNA) in the fifth-instar posterior silk gland of Bombyx mori. Here, using next-generation sequencing, we acquired 93.2 million processed reads from 10 small RNA libraries. In this paper, we tried to thoroughly describe how our dataset generated from deep sequencing which was recently published in BMC genomics. Results showed that our findings are largely enriched silkworm miRNA depository and may benefit us to reveal the miRNA functions in the process of silk production.

The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.

Role of Ca2+ on structures and properties of regenerated silk fibroin aqueous solutions and fibres

It has been known that the concentration of Ca2+ gradually increased along the silk gland of silkworm and it played an important role in the spinning process of silk fibre in vivo. In this work, Raman spectrometer, synchrotron radiation wide angle X-ray diffraction facility and material testing instrument were used to investigate the effect of Ca2+ on the structures and properties of regenerated silk fibroin (RSF) in its aqueous solutions and dry spun fibres. It was found that as the concentration of Ca2+ increased in the RSF aqueous solutions and fibres, the conformational transition to β-sheet was promoted, and the resultant post-treated RSF fibre showed higher crystallinity, higher crystalline orientation as well as better mechanical properties. Therefore, it could be concluded that the addition of Ca2+ had improved the structures of RSF aqueous solutions and the resultant fibres, and it might be a feasible approach to obtaining high performance RSF fibres.

중간엽줄기세포 증식을 위한 누에 실샘 유래 무혈청 배지 첨가제

Mesenchymal stem cells (MSCs) are must be cultured in chemically defined, xeno-free culture system for cell-based therapies. One major overcome for their clinical use is the unsafety of fetal bovine serum (FBS), which is a crucial part of all media currently used for the culture of MSCs. Previously, we developed novel methods the production of silkworm gland hydrolysate (SGH). Also, we showed the effect of the SGH on the cell growth promotion. We demonstrated whether the characteristics of MSCs were maintained in the serum-free media containing SGH during long-term cultivation. The results of microscopic observation showed that the morphology of stem cells were not different between the media containing 10% FBS and 1 mg/mL SGH. After the long-term culture of MSCs in serum-free media containing SGH, the rate of MSCs growth was well maintained. The analysis of anti-apoptotic effect of SGH showed that early apoptosis of MSCs was reduced by 3.7-fold. Furthermore, MSCs specific surface markers and stemness gene expression (Nanog, Sox2) were preserved as that of MSCs in the media containing 10% FBS. In conclusion, we demonstrate that the SGH can be used as useful supplement for MSCs cultivation in serumfree media.

Molecular mechanisms of silk gland damage caused by phoxim exposure and protection of phoxim‐induced damage by cerium chloride in Bombyx mori

It is known that exposure to organophosphorus pesticides (OP) including phoxim can produce oxidative stress, neurotoxicity, and greatly attenuate cocooning rate in the silkworm, Bombyx mori. Cerium treatment has been demonstrated to relieve phoxim-induced toxicity in B. mori; however, very little is known about the molecular mechanisms of silk gland injury due to OP exposure and protection of gland damage due to cerium pretreatment. The aim of this study was to evaluate silk gland damage and its molecular mechanisms in phoxim-induced silkworm toxicity and the protective mechanisms of cerium following exposure to phoxim. The results showed that phoxim exposure resulted in severe gland damage, reductions in protein synthesis and the cocooning rate of silkworms. Cerium (Ce) attenuated gland damage caused by phoxim, promoted protein synthesis, increased the antioxidant capacity of the gland and increased the cocooning rate of B. mori. Furthermore, digital gene expression data suggested that phoxim exposure led to significant up-regulation of 714 genes and down-regulation of 120 genes. Of these genes, 122 were related to protein metabolism, specifically, the down-regulated Ser2, Ser3, Fib-L, P25, and CYP450. Ce pretreatment resulted in up-regulation of 162 genes, and down-regulation of 141 genes, importantly, Ser2, Ser3, Fib-L, P25, and CYP333B8 were up-regulated. Treatment with CeCl3 + phoxim resulted in higher levels of Fib-L, P25, Ser2, Ser3, CAT, TPx, and CYP333B8 expression in the silk gland of silkworms. These findings indicated that Ce increased cocooning rate via the promotion of silk protein synthesis-related gene expression in the gland under phoxim-induced toxicity. These findings may expand the application of rare earths in sericulture.

Effects of environment parameters on sol-gel transition and dry-spinnability of regenerated silk fibroin aqueous solution

Protein concentration, pH, the types and concentrations of metallic ions, and extensional flow are thought to be important environment parameters affecting the natural spinning process. In this study, we investigate the effects of the types and concentrations of metallic ions (Ca2+, Mg2+, and K+ ions), pH, and silk fibroin concentration on the sol-gel transition and the rheological behavior of a regenerated silk fibroin (RSF) aqueous solution. The results show that with an increase in the silk fibroin concentration, the weak acidic RSF aqueous solutions containing Mg2+ or Ca2+ ions undergo a phase transition to a weak gel state. Moreover, the rheological characterization of RSF aqueous solutions shows a dramatic change, and their apparent viscosities increase by almost three orders of magnitude and approach the apparent viscosity of the native dope in the silkworm gland. By using conventional pressure equipment, we investigate the dry-spinnability of weak gels. Further, we observe that the as-spun fibers exhibit a smooth surface and have inferior mechanical properties. The structure of the as-spun fibers is predominantly in a random coil or Silk I conformation.