Silk Gland Of Silkworm Research Articles

Expression system for production of bioactive compounds, recombinant human adiponectin, in the silk glands of transgenic silkworms

Adiponectin is an adipocyte hormone involved in glucose and lipid metabolism. The aim of this study was to develop a human adiponectin expression system in transgenic silkworm using a human adiponectin expression vector. The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human adiponectin in the silk gland of transgenic silkworm, targeting vectors pB-A3-adiponectin-IRES-RFP and pB-Ser1-adiponectin-IRES-RFP were constructed and then introduced into the silkworm pupa. The transgenic silkworms were verified by PCR and then generated. The level of adiponectin in the transgenic silkworm was 6-10 ng/50 mg of freeze-dried powder, and western blotting using an antibody against human adiponectin demonstrated a specific band with a molecular weight of 30 kDa in the silkworm. These results showed that human adiponectin introduced into the silkworm genome was expressed successfully on a large-scale.

Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector

The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior silk glands of gene-targeted silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF-LPA-FibL-R was constructed, harboring a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and silkworm actin 3-promoter-driven gfp. The targeting vector was then introduced into the eggs of silkworm, and the transgenic silkworms were verified by PCR and DNA hybridization after being screened for the gfp gene. Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kD in the silk glands of the G3 generation transgenic silkworms. The level of expression of hGM-CSF in the posterior silk glands of the G3 generation transgenic silkworms was approximately 2.70 ng/g of freeze-dried powdered posterior silk gland. These results showed that the heterologous gene could be introduced into the silkworm genome and expressed successfully. Further more, the exogenous genes existing in the G5 transgenic silkworm identified by PCR confirmed its integration stability. In addition, the silk glands containing expressed hGM-CSF performed the function of significantly increasing leukocyte count of CY-treated mice in a time-and-dose-dependent manner.

The Fluorescent Pigment of Mulberry Transferred in the Body of Silkworm, <i>Bombyx Mori</i>

Using silkworm (Bombyx mori, Dazao strain) as material, the fluorescent pigment of mulberry, silkworm blood, silk gland, cocoon shell, silkworm excrement, silkworm urine and moth urine were analyzed by using the technology of thin layer chromatography (TLC). The results indicated that there were 7 bands of fluorescent pigment from the extracts of green cocoon shell after TLC analysis. While, 2 bands in silkworm blood and its urine extracts, 4 bands in the silk gland extracts, as well as 5 bands in moth urine extracts were detected, but no band detection in the silkworm excrement extracts. The blue-violet fluorescent pigment which has the same Rf (0.35) was detected from the extracts of green cocoon shell, silk gland, silkworm urine, moth urine and mulberry after TLC analysis, but it cannot be found in silkworm blood and silkworm excrement. It was revealed that the blue-violet fluorescent pigment may be transferred directly from mulberry leaves, and then accumulated in the silk gland. Most of the pigment remained in the cocoon shell. And only a small amount of this kind of pigment was excreted through urine. There was also some yellow-green fluorescent pigment detected in the silkworm blood, silk gland and cocoon shell extracts, but it cannot be detected in both the mulberry and silkworm urine extracts. It was suggested that yellow-green fluorescent pigment synthesis pathway exist in the body of silkworm (Bombyx mori, Dazao strain).

MicroRNAs of Bombyx mori identified by Solexa sequencing.

BackgroundMicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. We sequenced three small RNA libraries prepared from the whole body, and the anterior-middle and posterior silk glands of Bombyx mori, with a view to expanding the repertoire of silkworm miRNAs and exploring transcriptional differences in miRNAs between segments of the silk gland.ResultsWith the aid of large-scale Solexa sequencing technology, we validated 257 unique miRNA genes, including 202 novel and 55 previously reported genes, corresponding to 324 loci in the silkworm genome. Over 30 known silkworm miRNAs were further corrected in their sequence constitutes and length. A number of reads originated from the loop regions of the precursors of two previously reported miRNAs (bmo-miR-1920 and miR-1921). Interestingly, the majority of the newly identified miRNAs were silkworm-specific, 23 unique miRNAs were widely conserved from invertebrates to vertebrates, 13 unique miRNAs were limited to invertebrates, and 32 were confined to insects. We identified 24 closely positioned clusters and 45 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters were not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs were located in transposable elements, and displayed significant differences in abundance between the anterior-middle and posterior silk gland.ConclusionsConservative analysis revealed that miRNAs can serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs, and provide insights into miRNA evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior-middle and posterior silk glands supports their involvement as new levels in the regulation of the silkworm silk gland.

Preparation and characterization of regenerated fiber from the aqueous solution of Bombyx mori cocoon silk fibroin

The regenerated silk fibers with high strength and high biodegradability were prepared from the aqueous solution of Bombyx mori silk fibroin from cocoons with wet spinning method. Although the tensile strength of the regenerated silk fibroin fiber, 210 MPa is still half of the strength of native silk fiber, the diameter of the fiber is about 100 μm which is suitable for monofilament of suture together with high biodegradability. The high concentration (30%, w/v) of the aqueous solution of the silk fibroin which corresponds to the high concentration in the middle silkgland of silkworm was obtained. This was performed by adjusting the pH of the aqueous solution to 10.4 which corresponds to p K a value of the OH group of Tyr residues in the silk fibroin. The mixed solvent, methanol/acetic acid (7:3 in volume ratio) was used as coagulant solvent for preparing the regenerated fiber. The structural change of silk fibroin fiber by stretching was monitored with both 13C solid state NMR and X-ray diffraction methods, indicating that the high strength of the fiber is related with the long-range orientation of the silk fibroin chain with β-sheet structure.

New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm

We constructed three different fibroin H-chain expression systems to estimate the efficacy of producing recombinant proteins in the cocoon of transgenic silkworms. The results showed that the three different EGFP/H-chain fusion genes were all expressed selectively in the posterior silk gland of the transgenic silkworm. The recombinant protein content of transgenic silkworm cocoons is up to 15% (w/w) when using the most highly efficient H-chain expression system. To our knowledge, in comparison with silkworm silk gland expression systems in the literature, the highly efficient expression system developed in this study is the most efficient silkworm silk gland expression system to date. This expression system is the best candidate for foreign gene production and for creation of novel functional silk material. The results suggested the N-terminal domain and the intron of the H-chain gene are important in the secretion of fibroin and its transcription, respectively.

Cytopathology of Bombyx mori (Lepidoptera: Bombycidae) silk gland caused by multiple nucleopolyhedrovirus

A multiple nucleopolyhedrovirus previously isolated from infected Bombyx mori L. larvae (BmMNPV) in Paraná state, Brazil, was inoculated into B. mori larvae to examine susceptibility and cytopathology in silk gland cells. The anterior, middle and posterior silk glands were removed from the infected silkworm at different times post-inoculation and processed for cytopathology studies by light and transmission electron microscopy. BmMNPV infection was only detected at 72 h post-inoculation in cells of the middle and posterior silk glands. No sign of infection was found in the anterior silk gland. Cytopathological characteristics were similar to those found in other target tissues; initially, they consisted of nuclear hypertrophy and the formation of virogenic stroma (viroplasm), in which the progeny virions are produced. The virions are then enveloped and occluded in protein crystal structures, the polyhedra. After viral replication, cells undergo lysis and release a great number of BmMNPV polyhedra into the hemocoele. Histopathology showed early infection foci in regions surrounding trachea insertions, demonstrating that trachea is an infection-spreading organ in the insect body. Trachea penetrates the middle and posterior silk gland basal lamina, considered a barrier to virus, facilitating the penetration of budded virus. The anterior silk gland does not have tracheal insertions into the basal lamina, which reduces budded virus infection. Tracheal branches provide a conduit for non-occluded virions (budded virus) to pass through the basal lamina barrier and disseminate BmMNPV in the silkworm silk gland.

Intracellular mobilization of Ca 2+ by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands

20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca 2+ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca 2+ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP 3R) antagonist, and an L- or T-type Ca 2+ channel blocker. The T-type Ca 2+ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca 2+-free medium, indicating that the source of Ca 2+ is an intracellular reservoir. The IP 3R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca 2+ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca 2+ by activating IP 3R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP 3.

Simulation of Flow in the Silk Gland

Spiders and silkworms employ the complex flow of highly concentrated silk solution as part of silk fiber spinning. To understand the role of fluidic forces in this process, the flow of silk solution in the spider major ampullate and silkworm silk glands was investigated using numerical simulation. Our simulations demonstrate significant differences between flow in the spider and silkworm silk glands. In particular, shear flow effects are shown to be much greater in the spider than the silkworm, the silkworm gland exhibits a much different flow extension profile than the spider gland, and the residence time within the spider gland is eight times greater than in the silkworm gland. Lastly, simulations on the effect of spinning speed on the flow of silk solution suggest that a critical extension rate is the initiating factor for fiber formation from silk solution. These results provide new insight into silk spinning processes and will guide the future development of novel fiber spinning technologies.

Effects of UV-light on biochemical composition of silk gland of silkworm, Bombyx mori L. fed an artificial diet

The effects of UV-light on biochemical parameters of posterior silk gland of mulberry silkworm, Bombyx mori L. were investigated in two popular bivoltine hybrids, Jufang (Chinese) × Chenxin (Japanese) and Chenxin × Jufang fed an artificial diet. Larvae of the first day of fifth instar were used for the present investigation. The larvae were irradiated with 20 W of UV-light at the intensity of 40 cm above the larvae for one of three exposure times (30, 60 or 120 min) just before feeding. UV treatment produced no significant effects at 30 min. However, at 60 or 120 min there was a significant decrease in silk gland weight and the levels of total protein, free amino acids, RNA and carbohydrates. UV-light thus affects the biosynthetic activities of silk glands.

Hierarchical cluster analyses and heat map analyses of silkworm tissues using R-statistics

Proteins from silkworm (Bombyx mori L.) silk glands, fat bodies, and hemolymph were analyzed by matrix assisted laser desorption ionization time-of-flight mass spectrometry using sinapinic acid as a matrix. Peak percentages were calculated for each peak from m/z 2,000 to m/z 20,000 against the sum of all observed peak intensities; hierarchical cluster analysis and heat map analysis were performed using R-statistics software. For the silk glands, clusters obtained from MALDI-TOF-MS analysis were consistented with the morphological classification. Hierarchical clustering using MALDI-TOF-MS data was considered applicable to the small and sequential biopsies. In hemolymph, obvious clusters were observed from the anterior part to posterior part, and the proteins in hemolymph were not considered homogeneous throughout the body. While the clusters obtained from fat body analysis were weaker than those of silk glands and hemolymph, three clusters were observed. Based on the protein profile, fat bodies were also considered as differentiated tissue.

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm ( Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm ( Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland (PSG) of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75–80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis (2–DE) and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 (D1) and day 4 (D4) in the 5th instar stage from five different strains of silkworm ( Bombyx mori). While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Nongenomic action of an insect steroid hormone in steroid-induced programmed cell death

Programmed cell death (PCD) of the silkworm silk glands is triggered by the insect steroid hormone, 20-hydroxyecdysone (20E), and proceeds sequentially through cell shrinkage, nuclear condensation, DNA fragmentation, nuclear fragmentation and apoptotic body formation. A protein synthesis inhibitor, cycloheximide (CHX, 2 mM) induced a cell death that exhibited only nuclear and DNA fragmentation. A concentration of 0.2 mM CHX was ineffective at inducing the cell death when added alone, but in the presence of 20E, a cell death similar to that induced by 2 mM CHX was resulted with accompanying nuclear condensation. Since 2 and 0.2 mM CHX inhibited protein synthesis equally, the DNA and nuclear fragmentation appear to be mediated by a nongenomic action of 20E. In addition, we show a possible involvement of Ca 2+–PKC–caspase-3 like protease pathway in the nongenomic action. The data suggest that 20E-induced PCD is accomplished through the integration of genomic and nongenomic actions.

Structural Analysis of Fibroin Heavy Chain Signal Peptide of Silkworm <italic>Bombyx mori</italic>

To study the minimal length required for the secretion of recombinant proteins and silk proteins in posterior silk gland, the signal peptide (SP) of the fibroin heavy chain (FibH) of silkworm Bombyx mori was systematically shortened from the C-terminal. Its effect on the secretion of protein was observed using enhanced green fluorescent protein (EGFP) as a reporter. Secretion of EGFP fusion proteins was examined under fluorescence microscope. FibH SPs with lengths of 20, 18, 16 and 12 a.a. can direct the secretion of the reporter, yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP was shortened to 12 a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP. When 16 a.a. of the FibH SP were used, the secretion of fusion protein was normal and the cleavage site was between the Gly-Ser linker and Met, the starting amino acid of EGFP. These findings are applicable for the expression of foreign proteins in silkworm silk gland. The cleavage site of the SP is discussed and compared with the predictive results of the SignalP 3.0 online prediction program.

Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (Bombyx mori L.) using two-dimensional polyacrylamide gel electrophoresis.

Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4-8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype

Exogenous prostaglandin F 2α as a mediator in the regulation of silkworm growth and silk gland genome

Prostaglandins are locally acting hormones that have remarkable variety of physiological functions. They are rapidly synthesized in several types of vertebrate cells as oxygenated metabolites of arachidonic acid in response to various stimuli. In many insect species they are biosynthesized in fat body and hemocytes mainly in response to bacterial infections. In the present study, we administered synthetic analog of prostaglandin F 2α, the most prominent of the prostaglandins to the 48 h old fifth instar silkworm, Bombyx mori L. at a single dose of 4 μg per larva to study its effects on the larval growth pattern and silk synthesis. The possible role of PGF 2α at altering the quantum of silk synthesis by controlling the silk gene expression was also studied. The genomic DNA was isolated from the posterior silk gland on Days 5 and 7 of the fifth instar from the prostaglandin treated and the control larvae and were random amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of PGF 2α in the silk biosynthesis controlling the silk gene expression. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of PGF 2α analog in the silk biosynthetic process with its physiological and molecular implications are discussed.

Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene , coupled with the constitutive cytoplasmic actin gene A3 promoter , was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.

Liquid crystallinity of natural silk secretions

NATURAL silk exhibits a strength and stiffness similar to, and a toughness up to ten times greater than, that of artificial high-performance fibres1–5. These exceptional tensile properties, the optical birefringence of some silk secretions6–9 and the molecular order exhibited by some synthetic polypeptides in solution10 all suggest that natural silk secretions might form liquid-crystalline phases. We have now used polarized-light microscopy to study the secretions from major ampullae of spiders (Nephila clavipes) and from silk glands of silkworms (Bombyx mori). As the concentration is increased by evaporation of water, nematic liquid-crystalline microstructures develop. We deduce that natural silk secretions become liquid crystalline after leaving the gland but before solidifying into a fibre, thus promoting global molecular alignment in the fibre. Our hand-drawn fibres from droplets of secretion, as well as sheared thin films, show a banded microstructure which is indicative of a periodic variation in the direction of molecular alignment11. Both B. mori and N. clavipes, on the other hand, have apparently developed processing routes that ensure uniform molecular alignment: the threads and draglines, respectively, of these species do not show banded microstructures.

Elongation factor 1 from the silk gland of silkworm: Reconstruction of EF-1 M from its subunits, EF-1a and EF-1bc

Elongation factor 1 from the silk gland of silkworm: Reconstruction of EF-1 M from its subunits, EF-1a and EF-1bc