Search Results (9)

Spider silk is part of a special class of natural protein fibers that have high strength and toughness: these materials have excellent comprehensive properties that are not found in other natural fibers (including silk) or most synthetic fibers. Spider egg case filaments have good hardness, can resist water, can protect spider eggs from external threats, have a significantly high initial modulus and high moisture absorption rate, and are expected to be used as a new generation of environmentally friendly natural polymer fibers and biomaterials. However, spiders are predatory and difficult to rear in large numbers, and it is also difficult to obtain spider egg case filaments in large quantities. Silkworms and spiders have a similar spinning system, and the use of transgenic technology in silkworms can obtain stable and high-yield exogenous gene proteins for a long time, representing an ideal bioreactor for the production of spider silk. In this study, the eukaryotic bioreactor and piggyBac transposon system were employed to recombinantly introduce the egg case silk protein of Nephila clavata (Nc-CYSP1) into the silkworm in the silkworm heavy-chain expression system. The results revealed that the silk glands produced a new type of transgenic silk with a significantly high initial modulus and high moisture absorption. In summary, this study provides an experimental reference for future research on the large-scale production and application of spider egg case filamentous protein, with great application prospects in the development of new environmentally friendly materials. Full article

The efficient production of silkworm silk is crucial to the silk industry. Silk protein synthesis is regulated by the juvenile hormone (JH) and 20-Hydroxyecdysone (20E). Therefore, the genetic regulation of silk production is a priority. JH binding protein (JHBP) transports JH from the hemolymph to target organs and cells and protects it. In a previous study, we identified 41 genes containing a JHBP domain in the Bombyx mori genome. Only one JHBP gene, BmJHBPd2, is highly expressed in the posterior silk gland (PSG), and its function remains unknown. In the present study, we investigated the expression levels of BmJHBPd2 and the major silk protein genes in the high-silk-producing practical strain 872 (S872) and the low-silk-producing local strain Dazao. We found that BmJHBPd2 was more highly expressed in S872 than in the Dazao strain, which is consistent with the expression pattern of fibroin genes. A subcellular localization assay indicated that BmJHBPd2 is located in the cytoplasm. In vitro hormone induction experiments showed that BmJHBPd2 was upregulated by juvenile hormone analogue (JHA) treatment. BmKr-h1 upregulation was significantly inhibited by the overexpression of BmJHBPd2 (BmJHBPd2OE) at the cell level when induced by JHA. However, overexpression of BmJHBPd2 in the PSG by transgenic methods led to the inhibition of silk fibroin gene expression, resulting in a reduction in silk yield. Further investigation showed that in the transgenic BmJHBPd2OE silkworm, the key transcription factor of the JH signaling pathway, Krüppel homolog 1 (Kr-h1), was inhibited, and 20E signaling pathway genes, such as broad complex (Brc), E74A, and ultraspiracle protein (USP), were upregulated. Our results indicate that BmJHBPd2 plays an important role in the JH signaling pathway and is important for silk protein synthesis. Furthermore, our findings help to elucidate the mechanisms by which JH regulates silk protein synthesis. Full article

The transgenesis of silkworms is an important way to innovate genetic resources and silk function. However, the silk-gland (SG) of transgenic silkworms, which is the most concerned target tissue of sericulture, often suffers from low vitality, stunting and other problems, and the reasons are still unknown. This study trans engineered recombinant Ser3, a middle silk gland (MSG) specific expression gene, in the posterior silk gland (PSG) of the silkworm, and studied hemolymph immune melanization response changes in mutant pure line SER (Ser3^+/+). The results showed that although the mutant had normal vitality, the melanin content and phenoloxidase (PO) activity in hemolymph related to humoral immunity were significantly reduced, and caused significantly slower blood melanization and weaker sterilization ability. The mechanism investigation showed that the mRNA levels and enzymatic activities of phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and dopamine decarboxylase (DDC) in the melanin synthesis pathway in mutant hemolymph, as well as the transcription levels of the PPAE, SP21 and serpins genes in the serine protease cascade were significantly affected. Moreover, the total antioxidant capacity, superoxide anion inhibition capacity and catalase (CAT) level related to the redox metabolic capacity of hemolymph were significantly increased, while the activities of superoxide dismutase (SOD) and glutathione reductase (GR), as well as the levels of hydrogen peroxide (H₂O₂) and glutathione (GSH), were significantly decreased. In conclusion, the anabolism of melanin in the hemolymph of PSG transgenic silkworm SER was inhibited, while the basic response level of oxidative stress was increased, and the hemolymph immune melanization response was decreased. The results will significantly improve the safe assessment and development of genetically modified organisms. Full article

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1–5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses. Full article

The expression of trehalase in the midgut of insects plays an important role in glucose supply to the hemolymph. Energy metabolism is usually regulated by the estrogen-related receptor (ERR). A decrease in ATP levels is caused by the ERR hindering glycolysis. However, the relationship between trehalose accumulation and ERR expression is still unclear. Here, we found that silkworm ERR (BmERR) is concentrated and BmERR expression is strongly correlated with trehalase in the midgut during the last instar silkworm larval stage. We cloned the promoter of the trehalase from Bombyx mori (BmTreh) and found that the ERR bound directly to the core response elements of the promoter. Cell level interference and the overexpression of ERR can reduce or enhance BmTreh transcription and promoter activity. Overexpressed transgenic BmERR can significantly increase the expression of BmTreh in the midgut of the last instar silkworm larvae, thereby hydrolyzing trehalose into glucose and releasing it into the hemolymph. Additionally, increased hemolymph glucose content reduces silkworm pupa weight but does not affect silk protein production from the silk gland. Our results suggest a novel function for BmERR through its involvement in BmTreh regulation and expand the understanding of ERR functions in insect trehalose metabolism. Full article

Silkworm is an economically important insect that synthetizes silk proteins for silk production in silk gland, and silk gland cells undergo endoreplication during larval period. Transcription factor Myc is essential for cell growth and proliferation. Although silkworm Myc gene has been identified previously, its biological functions in silkworm silk gland are still largely unknown. In this study, we examined whether enhanced Myc expression in silk gland could facilitate cell growth and silk production. Based on a transgenic approach, Myc was driven by the promoter of the fibroin heavy chain (FibH) gene to be successfully overexpressed in posterior silk gland. Enhanced Myc expression in the PSG elevated FibH expression by about 20% compared to the control, and also increased the weight and shell rate of the cocoon shell. Further investigation confirmed that Myc overexpression increased nucleus size and DNA content of the PSG cells by promoting the transcription of the genes involved in DNA replication. Therefore, we conclude that enhanced Myc expression promotes DNA replication and silk protein expression in endoreplicating silk gland cells, which subsequently raises silk yield. Full article

Bombyx mori silk protein genes are strictly turned on and off in different developmental stages under the hormone periodically change. The broad complex (BrC) is a transcription factor mediating 20-hydroxyecdysone action, which plays important roles during metamorphosis. Here, we observed that two isoforms of BmBrC (BmBrC-Z2 and BmBrC-Z4) exhibited contrasting expression patterns with fibroin genes (FibH, FibL and P25) in the posterior silk gland (PSG), suggesting that BmBrC may negatively regulate fibroin genes. Transgenic lines were constructed to ectopically overexpress BmBrC-Z2 in the PSG. The silk protein genes in the transgenic line were decreased to almost half of that in the wild type. The silk yield was decreased significantly. In addition, the expression levels of regulatory factors (BmKr-h1 and BmDimm) response to juvenile hormone (JH) signal were inhibited significantly. Then exogenous JH in the BmBrC-Z2 overexpressed lines can inhibit the expression of BmBrC-Z2 and activate the expression of silk protein genes and restore the silk yield to the level of the wild type. These results indicated that BmBrC may inhibit fibroin genes by repressing the JH signal pathway, which would assist in deciphering the comprehensive regulation mechanism of silk protein genes. Full article

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. Full article

Human platelet derived growth factor (PDGF) is a major therapeutic protein with great demand in the clinical setting; however, its rate of supply is far from meeting needs. Here, we provide an effective strategy to produce PDGF-BB in large quantities using a transgenic silkworm. The codon-optimized PDGF-B gene regulated by the highly efficient sericin-1 expression system was integrated into the genome of a silkworm. The high transcriptional expression of the PDGF-BB gene in the transgenic silkworm competitively inhibited the transcription expression of the endogenous sericin-1 gene which caused a significant 37.5% decline. The PDGF-BB synthesized in the middle silk gland (MSG) of transgenic silkworms could form a homodimer through intermolecular disulfide bonds, which is then secreted into sericin lumen and finally, distributed in the sericin layer of the cocoon. In this study, a protein quantity of approximately 0.33 mg/g was found in the cocoon. Following a purification process, approximately 150.7 μg of recombinant PDGF-BB with a purity of 82% was purified from 1 g of cocoons. Furthermore, the bioactivity assays showed that the purified recombinant PDGF-BB was able to promote the growth, proliferation and migration of NIH/3T3 cells significantly. These results suggest that the silk gland bioreactor can produce active recombinant PDGF-BB as an efficient mitogen and wound healing agent. Full article