Fibroin Gene Research Articles

Bmgsb is involved in the determination of cell fate by affecting the cell cycle genes in the silk gland of Bombyx mori

Silk gland is the only organ of silkworm that can produce silk protein, which is a natural macromolecular protein complex and widely utilized in various fields such as biomaterials and biomedicine. The development of silk gland and the expression patterns of silk protein crucial for the silk industry. In this study, the function of a transcription factor Bmgsb was investigated with CRISPR/Cas9 and transgenic system. It was found that the homozygous individuals in the Bmgsb KO line experienced spinning failure and pupae death, the AMSG exhibited defects, and the ASG displayed abnormal curvature. These phenotypes were accompanied by increased DNA endoreplication and significantly upregulated expression of fibroin genes in the ASG. RT-qPCR results confirmed significant upregulation of cell cycle-related genes, including cyclin G and cyclin T in the Bmgsb KO line. Furthermore, ectopic expression of Bmgsb in the PSG weakened PSG curvature, inhibited DNA endoreplication, and downregulated the expression of fibroin genes. These findings strongly suggest that Bmgsb plays a crucial role in determining cell fate in the silk gland and regulating the expression of silk protein through the cyclin pathway. Our research provides a theoretical foundation for further studies on organ differentiation and have implications for the silk industry.

Identification and functional study of Fib-L, a major silk fibroin gene component in rice leaf folders.

The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), is a major migratory pest in rice agriculture. This pest is characterised by its larvae's ability to fold rice leaves using silk, a behaviour that culminates in the formation of a silken cocoon during the pupal stage. The fibroin light chain (CmFib-L) gene is crucial for silk production, yet its specific function in C. medinalis has reminded elusive. This study presents a comprehensive analysis of the CmFib-L gene, revealing its complete open reading frame (ORF) and expression patterns. Notably, the gene is highly expressed in the fifth-instar larvae and the silk gland, which are critical stages for silk production. Our experiments demonstrate that silencing the CmFib-L gene leads to a reduction in pupal weight, an extension of the pupal stage and a disorganised silk cocoon. Furthermore, the larval behaviour of leaf folding and spinning is significantly impaired when the expression of CmFib-L is downregulated. These findings not only show the importance of fibroin light chain in silk production but also reveal a new target gene to regulate and control the behaviour and development of C. medinalis.

Screening of Economically Sustainable Strains of Bivoltine Silkworm, Bombyx mori L. by Assessing the Comparative Fibroin Gene Expression

Screening of Economically Sustainable Strains of Bivoltine Silkworm, Bombyx mori L. by Assessing the Comparative Fibroin Gene Expression

Developmental and nuclear proteomic signatures characterize the temporal regulation of fibroin synthesis during the last molting-feeding transition of silkworm

Silkworm fibroins are natural proteinaceous macromolecules and provide core mechanical properties to silk fibers. The synthesis process of fibroins is posterior silk gland (PSG)-exclusive and appears active at the feeding stage and inactive at the molting stage. However, the molecular mechanisms controlling it remain elusive. Here, the silk gland's physiological and nuclear proteomic features were used to characterize changes in its structure and development from molting to feeding stages. The temporal expression profile and immunofluorescence analyses revealed a synchronous transcriptional on-off mode of fibroin genes. Next, the comparative nuclear proteome of the PSG during the last molting-feeding transition identified 798 differentially abundant proteins (DAPs), including 42 transcription factors and 15 epigenetic factors. Protein-protein interaction network analysis showed a “CTCF-FOX-HOX-SOX” association with activated expressions at the molting stage, suggesting a relatively complex and multifactorial regulation of the PSG at the molting stage. In addition, FAIRE-seq verification indicated “closed” and “open” conformations of fibroin gene promoters at the molting and feeding stages, respectively. Such proteome combined with chromatin accessibility analysis revealed the detailed signature of protein factors involved in the temporal regulation of fibroin synthesis and provided insights into silk gland development as well as silk production in silkworms.

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.

Rapid production of chimeric silkworm/spider silk with improved mechanical properties by infection of nonpermissive Bombyx mori with recombinant AcMNPV harboring native-size of spidroin genes

Spider silks with excellent mechanical properties attract more attention from scientists worldwide, and the dragline silk that serves as the framework of the spider's web is considered one of the strongest fibers. However, it is unfeasible for large-scale production of spider silk due to its highly territorial, cannibalistic, predatory, and solitary behavior. Herein, to alleviate some of these problems and explore aneasy way to produce spider fibers, we constructed recombinant baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) simultaneously expressing Trichonephila clavipes native ampullate spidroin 2 (MaSp-G) and spidroin 1 (MaSp-C) driven by the promoters of silkworm fibroin genes, to infect the nonpermissive Bombyx mori larvae at the fifth instar. MaSp-G and MaSp-C were co-expressed in the posterior silk glands (PSGs) of infected silkworms and successfully secreted into the lumen of the silk gland for fibroin globule assembly. The integration of MaSp-G and MaSp-C into silkworm silk fibers significantly improved the mechanical properties of these chimeric silk fibers, especially the strength and extensibility, which may be caused by the increment of β-sheet in the chimeric silkworm/spider silk fiber. These results demonstrated that silkworms could be developed as the nonpermissive heterologous host for the mass production of chimeric silkworm/spider silk fibers via the recombinant baculovirus AcMNPV.

Overexpression of BmJHBPd2 Repressed Silk Synthesis by Inhibiting the JH/Kr-h1 Signaling Pathway in Bombyx mori.

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.

Cold-stored mulberry leaves affect antioxidant system and silk proteins of silkworm (Bombyx mori) larva.

Cold storage has been widely used to maintain the quality of vegetables, but whether eating cold-stored vegetables affects health remains unknown. This study used silkworms as an animal model to evaluate the effects of nutrient changes in cold-stored mulberry leaves (CSML) on health. Compared with fresh mulberry leaves (FML), CSML contained lower vitamin C, soluble sugars and proteins, and higher H2 O2 , suggesting decreased antioxidant ability and nutrition. The CSML did not obviously affect larval survival rate, body weight or dry matter rate, cocoon shape, weight and size, or final rates of cluster and cocooning relative to the FML, suggesting CSML did not alter overall growth and development. However, the CSML increased the initial rates of cluster and cocooning and upregulated BmRpd3, suggesting CSML shortened larval lifespan and enhanced senescence. CSML upregulated BmNOX4, downregulated BmCAT, BmSOD and BmGSH-Px and increased H2 O2 in silkworms, suggesting CSML caused oxidative stress. CSML upregulated ecdysone biosynthesis and inactivation genes and elevated ecdysone concentration in silkworms, suggesting that CSML affected hormone homeostasis. CSML upregulated apoptosis-related genes, downregulated sericin and silk fibroin genes and decreased sericin content rate in silkworms, suggesting oxidative stress and protein deficiency. Cold storage reduced nutrition and antioxidant capability of mulberry leaves. CSML did not influence growth and development of silkworm larva, but affected health by causing oxidative stress and reducing protein synthesis. The findings show that the ingredient changes in CSML had negative effects on health of silkworms. © 2023 Society of Chemical Industry.

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties.

Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H-chain and L-chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long-lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50 =9.7Mbp/32.4Mbp, L50 =13/11) and complete (BUSCO complete =99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

Identification of the fibroin of Stigmaeopsis nanjingensis by a nanocarrier-based transdermal dsRNA delivery system

Stigmaeopsis nanjingensis (Ma and Yuan) (Acari: Tetranychidae) is an important pest of bamboo—feeding behavior and silk production by the female adult mites is seriously harmful to bamboo leaves. Due to its small size, silking and cocooning, its management is difficult. This study discusses a fast and easy method for management of the pest by disturbing the spinning behavior. Stigmaeopsis nanjingensis is host specific and feeds only on bamboo leaves. Leaf margins of bamboo are highly hydrophobic, which makes dsRNA difficult to immerse. Hence, it is a challenge to apply the commonly used feeding method to inhibit gene expression in mites. In this study, we deliver dsRNA to interfere with the expression of fibroin by body wall permeation with a nanocarrier-based delivery system. The dsRNA/nanocarrier formulation droplets could enter the body cavity within 2 min after falling on the mite. The fibroin silencing efficiency was 75.4%, and the results of electron microscopy showed that dsRNA/nanocarrier damage the morphological structure of the silk thread. This study demonstrated the effectiveness of a nanocarrier-based percutaneous dsRNA delivery system in S. nanjingensis and its effect on the fibroin gene that influences the spinning behavior of S. nanjingensis. These findings may provide a new delivery system for RNAi-based control of spider mites that utilize protective webbing in the field.

New insights into the proteins interacting with the promoters of silkworm fibroin genes

The silkworm, Bombyx mori, is a silk-producing insect that has contributed greatly to human society. The silk gland of B. mori is a specialized organ responsible for synthesizing silk fibroin and sericin proteins under control of numerous factors. However, which factors are involved in direct silk protein synthesis regulation remains largely unknown. We report the identification of promoter-interacting proteins (PIPs) necessary for the regulation of genes encoding fibroin proteins, including the fibroin heavy chain (fibH), fibroin light chain (fibL), and a 25-kD polypeptide protein (P25). In the fourth larval molting stage (M4) or day 5 fifth-instar larvae (L5D5), a total of 198, 292, and 247 or 330, 305, and 460 proteins interacting with the promoter region of fibH, fibL and P25, respectively, were identified from the posterior silk gland by DNA pull-down combined with mass spectrometry. Many PIPs were particularly involved in ribosome- and metabolism-related pathways. Additionally, 135 and 212 proteins were identified as common PIPs of fibH, fibL and P25 in M4 and L5D5, respectively. Among all PIPs, we identified 31 potential transcription factors, such as Y-box and poly A-binding proteins, which play roles in nucleotide binding, ATP binding, or protein folding. This study provides the first in-depth profile of proteins interacting with fibroin gene promoters and contributes to a better understanding of silk protein synthesis regulation.

The balance of crystalline and amorphous regions in the fibroin structure underpins the tensile strength of bagworm silk

Protein-based materials are considered versatile biomaterials, and their biodegradability is an advantage for sustainable development. Bagworm produces strong silk for use in unique situations throughout its life stages. Rigorous molecular analyses of Eumeta variegata suggested that the particular mechanical properties of its silk are due to the coexistence of poly-A and GA motifs. However, little molecular information on closely related species is available, and it is not understood how these properties were acquired evolutionarily or whether the motif combination is a conserved trait in other bagworms. Here, we performed a transcriptome analysis of two other bagworm species (Canephora pungelerii and Bambalina sp.) belonging to the family Psychidae to elucidate the relationship between the fibroin gene and silk properties. The obtained transcriptome assemblies and tensile tests indicated that the motif combination and silk properties were conserved among the bagworms. Furthermore, our analysis showed that C. pungelerii produces extraordinarily strong silk (breaking strength of 1.4 GPa) and indicated that the cause may be the C. pungelerii -specific balance of crystalline/amorphous regions in the H-fibroin repetitive domain. This particular H-fibroin architecture may have been evolutionarily acquired to produce strong thread to maintain bag stability during the relatively long development period of Canephora species relative to other bagworms.

Proteomic evidence for the silk fibroin genes of spider mites (Order Trombidiformes: Family Tetranychidae)

Spider mites are a group of arachnids belonging to Acari (mites and ticks), family Tetranychidae, known to produce nanoscale silk fibers characterized by a high Young's modulus. The silk fibroin gene of spider mites has been computationally predicted through genomic analysis of Tetranychus urticae Koch, but it has yet to be confirmed by proteomic evidence. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, Tetranychus kanzawai Kishida and Panonychus citri (McGregor), and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. As a result, two fibroins were identified, which were different genes than those previously predicted by computational methods. The amino acid composition and secondary structure suggest similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical properties, exhibiting a high Young's modulus. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that sometimes emerge in multiple lineages in a convergent manner and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks. SignificanceHere we sequenced and assembled the transcriptome from two genera of spider mites, T. kanzawai and P. citri, and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. Spider mite silk is especially characterized by its extremely fine nano-scale diameter and high Young's modulus, even exceeding those of spider silks. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes, which independently evolved in mites, insects, and spiders but yet show sequence convergence, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks.

Sage controls silk gland development by regulating Dfd in Bombyx mori

Silk gland is an organ that produces and secretes silk proteins. The development of the silk gland is essential for high silk production yield and silk quality. Although Sage reportedly plays a pivotal role in embryonic silk gland development, the mechanism underlying its action remains unclear. Our study aimed to determine the genes downstream of Sage through which it regulates the development of the silk gland. After chromatin immunoprecipitation and sequencing, Dfd was identified as a downstream target gene of Sage and it was confirmed that Sage could inhibit Dfd expression by competing with SGF1. When Dfd was knocked down through RNA interference (RNAi), the number of cells in the middle silk gland decreased, and the posterior silk gland was straightened. Simultaneously, the expression of Ser1 and silk fibroin genes was no longer strictly regional. These changes eventually led to an alteration in the composition of the Dfd RNAi cocoon. In conclusion, our research contributes to a deeper understanding of the development of silk glands.

Cocoon-Spinning Behavior and 20-Hydroxyecdysone Regulation of Fibroin Genes in Plutella xylostella.

The diamondback moth Plutella xylostella is a serious pest of crucifers. It has high reproductive potential and is resistant to many insecticides. Typically, the last-instar larvae of P. xylostella, before pupation, move to the lower or outer plant leaves to make a loose silk cocoon and pupate inside for adult formation. To better understand this pivotal stage we studied the cocoon-spinning behavior of P. xylostella and measured three successive phases by video-recording, namely the selection of a pupation site, spinning a loose cocoon and padding the scaffold cocoon. Subsequently, we cloned three fibroin genes related to cocoon production, i.e., fibroin light chain (Fib-L), fibroin heavy chain (Fib-H), and glycoprotein P25. A spatio-temporal study of these three fibroin genes confirmed a high expression in the silk glands during the final larval instar silk-producing stage. In parallel, we did an exogenous treatment of the insect molting hormone 20-hydroxyecdysone (20E), and this suppressed fibroin gene expression, reduced the normal time needed for cocoon spinning, and we also observed a looser cocoon structure under the scanning electron microscope. Hence, we demonstrated that the expression levels of key genes related to the synthesis of 20E [the three Halloween genes Spook (Spo), Shadow (Sad), and Shade (Shd)] decreased significantly during spinning, the expression of the 20E receptor (EcR and USP) was significantly lower during spinning than before spinning, and that the expression levels of CYP18-A1 related to 20E degradation were significantly up-regulated during spinning. The significance of the cocoon and the effects of 20E on the cocoon-spinning behavior of P. xylostella are discussed.

The genome sequence of Samia ricini, a new model species of lepidopteran insect.

Samia ricini, a gigantic saturniid moth, has the potential to be a novel lepidopteran model species. Samia ricini is far more resistant to diseases than the current model species Bombyx mori, and therefore can be more easily reared. In addition, genetic resources available for S.ricini rival those for B.mori: at least 26 ecoraces of S.ricini are reported and S.ricini can hybridize with wild Samia species, which are distributed throughout Asian countries, and produce fertile progenies. Physiological traits such as food preference, integument colour and larval spot pattern differ among S.ricini strains and wild Samia species so that those traits can be targeted in forward genetic analyses. To facilitate genetic research in S.ricini, we determined its whole genome sequence. The assembled genome of S.ricini was 458Mb with 155 scaffolds, and the scaffold N50 length of the assembly was ~21Mb. In total, 16,702 protein coding genes were predicted. While the S.ricini genome was mostly collinear with that of B.mori with some rearrangements and few S.ricini-specific genes were discovered, chorion genes and fibroin genes seemed to have expanded in the S.ricini lineage. As the first step of genetic analyses, causal genes for "Blue," "Yellow," "Spot," and "Red cocoon" phenotypes were mapped to chromosomes.

Transgenic Ectopic Overexpression of Broad Complex (BrC-Z2) in the Silk Gland Inhibits the Expression of Silk Fibroin Genes of Bombyx mori.

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.

Fibroin gene expression and antioxidant enzymes are elevated in Bombyx mori when reared on preferred host plants

Silk fibroin filaments are natural proteins produced by silkworm, Bombyx mori, which is bounded by sericin polymers in silk thread used in the cocoon. Fibroin gene expression and antioxidant enzymes were studied in the midgut of Bombyx mori larvae when reared on tropical mulberry plant varieties (Jorhat, TR10, BC2-59, and Hmute). In this study, the B. mori strain FC2 x FC1 showed a high expression of fibroin at both mRNA and protein levels when reared on four plant varieties. B. mori strains FC1 x FC2 (reared on Local, Jorhat and BC2-59) and CSR4 x CSR2 (reared on Jorhat, TR10 and BC2-59) had a higher level of expression of fibroin protein. The strain FC2 X FC1 showed relatively higher SOD and moderate catalase activities. This study reveals the differential expression of fibroin and tolerance to stress in B. mori strains which might justify the rationality of using the most suitable host plants and economically viable strains (FC2 X FC1) for producing commercially the best quality silk threads.

Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system.

The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons. The mechanism by which the fibroin protein is efficiently synthesized and precisely regulated is an important aspect that has yet to be fully elucidated. Here, we describe the regulatory characteristics of the promoters of fibroin protein-encoding genes, namely, fibroin heavy chain (fibH) and fibroin light chain (fibL), using an optimized Gal4/UAS binary system. We found that (1) UAS-linked enhanced green fluorescent protein (EGFP) was effectively activated in the PSGs of Gal4/UAS transgenic silkworms, and fluorescence was continuously detected in the PSGs after complete formation of silk glands. (2) In the PSGs of fourth- and fifth-instar larvae of transgenic silkworms driven by fibL-Gal4 (LG4) or fibH-Gal4 (HG4), EGFP mRNA was detected in only day-3 to day-6 fifth-instar larvae, while the EGFP protein could be detected at each day of both larval stages. (3) High-level expression of Gal4 and UAS-linked EGFP caused a delay in PSG degradation in Gal4/UAS transgenic silkworms. (4) At the early pupal stage, EGFP fluorescence was also detected in fat bodies of Gal4/UAS transgenic silkworms, indicating that the PSG-specific EGFP was transported into fat bodies during PSG degeneration; however, the underlying mechanism needs to be further elucidated. This study provides a modified Gal4/UAS system used for efficient tissue-specific expression of target genes in the PSGs of silkworms and provides new insights into the regulatory characteristics of the promoters of key fibroin protein-encoding genes.