Transgenic Silkworms Research Articles

Bombyx mori nucleopolyhedrovirus induces BmFABP1 downregulation to promote viral proliferation.

Fatty acid binding proteins (FABPs) play an important role as endogenous cytoprotectants. However, studies on FABPs in invertebrates are scarce. Previously, we discovered Bombyx mori fatty acid binding protein 1 (BmFABP1) through co-immunoprecipitation. Here, we cloned and identified BmFABP1 from BmN cells. The results of immunofluorescence indicated that BmFABP1 was localized in the cytoplasm. The tissue expression profile of silkworms showed that BmFABP1 was expressed in all tissues except hemocytes. The expression level of BmFABP1 gradually decreases in BmN cells and B. mori larvae after infection with B. mori nucleopolyhedrovirus (BmNPV). Upregulation of BmFABP1 expression through overexpression or WY14643 treatment significantly inhibited the replication of BmNPV, while downregulation of BmFABP1 expression by RNA interference promoted the replication of BmNPV. The same results were obtained in experiments on silkworm larvae. These results suggest that BmNPV induces BmFABP1 downregulation to promote its proliferation and that BmFABP1 has a potential anti-BmNPV role. This is the first report on the antiviral effect of BmFABP1 in silkworms and provides new insights into the study of the FABP protein family. Also, it is important to study BmNPV resistance in silkworms to breed transgenic silkworms with BmNPV resistance.

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

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

Effects of RGD-fused silk fibroin in a solution format on fibroblast proliferation and collagen production.

Collagen production in fibroblasts is important for skin tissue repair. Cell-adhesive Arg-Gly-Asp (RGD) peptides immobilized on scaffolds stimulate fibroblast collagen production, but RGD peptides in solution exhibit opposite effects. Transgenic silkworm technology enables the design of fusion positions for RGD peptides in silk fibroin molecules. The effect of RGD-fused silk fibroin in solution on fibroblast cell activity remains unclear. To clarify the effects of RGD peptides fused to silk fibroin heavy (H)-chain or light (L)-chain on fibroblast proliferation and collagen production when RGD-fused silk fibroin proteins were added to the culture medium. Silk fibers with RGD-fused H-chains (H-RGD) or L-chains (L-RGD) were degummed, dissolved, and dialyzed to prepare H-RGD or L-RGD aqueous solutions, respectively. These solutions were added to the fibroblast medium, and their proliferation and collagen production were quantified. Both L- and H-RGD stimulated fibroblast proliferation at a similar level, even in a solution format, but L-RGD promoted fibroblast collagen production significantly, indicating the synergistic effect of the native H-chain and RGD-fused L-chain. RGD-fused silk fibroin in solution stimulated fibroblast proliferation and collagen production, depending on the fusion position of the peptides.

Drosophila melanogaster resilin improves the mechanical properties of transgenic silk.

Resilin is a natural protein with high extensibility and resilience that plays a key role in the biological processes of insects, such as flight, bouncing, and vocalization. This study used piggyBac-mediated transgenic technology to stably insert the Drosophila melanogaster resilin gene into the silkworm genome to investigate whether exogenous protein structures improve the mechanical properties of silkworm silk. Molecular detection showed that recombinant resilin was expressed and secreted into silk. Secondary structure and mechanical property analysis showed that the β-sheet content in silk from transgenic silkworms was higher than in wild-type silk. The fracture strength of silk fused with resilin protein was 7.2% higher than wild-type silk. The resilience of recombinant silk after one-time stretching and cyclic stretching was 20.5% and 18.7% higher than wild-type silk, respectively. In summary, Drosophila resilin can enhance the mechanical properties of silk, and this study is the first to improve the mechanical properties of silk using proteins other than spider silk, which broadens the possibilities for the design and application of biomimetic silk materials.

Sericin Ser3 Ectopic Expressed in Posterior Silk Gland Affects Hemolymph Immune Melanization Response via Reducing Melanin Synthesis in Silkworm

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.

Tandem Multimerization Can Enhance the Structural Homogeneity and Antifungal Activity of the Silkworm Protease Inhibitor BmSPI39

Previous studies have shown that BmSPI39, a serine protease inhibitor of silkworm, can inhibit virulence-related proteases and the conidial germination of insect pathogenic fungi, thereby enhancing the antifungal capacity of Bombyx mori. The recombinant BmSPI39 expressed in Escherichia coli has poor structural homogeneity and is prone to spontaneous multimerization, which greatly limits its development and application. To date, the effect of multimerization on the inhibitory activity and antifungal ability of BmSPI39 remains unknown. It is urgent to explore whether a BmSPI39 tandem multimer with better structural homogeneity, higher activity and a stronger antifungal ability can be obtained by protein engineering. In this study, the expression vectors of BmSPI39 homotype tandem multimers were constructed using the isocaudomer method, and the recombinant proteins of tandem multimers were obtained by prokaryotic expression. The effects of BmSPI39 multimerization on its inhibitory activity and antifungal ability were investigated by protease inhibition and fungal growth inhibition experiments. In-gel activity staining and protease inhibition assays showed that tandem multimerization could not only greatly improve the structural homogeneity of the BmSPI39 protein, but also significantly increase its inhibitory activity against subtilisin and proteinase K. The results of conidial germination assays showed that tandem multimerization could effectively enhance the inhibitory ability of BmSPI39 on the conidial germination of Beauveria bassiana. A fungal growth inhibition assay showed that BmSPI39 tandem multimers had certain inhibitory effects on both Saccharomyces cerevisiae and Candida albicans. The inhibitory ability of BmSPI39 against these the above two fungi could be enhanced by tandem multimerization. In conclusion, this study successfully achieved the soluble expression of tandem multimers of the silkworm protease inhibitor BmSPI39 in E. coli and confirmed that tandem multimerization can improve the structural homogeneity and antifungal ability of BmSPI39. This study will not only help to deepen our understanding of the action mechanism of BmSPI39, but also provide an important theoretical basis and new strategy for cultivating antifungal transgenic silkworms. It will also promote its exogenous production and development and application in the medical field.

Transcriptome analysis reveals that knocking out BmNPV iap2 induces apoptosis by inhibiting the oxidative phosphorylation pathway

Apoptosis is essential for the normal growth, development, and immunity defense of living organisms, and its function and mechanisms have been intensively studied. When viral infection occurs, apoptosis is triggered, causing programmed death of the infected cells. Meanwhile, viruses have also evolved countermeasures to inhibit apoptosis in host cells. We previously constructed a transgenic silkworm line with significantly improved resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) by knocking out the BmNPV inhibitor of apoptosis 2 (iap2) gene. However, the mechanism of how IAP2 induces apoptosis still needs to be further investigated. Here, the transcriptomes of Cas9(−)/sgiap2 (−) and Cas9(+)/sgiap2(+) strains were analyzed at 48 h after BmNPV infection, and a total of 709 differential genes were obtained. A KEGG analysis revealed that the differentially expressed genes were enriched in the oxidative phosphorylation, proteasome, and ribosome pathways. In the oxidative phosphorylation pathway, 41 differentially expressed genes were downregulated, and 12 of these genes were verified by qRT-PCR. More importantly, the knockout of BmNPV iap2 led to the inhibition of the oxidative phosphorylation pathway, followed by activated oxidative stress triggered apoptosis, thereby inhibiting the replication of BmNPV in vitro and vivo. The results provide a basis for the analysis of the initiation of apoptosis that can inhibit virus proliferation, and the study presents new ideas for the subsequent creation of resistant material.

Enhanced locomotor behaviour is mediated by activation of tyrosine hydroxylase in the silkworm brain.

Animal behaviour regulation is a complex process involving many factors, and the nervous system is an essential factor in this process. In many species, pathogens can alter host behaviour by affecting the host's nervous system. An interesting example is that the silkworm shows enhanced locomotor behaviour after being infected with the nucleopolyhedrosis virus. In this study, we analysed the transcriptome of the silkworm brain at different time points after infection and found that various genes related to behaviour regulation changed after infection. In-depth analysis showed that the tyrosine hydroxylase gene might be a key candidate gene, and the content of dopamine, its downstream metabolite, increased significantly in the brain of silkworms infected with the virus. After the injection of tyrosine hydroxylase inhibitor into the infected silkworm, the dopamine content in the silkworm brain decreased and the locomotor behaviour caused by the virus was blocked successfully. These results confirm that tyrosine hydroxylase is involved in regulating enhanced locomotor behaviour after virus infection in silkworms. Furthermore, the tyrosine hydroxylase gene was specifically overexpressed in the brain of the silkworm, and the transgenic silkworm was enhanced in locomotor behaviour and foraging behaviour. These results suggest that the tyrosine hydroxylase gene plays a vital role in regulating insect behaviour.

Insights into the quality of recombinant proteins produced by two different Bombyx mori expression systems

The silkworm, Bombyx mori, is an attractive host for recombinant protein production due to its high expression efficiency, quality, and quantity. Two expression systems have been widely used for recombinant protein production in B. mori: baculovirus/silkworm expression system and transgenic silkworm expression system. Both expression systems enable high protein production, but the qualities of the resulting recombinant proteins have not been well evaluated. In this study, we expressed bovine interferon γ (IFN-γ) using the two systems and examined the quality of the resulting proteins in terms of N-glycosylation and protein cleavage. Both expression systems successfully produced IFN-γ as an N-glycoprotein. Although the production in the baculovirus/silkworm expression system was much more efficient than that in the transgenic silkworm expression system, unexpected variants of IFN-γ were also produced in the former system due to the different N-glycosylation and C-terminal truncations. These results indicate that while high protein production could be achieved in the baculovirus/silkworm expression system, unintentional protein modification might occur, and therefore protein expression in the transgenic silkworm expression system is preferable from the point-of-view of N-glycosylation of the recombinant protein and evasion of unexpected attack by a protease in B. mori.

Establishment and application of a silkworm CRISPR/Cas9 tool for conditionally manipulating gene disruption in the epidermis

Spatial or temporal specific gene knockout system is a valuable tool for studying the molecular mechanisms underlying developmental processes. The integument is essential for insect fitness and survival, but tools for dissecting function of genes in this tissue are lacking. In this study, we firstly identified an epidermis specifically expressed gene of the domesticated silkworm, BmCPG25, by comparative transcriptomic analysis. Furthermore, a transgenic silkworm expressing the RNA dependent CRISPR-Cas9 protein driven by the regulatory region of the BmCPG25 was established. Immunochemistry analysis showed the endonuclease was specifically expressed in the nuclear of epidermal cells. We also validated the efficiency of this system by disrupting the function of an epidermis specifically expressed cuticular protein gene (Cpr21) and a ubiquitously expressed cuticular gene (Cph18), respectively. In summary, we successfully constructed a conditional knockout toolkit to manipulate the gene editing in epidermal cells, which provides a valuable approach to study the molecular mechanism of integument development.

A Bioengineering Approach for the Development of Fibroblast Growth Factor-7-Functionalized Sericin Biomaterial Applicable for the Cultivation of Keratinocytes.

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.

Cytokine receptor DOME controls wing disc development in Bombyx mori

In multicellular organisms, the JAK/STAT signaling pathway is involved in cell proliferation, differentiation, apoptosis, and immune regulation. Through activation of the Stat92E transcription factor, JAK/STAT signaling induced proper wing development in Drosophila. Domeless (DOME) was the first identified invertebrate JAK/STAT receptor. However, the function of DOME in Bombyx mori development remains unclear, especially in wing morphogenesis. In this study, we isolated the cytokine receptor DOME gene in B. mori and evaluated its function in DOME-knockout models. We found that overexpression of DOME at the cellular level upregulated the expression of JAK/STAT pathway-related genes, promoted proliferation, and inhibited apoptosis. The results of the interference with DOME had the opposite effects with those of overexpression at the cellular level. Using CRISPR/Cas9 technology, we constructed a DOME-knockout transgenic silkworm strain (KO-DOME) and found that the wings of the pupa and moth stages were vesicle-shaped and smaller than those of the wild-type silkworm. Some KO-DOME silkworms were unable to extend their wings from the pupal case after eclosion. We detected the expression of cyclin and apoptosis-related genes in the wing disc of the moth stage and found that some cyclin genes, such as CyclinA, CyclinB, and CyclinD, were downregulated, whereas apoptotic genes, such as Caspase1, Caspase3, and Caspase8, were upregulated. We propose that DOME regulates cell proliferation and apoptosis by affecting the JAK/STAT signaling pathway, ultimately influencing the development of wing discs. Our study provides empirical evidence for the biological function of the silkworm DOME gene, which is essential for the normal development of wings.

Bioengineered Silkworm for Producing Cocoons with High Fibroin Content for Regenerated Fibroin Biomaterial-Based Applications.

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.

Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk.

Infectious disease is one of the most serious problems in the aquaculture industry for ornamental or edible fish. This study attempted to develop a new device for preventing an aquatic bacterial disease, ulcer disease, caused by Aeromonas salmonicida (As), using “affinity silk”. Affinity silk is a silk protein-containing fibroin L-chain (FibL) fused to the single-chain variable fragment (scFv). It can be easily processed into different formats such as fibers, gels, sponges, or films. A transgenic silkworm that could express a cDNA construct containing FibL fused to an scFv derived from a monoclonal antibody (MAb) against As was successfully generated. An enzyme-linked immunosorbent assay was used to detect As by employing 96-well plates coated with scFv-conjugated affinity silk. As could be captured efficiently by glass wool coated with affinity silk in the column. Furthermore, the air-lift water filter equipped with the affinity silk-coated wool could considerably reduce the concentration of As in water and was estimated to have sufficient ability to trap a lethal dose of As. These findings show that the “affinity silk filter” is a potential device for the prophylaxis of aquatic animal diseases.

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.

Biocompatible fluorescent silk fibroin bioink for digital light processing 3D printing

Chemically modified silk fibroin (SF) bioink has been used for three-dimensional (3D) bioprinting in tissue engineering because of its biocompatibility and printability. Also, fluorescent silk fibroin (FSF) from transgenic silkworms has been recently applied in biomedicine because of its fluorescence property. However, the fabrication of fluorescent hydrogel from FSF has not been elucidated. In this study, we showed the fabrication of a digital light processing (DLP) printable bioink from a chemically modified FSF. This bioink was fabricated by covalent conjugation of FSF and glycidyl methacrylate (GMA) and can be printed into various structures, such as the brain, ear, hand, lung, and internal organs. The physical properties of glycidyl methacrylated fluorescent silk fibroin (FSGMA) hydrogel was like the glycidyl methacrylated non-fluorescent silk fibroin (SGMA) hydrogel. The FSGMA hydrogel significantly retains its fluorescence property and has excellent biocompatibility. All these properties make FSGMA hydrogel a potent tool in encapsulated cell tracking and observing the scaffolds' degradation in vivo. This study suggested that our 3D DLP printable FSF bioink could play a promising role in the biomedical field.

Genetically engineered pH-responsive silk sericin nanospheres with efficient therapeutic effect on ulcerative colitis

Ulcerative colitis (UC) is one type of inflammatory bowel disease (IBD) and lactoferrin (LF) is a promising protein drug to treat UC. However, targeted LF delivery to optimize bioavailability, targeting and effectiveness remains a challenge. Here, we report an effective strategy to fabricate silk sericin nanospheres systems for the delivery of recombinant human lactoferrin (SS-NS-rhLF). The system is based on the use of optimized transgenic silkworms to generate genetically engineered silk fibers (rhLF-silks). The rhLF silks were used for fabricating SS-NS-rhLF by ethanol precipitation. The SS-NS-rhLF were stable with a spherical morphology with an average diameter of 123 nm. The negatively charged sericins in a pH ≥ 5.5 environment achieved specific targeting of the SS-NS-rhLF to positively charged colonic sites. The SS-NS-rhLF achieved efficient uptake by cells in the inflamed colon of mice when compared to free lactoferrin in solution (SOL-rhLF). Furthermore, oral administration of the SS-NS-rhLF with low dose of rhLF significantly relived symptoms of UC in mice and achieved comparable therapeutic effect to the high dose of SOL-rhLF by supporting the reformation of cell structure and length of colon tissue, reducing the release of inflammatory factors, inhibiting the activation of the NF-κB inflammatory pathway, and maintaining a stable intestinal microbial population in mice. These results showed that the SS-NS-rhLF is a promising system for colitis treatment. Statement of significanceTargeting and effective delivery of multiple biological functional protein human lactoferrin (rhLF) is a promising strategy to treat ulcerative colitis in the clinic. Here, rhLF-transgenic silk cocoons were used to fabricate a rhLF-sericin nanosphere delivery system (SS-NS-rhLF). The fabricated SS-NS-rhLF showed identical spherical morphology, stable structure, sustainable rhLF release, efficient cell uptake and negative charge in an environment of pH above 5.5, thus realized the specific targeting to the positively charged colonic sites to treat UC mice through oral administration. The therapeutic effect of SS-NS-rhLF with a low rhLF dose in the UC mice was comparable to the high dose of free rhLF treatment in solution form, suggesting that the SS-NS-rhLF is a promising system for colitis treatment.

Effects of Serine/Arginine Enriched Protein BmUP on the Development of Male Silkworm Reproductive Organs

Serine/arginine-rich proteins are a class of highly conserved splicing factor proteins involved in constitutive and alternative splicing. We screened a low molecular weight serine/arginine rich protein from silkworms and named it BmUP. Temporal and spatial expression analysis indicated that the BmUP gene was specifically expressed in the silkworm testis, and the highest expression occurred in the pre-pupa stage from the fifth instar to the moth stages. Here, we generated BmUP knockout individuals with the CRISPR/Cas9 system. Both the internal and external genitalia of knockout individuals were abnormal in knockout compared with wild-type male silkworms. In transgenic silkworms overexpressing BmUP, male silkworms showed a phenotype similar to that of the knockout individuals, whereas female individuals showed no significant differences from the wild type. In addition, by conducting promoter analysis, we identified Bmachi, a transcription factor that regulates the BmUP gene. Gel migration experiments revealed that BmAchi specifically binds the BmUP promoter. Quantitative real-time PCR showed that an increase in Bmachi expression up-regulated the expression of BmUP. In contrast, when the expression of Bmachi decreased, the expression of BmUP also downregulated in the experimental group compared with the control group. These results provide new insights for studying the effects of serine/arginine-rich proteins on the development of silkworm genitals.

Long Non-Coding RNA Bmdsx-AS1 Effects on Male External Genital Development in Silkworm.

Simple SummaryLncRNAs are a class of non-coding RNAs longer than 200 nt that are involved in a variety of biological processes. Studies on lncRNAs in Bombyx mori have shown that some lncRNAs are involved in brain development, silk production and the response to virus infection of the host. However, the roles of lncRNAs are still largely unknown in the silkworm. In this study, we analyzed the function of lncRNAs Bmdsx-AS1 in silkworm by transgenic overexpression, which not only affects the development of male silkworm external genitalia, but also participates in the regulation of EGFR signaling pathway. Moreover, we studied the upstream promoter of Bmdsx-AS1 and found that the BmAbd-B transcription factor of the Hox gene family can negatively regulate the expression of Bmdsx-AS1. These results laid a substantial foundation for in-depth study of the function of lncRNAs in the silkworm.Long non-coding RNAs (lncRNAs) have been suggested to play important roles in some biological processes. However, the detailed mechanisms are not fully understood. We previously identified an antisense lncRNA, Bmdsx-AS1, that is involved in pre-mRNA splicing of the sex-determining gene Bmdsx in the silkworm. In this study, we analyzed the changes in the male external genitalia of transgenic overexpressed Bmdsx-AS1 silkworm lines and analyzed downstream and upstream responses. We found that Bmdsx-AS1 transgenic silkworms, compared with wild type, showed more claspers in the male external genitalia. Quantitative real-time PCR (qPCR) results indicated that overexpression of Bmdsx-AS1 decreased the expression of genes in the EGFR signaling pathway. Knockdown of Bmdsx-AS1 increased the activity of the EGFR pathway. Through promoter prediction, promoter truncation and electrophoretic mobility shift assay (EMSA) analyses, we found that the protein encoded by the Hox gene BmAbd-B specifically binds to the promoter of Bmdsx-AS1. Moreover, overexpression of BmAbd-B in the silkworm BmE cell line indicated that BmAbd-B negatively regulates the mRNA expression of Bmdsx-AS1. Our study provides insights into the regulatory mechanism of the lncRNA in the silkworm.

Very Early Corona Treatment-Mediated Artificial Incubation of Silkworm Eggs and Germline Transformation of Diapause Silkworm Strains

Diapause is an important biological characteristic for many insect species to adapt to adverse environmental conditions and maintain the continuity of the race. Compared with the traditional hydrochloric acid or/and cold storage treatment methods, the artificial corona incubation technology of silkworm (Bombyx mori) eggs has many advantages including, the absence of pollution, easy operation and safety. However, this technology has not yet been applied in sericulture. In this study, we developed a novel artificial corona instrument to successfully disrupt the diapause of newly laid and refrigerated eggs from various Chinese and Japanese lineage silkworm strains. Subsequently, we invented a very early corona treatment (VECT) strategy to prevent the diapause of newly laid silkworm eggs within 4 h of oviposition. The hatching rates of the larvae were more than 95% in all diapause silkworm strains, which was comparable to the effect of the traditional HCl treatment strategy. In addition, we developed a combination strategy of VECT and pre-blastoderm microinjection and successfully created transgenic silkworms in various diapause strains. The results of the current study can aid in improving the corona artificial incubation technology and promote its application in sericulture.