RESUMO
Viral diseases pose a significant threat to livestock husbandry and plant cultivation. CRISPR/Cas9-mediated targeted editing of viral genes offers a promising approach to antiviral therapy. The silkworm, Bombyx mori, is an economically important insect susceptible to infection by B. mori nucleopolyhedrovirus (BmNPV), and viral outbreaks cause severe economic losses to the sericulture industry. Here, we identified BmNPV orf76 as a viral late gene that is highly similar to Autographa californica multiple nucleopolyhedrovirus Ac93. The deletion of orf76 abolished BmNPV proliferation and hindered the production of infectious budded viruses. We generated a transgenic line, Cas9(+)/sgorf76(+), that did not affect the growth or development of the silkworm and demonstrated that the transgenic line Cas9(+)/sgorf76(+) efficiently cleaved orf76 at the sgorf76 site, resulting in large deletions at 120 h post-infection, with no observed off-target effects. Survival analyses revealed that the transgenic line Cas9(+)/sgorf76(+) exhibited significantly higher survival rates than the control lines Cas9(-)/sgorf76(-), regardless of the BmNPV inoculation dose. Additionally, the number of BmNPV DNA copies and the expression levels of viral genes were markedly inhibited in the transgenic line Cas9(+)/sgorf76(+) compared with the control line Cas9(-)/sgorf76(-). The results provide a promising target for Cas9-mediated antiviral therapy against BmNPV, and the findings provide new insights for baculovirus gene function studies and lepidopteran pest control.
RESUMO
The 871C silkworm strain exhibits a high level of resistance to Bombyx mori nucleopolyhedrovirus (BmNPV), making it a valuable variety for the sericulture industry. Understanding the underlying mechanism of its resistance holds great biological significance and economic value in addressing viral disease risks in sericulture. Initially, we infected the resistant strain 871C and its control strain 871 with BmNPV and conducted secondary infection experiments using the progeny occlusion bodies (OBs). As a result, a significant decrease in pathogenicity was observed. Electron microscopy analysis revealed that 871C produces progeny virions with defective DNA packaging, reducing virulence following BmNPV infection. Blood proteomic identification of the silkworm variety 871C and control 871 after BmNPV infection demonstrated the crucial role of the viral proteins P6.9 and VLF-1 in the production of defective viruses by impeding the proper encapsulation of viral DNA. Additionally, we discovered that BmHSP19.9 interacts with P6.9 and VLF-1 and that its expression is significantly upregulated after BmNPV infection. BmHSP19.9 exhibits strong antiviral activity, in part by preventing the entry of the proteins P6.9 and VLF-1 into the nucleus, thereby hindering viral nucleocapsid and viral DNA assembly. Our findings indicate that the antiviral silkworm strain 871C inhibits BmNPV proliferation by upregulating Bmhsp19.9 and impeding the nuclear localization of the viral proteins P6.9 and VLF-1, leading to the production of defective viral particles. This study offers a comprehensive analysis of the antiviral mechanism in silkworms from a viral perspective, providing a crucial theoretical foundation for future antiviral research and the breeding of resistant silkworm strains.
Assuntos
Bombyx , Nucleopoliedrovírus , Bombyx/virologia , Animais , Nucleopoliedrovírus/fisiologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Vírion/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteômica/métodosRESUMO
BACKGROUND: Bombyx mori nuclear polyhedrosis virus (BmNPV), as a typical baculovirus, is the primary pathogen that infects the silkworm B. mori, a lepidopteran species. Owing to the high biological safety of BmNPV in infecting insects, it is commonly utilized as a biological insecticide for pest control. Apoptosis is important in the interaction between the host and pathogenic microorganisms. MicroRNAs (miRNAs) influence immune responses and promote stability of the immune system via apoptosis. Therefore, the study of apoptosis-related miRNA in silkworms during virus infection can not only provide support for standardizing the prevention and control of diseases and insect pests, but also reduce the economic losses to sericulture caused by the misuse of biological pesticides. RESULTS: Through transcriptome sequencing, we identified a miRNA, miR-31-5p, and demonstrated that it can inhibit apoptosis in silkworm cells and promote the proliferation of BmNPV in BmE-SWU1 cells. We identified a target gene of miR-31-5p, B. mori cytochrome P450 9e2 (BmCYP9e2), and demonstrated that it can promote apoptosis in silkworm cells and inhibit the proliferation of BmNPV. Moreover, we constructed transgenic silkworm strains with miR-31-5p knockout and confirmed that they can inhibit the proliferation of BmNPV. CONCLUSION: These data indicate that miR-31-5p may exert functions of inhibiting apoptosis and promoting virus proliferation by regulating BmCYP9e2. The findings demonstrate how miRNAs influence host cell apoptosis and how they are involved in the host immune system response to viruses, providing important insights into the applications of biological insecticides for pest control. © 2024 Society of Chemical Industry.
Assuntos
Apoptose , Bombyx , Sistema Enzimático do Citocromo P-450 , Proteínas de Insetos , MicroRNAs , Nucleopoliedrovírus , Animais , Bombyx/genética , Bombyx/virologia , Bombyx/crescimento & desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Nucleopoliedrovírus/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Replicação Viral/efeitos dos fármacos , Linhagem CelularRESUMO
The Notch signaling pathway is important in cell cycle regulation and cell proliferation. The transcriptional repressor Suppressor of Hairless [Su(H)] is a molecular switch for downstream target genes of the Notch signaling pathway but the regulatory mechanism of the Su(H) gene in the cell cycle is unclear. We determined the function of the Notch signaling pathway and Bombyx mori Su(H) [BmSu(H)] in the regulation of the silkworm cell cycle. Inhibition of Notch signaling promoted the replication of DNA in silkworm gland cells and expression of the BmSu(H) gene was significantly reduced. Overexpression of the BmSu(H) gene inhibited DNA replication and cell proliferation of silkworm cells, whereas knockout of the BmSu(H) gene promoted DNA replication and cell proliferation. Knockout of the BmSu(H) in silkworms improved the efficiency of silk gland cell endoreplication and increased important economic traits. We demonstrated that BmSu(H) protein can directly bind to the promoters of BmCyclinA, BmCyclinE and BmCDK1 genes, inhibiting or promoting their transcription at the cell and individual level. This study identified molecular targets for genetic improvement of the silkworm and also provided insights into the regulatory mechanism of the cell cycle.
Assuntos
Bombyx , Ciclo Celular , Proteínas de Insetos , Animais , Bombyx/genética , Bombyx/metabolismo , Ciclo Celular/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Transdução de Sinais , Seda/genética , Proliferação de Células/genética , Replicação do DNA , Regiões Promotoras Genéticas/genética , Endorreduplicação , Regulação da Expressão Gênica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.
Assuntos
Bombyx , Nucleopoliedrovírus , Animais , Bombyx/metabolismo , Nucleopoliedrovírus/fisiologia , Genes Virais , Proliferação de Células , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
The ovary generally undergoes tissue remodeling during larval to pupal transition, which includes membrane degeneration and ovariole growth. At the same time, the hormones produced by insects significantly change during metamorphosis. However, the regulatory mechanism for ovarian development and hormones is not fully understood in insects. Herein, we found that matrix metalloproteinase 2 (MMP2) was highly expressed in the ovarian capsules and ovarioles, and the development was abnormal after knocking out MMP2 in Bombyx mori. The process of abnormal degradation of collagen I due to MMP2 deletion, which resulted in abnormal development of ovarioles and eggs, was analyzed in detail. The proteomics of ovaries in the MMP2-knock out and wild type strains showed a critically significant difference in the expression of a protein, insulin-like peptide (ILP). Additional analysis revealed significant alteration of ILP during ovarian development, and abnormal expression of ILP significantly affected ovarian development in vivo and MMP2 expression in vitro and in vivo. These results showed that MMP2 regulation of ovarian tissue remodeling is closely related to ILP expression. Our study provides new insights into the regulatory mechanism of MMP2 and ovarian development in B. mori.
RESUMO
Endomitosis is involved in developmental processes associated with an increase in metabolic cell activity, which is characterized by repeated rounds of DNA replication without cytokinesis. Endomitosis cells are widespread in protozoa, plants, animals and humans. Endomitosis cell cycle is currently viewed as a variation of the canonical cell cycle and transformed from mitotic cell cycle. However, the meaningful question about how endomitosis transformed from mitosis is still unclear. Herein, we identified a novel transcription factor in silk glands, ZFP67, which is gradually reduced in silk glands during the transition of mitosis to endomitosis. In addition, over-expressed ZFP67 in silk glands led to the transition delayed. And, knock-out of ZFP67 led to abnormal chromatin division and unsuccessful cell division. These data reveled that ZFP67 played an important role in transition of mitosis to endomitosis. Furthermore, ZFP67 can regulate the transcription of cyclin B, a key cyclin related to cell division and G2/M phase, which is demonstrated by chromatin immunoprecipitation and dual luciferase reporter system in this article. In conclusion, it can be speculated that the decreasing expression of ZFP67 in silk glands during the transition stage of mitosis-to-endomitosis resulted in the lack of cyclin B, which further led to unsuccessful cytokinesis and then promoted the transition from mitosis to endomitosis of silk gland cells.
Assuntos
Mitose , Fatores de Transcrição , Humanos , Animais , Fatores de Transcrição/genética , Ciclina B1/genética , Ciclo Celular , Replicação do DNA , Seda/genéticaRESUMO
Apoptosis, as an important part of the immune response, is one of the core events in the host-virus interaction. Studies have shown that long non-coding RNAs (lncRNAs) play important roles in the process of cell apoptosis and pathophysiology. To investigate the apoptosis-related lncRNAs involved in Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworms, transcriptome sequencing was conducted based on silkworm cells infected with BmNPV before and after B. mori inhibitor of apoptosis (Bmiap) gene knockout. A total of 23 differentially expressed lncRNAs were identified as being associated with the mitochondrial apoptosis pathway. Moreover, we demonstrated that B. mori LINC5438 has the function of inhibiting apoptosis in silkworm cells. Overexpression of LINC5438 promoted the proliferation of BmNPV, while interference with LINC5438 inhibited its proliferation, indicating that LINC5438 plays an important role in BmNPV infection. Our results also showed that LINC5438 can regulate the expression of Bmiap, BmDronc, BmICE, and its predicted target gene BmAIF, suggesting that LINC5438 may function through the mitochondrial pathway. These findings provide important insights into the mechanisms of virus-host interaction and the applications of baculoviruses as biological insecticides.
Assuntos
Bombyx , RNA Longo não Codificante , Animais , Bombyx/metabolismo , RNA Longo não Codificante/genética , Apoptose , Proliferação de Células , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
The immediate early protein 1 (IE1) acts as a transcriptional activator and is essential for viral gene transcription and viral DNA replication. However, the key regulatory domains of IE1 remain poorly understood. Here, we analyzed the sequence characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) IE1 and identified the key functional domains of BmNPV IE1 by stepwise truncation. Our results showed that BmNPV IE1 was highly similar to Autographa californica nucleopolyhedrovirus (AcMNPV) IE1, but was less conserved with IE1 of other baculoviruses, the C-terminus of IE1 was more conserved than the N-terminus, and BmNPV IE1 was also necessary for BmNPV proliferation. Moreover, we found that IE1158-208 was a major nuclear localization element, and IE11-157 and IE1539-559 were minor nuclear localization elements, but the combination of these two minor elements was equally sufficient to fully mediate the nuclear entry of IE1. Meanwhile, IE11-258, IE1560-584, and the association of amino acids 258 and 259 were indispensable for the transactivation activity of BmNPV IE1. These results systematically resolve the functional domains of BmNPV IE1, which contribute to the understanding of the mechanism of baculovirus infection and provide a possibility to synthesize a small molecule IE1-truncated mutant as an agonist or antagonist.
Assuntos
Bombyx , Replicação do DNA , Aminoácidos/metabolismo , Animais , Bombyx/metabolismo , DNA Viral , Regulação Viral da Expressão Gênica , Proteínas de Insetos/genética , Nucleopoliedrovírus , Transativadores/metabolismo , Replicação ViralRESUMO
The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions. Although E2Fs have been widely studied in mammals, there have been few studies performed in insects. Here, we determined the function of E2F4 in the silkworm, Bombyx mori. We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals. Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase, whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase. These results indicate that BmE2F4 is important in cell cycle regulation. We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland. Furthermore, we identified a protein called Bm14-3-3ζ that can interact with BmE2F4 and allow it to localize in the nucleus. Overexpression of the Bm14-3-3ζ gene led to cell cycle arrest in the G1 phase, while knocking down the gene increased the proportion of cells in S phase. These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.
Assuntos
Bombyx , Animais , Bombyx/metabolismo , Ciclo Celular , Replicação do DNA , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mamíferos/metabolismo , Fatores de Transcrição/genéticaRESUMO
The matrix metalloproteinases (MMPs) and their endogenous inhibitory factors, tissue inhibitors of metalloproteinases (TIMPs), are implicated in many diseases. However, the mammalian MMPs (> 20) and TIMPs (> 3) are larger in number, and so little is known about their individual roles in organisms. Hence, we have systematically studied the roles of all three MMPs and one TIMP in silkworm innate immunity and metamorphosis. We observed that MMPs and TIMP are highly expressed during the pupation stage of the silkworms, and TIMP could interact with each MMPs. High-activity MMPs and low-activity TIMP may enhance the infection of B. mori nucleopolyhedrovirus in both in vitro and in vivo. MMPs' knockout and TIMP overexpression delayed silkworm development and even caused death. Interestingly, different MMPs' knockout led to different tubular tissue dysplasia. These findings provide insights into the conserved functions of MMPs and TIMP in human organogenesis and immunoregulation.
Assuntos
Bombyx , Imunidade Inata , Metaloproteinases da Matriz , Metamorfose Biológica , Inibidores Teciduais de Metaloproteinases , Animais , Bombyx/imunologia , Bombyx/fisiologia , Mamíferos , Metaloproteinases da Matriz/fisiologia , Inibidores Teciduais de Metaloproteinases/fisiologiaRESUMO
Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.
Assuntos
Bombyx/genética , Replicação do DNA/genética , Geminina/genética , Seda/genética , Animais , Bombyx/metabolismo , Ciclo Celular/genética , Geminina/antagonistas & inibidores , Mitose/genética , Interferência de RNA , Seda/biossínteseRESUMO
Understanding virus-host interaction is very important for delineating the mechanism involved in viral replication and host resistance. Baculovirus, an insect virus, can cause S or G2/M phase arrest in insect cells. However, the roles and mechanism of Baculovirus-mediated S or G2/M phase arrest are not fully understood. Our results, obtained using flow cytometry (FCM), tubulin-labeling, BrdU-labeling, and CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), showed that Bombyx mori nucleopolyhedrovirus (BmNPV) induced G2/M phase arrest and inhibited cellular DNA replication as well as cell proliferation in BmN-SWU1 cells. We found that BmNPV induced G2/M arrest to support its replication and proliferation by reducing the expression of BmCDK1 and BmCyclin B. Co-immunoprecipitation assays confirmed that BmNPV IAP1 interacted with BmCDK1. BmNPV iap1 was involved in the process of BmNPV-induced G2/M arrest by reducing the content of BmCDK1. Taken together, our results improve the understanding of the virus-host interaction network, and provide a potential target gene that connects apoptosis and the cell cycle.
RESUMO
Cell division cycle protein 37 (Cdc37) is a molecular chaperone that actively participates in many intracellular physiological and biochemical processes as well as pathogen infection. However, the function of Cdc37 in silkworm cells under Bombyx mori nucleopolyhedrovirus (BmNPV) infection is unknown. We cloned and identified BmCdc37, a Cdc37 gene from B. mori, which is highly conserved among other species. After BmNPV infection, the expression level of the BmCdc37 gene was up-regulated and showed an expression pattern similar to the BmHsp90 gene, which relies on Cdc37 to stabilize and activate specific protein kinases. The immunofluorescence, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP) assays all indicated that BmCdc37 interacts with BmHsp90 in silkworm cells. Both BmCdc37 and BmHsp90 promote the reproduction of BmNPV. Co-expression of BmCdc37 and BmHsp90 was better at promoting virus proliferation than overexpression alone. These findings all indicate that BmCdc37 plays an active role in the proliferation of BmNPV.
Assuntos
Bombyx , Animais , Bombyx/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células , Interações Hospedeiro-Patógeno , Proteínas de Insetos/genética , NucleopoliedrovírusRESUMO
The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori. In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin ß1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2'-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin ß1.
Assuntos
Bombyx , Proteínas de Insetos , Metaloproteinase 1 da Matriz , Organogênese , Traqueia/enzimologia , Animais , Bombyx/enzimologia , Bombyx/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismoRESUMO
Energy metabolism is important for the proliferation of microsporidia in infected host cells, but there is limited information on the host response. The energy metabolism response of silkworm (Bombyx mori) to microsporidia may help manage Nosema bombycis infections. We analyzed differentially expressed genes in the B.mori midgut transcriptome at two significant time points of microsporidia infection. A total of 1448 genes were up-regulated, while 315 genes were down-regulated. A high proportion of genes were involved in the phosphatidylinositol signaling system, protein processing in the endoplasmic reticulum, and glycerolipid metabolism at 48 h post infection (h p.i.), and a large number of genes were involved in the TCA cycle and protein processing at 120 h p.i. These results showed that the early stages of microsporidia infection affected the basic metabolism and biosynthesis processes of the silkworm. Knockout of Bm_nscaf2860_46 (Bombyx mori isocitrate dehydrogenase, BmIDH) and Bm_nscaf3027_062 (Bombyx mori hexokinase, BmHXK) reduced the production of ATP and inhibited microsporidia proliferation. Host fatty acid degradation, glycerol metabolism, glycolysis pathway, and TCA cycle response to microsporidia infection were also analyzed, and their importance to microsporidia proliferation was verified. These results increase our understanding of the molecular mechanisms involved in N. bombycis infection and provide new insights for research on microsporidia control. IMPORTANCE: Nosema bombycis can be vertically transmitted in silkworm eggs. The traditional prevention and control strategies for microsporidia are difficult and time-consuming, and this is a problem in silkworm culture. Research has mainly focused on host gene functions related to microsporidia infection and host immune responses after microsporidia infection. Little is known about the metabolic changes occurring in the host after infection. Understanding the metabolic changes in the silkworm host could aid in the recognition of host genes important for microsporidia infection and growth. We analyzed host metabolic changes and the main participating pathways at two time points after microsporidia infection and screened the microsporidia-dependent host energy metabolism genes BmIDH and BmHXK. The results revealed genes that are important for the proliferation of Nosema bombycis. These results illustrate how microsporidia hijack the host genome for their growth and reproduction.
Assuntos
Bombyx , Nosema , Animais , Bombyx/genética , Metabolismo Energético/genética , Perfilação da Expressão Gênica , Nosema/genéticaRESUMO
Apoptosis is a cellular defense mechanism used for the elimination of host cells infected by viruses. Viruses have evolved corresponding inhibitors of apoptosis genes to promote their replication. Anti-apoptosis-related genes, involved in baculovirus proliferation, have been proposed but it is unclear whether these genes can be manipulated in gene therapy. We constructed a transgenic silkworm, using the CRISPR/Cas9 system to knock out the BmNPV inhibitor of apoptosis 2 (iap2). The sequencing results showed that all the sequences could edit the target site of BmNPV iap2 gene. There were no differences in economic traits and growth tests between the BmNPV iap2 knockout strain transgenic silkworm lines and the control groups. However, the mortality rate was significantly reduced, the median lethal dose (LD50) was about 100 times higher than the control group, and the onset time was prolonged by 1-2 days after knocking out BmNPV iap2. In addition, the expression levels of apoptotic-related genes Bmiap2, BmICE and BmDreed were significantly affected and the activity of caspase 9 was increased after BmNPV iap2 being edited in transgenic silkworm. These results demonstrated that gene editing BmNPV iap2 could significantly inhibit BmNPV replication and proliferation. This approach provides a new strategy for antiviral research.
Assuntos
Antivirais/metabolismo , Bombyx/virologia , Edição de Genes , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Sequência de Bases , Interações Hospedeiro-Patógeno/genética , Nucleopoliedrovírus/fisiologia , Replicação ViralRESUMO
DNA methylation is an important epigenetic modification and has been shown to be involved in the response to abiotic stress. However, there are few studies on DNA methylation in insect response to environmental signals. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles between two silkworm strains with significantly different resistance to heat and humidity by whole-genome bisulfite sequencing (WGBS). We identified, in total, 2934 differentially methylated regions (DMRs) between RT_48h (resistant strain with high-temperature/humidity treatment for 48 h) and ST_48h (sensitive strain with high-temperature/humidity treatment for 48 h) under cytosine context (CG), which corresponded to 1230 DMR-related genes (DMGs), and the DMRs were primarily located in the gene body (exon and intron) region. Gene ontology (GO) and KEGG analysis showed that these DMGs were most significantly enriched in binding, cellular metabolic process, and RNA transport pathways. Moreover, 10 DMGs have been revealed to be involved in the heat-humidity stress response in the silkworm. The results of this study indicated that DNA methylation plays crucial roles in silkworm response to environmental stressors and provides important clues to identify key resistance genes in silkworm under high-temperature/humidity stress response.
Assuntos
Bombyx/genética , Metilação de DNA/genética , Epigênese Genética/genética , Estresse Fisiológico/genética , Animais , Bombyx/fisiologia , Genoma de Inseto/genética , Resposta ao Choque Térmico/genética , Temperatura Alta/efeitos adversos , Umidade/efeitos adversos , Sulfitos/metabolismo , Sequenciamento Completo do GenomaRESUMO
OBJECTIVE: Rapid and convenient detection of protein-protein interactions (PPIs) is of great significance for understanding function of protein. RESULTS: For efficiently detecting PPIs, we used the changes of proteins fluorescence localization to design a novel system, fluorescence translocation co-localization (FTCL), based on nuclear localization signal (NLS) in living cells. Depending on the original state of protein localization (both in the cytoplasm, both in the nucleus, one in the nucleus and another in the cytoplasm), two target proteins can be partitioned into the cytoplasm and nucleus by adding a NLS or mutating an existing NLS. Three independent results display that the changes of protein fluorescence co-localization were observed following co-expression of the two target proteins. At the same time, we verified the accuracy of fluorescence co-localization by co-immunoprecipitation. CONCLUSIONS: There FTCL system provided a novel detection method for PPIs, regardless of protein localization in the nucleus or cytoplasm. More importantly, this study provides a new strategy for future protein interaction studies through organelle localization (such as mitochondria, Golgi and cytomembrane, etc.).
Assuntos
Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Luminescentes/genética , Sinais de Localização Nuclear/metabolismo , Animais , Linhagem Celular , Núcleo Celular/química , Citoplasma/química , Feminino , Imunoprecipitação , Proteínas de Insetos/química , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Plasmídeos/genética , Mapas de Interação de Proteínas , Transporte ProteicoRESUMO
As an important insect immune response, apoptosis plays a critical role in the interaction between baculoviruses and insect hosts. Previous reports have identified inhibitor of apoptosis (IAP) proteins in both insects and baculoviruses, but the relationship between these proteins is still not clearly understood. Here, we found that insect IAP proteins were clustered with baculovirus IAP3, suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera. We demonstrated that Bombyx mori inhibitor of apoptosis (Bmiap) gene had an inhibitory effect on apoptosis in silkworm cells. Further analysis of the effects of Bmiap genes on the proliferation of B. mori nucleopolyhedrovirus (BmNPV) showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells. Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells, implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection. Taken together, our results provide important insights into the functional relationships of iap genes, and improve our knowledge of apoptosis in baculoviruses and insect hosts.