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1.
Insect Biochem Mol Biol ; 116: 103264, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707207

RESUMO

Elaborate regulation of tissue- and stage-specific expression of genes is prerequisite for insect development. The hormone 20-hydroxyecdysone (20E) initiates metamorphosis by regulating the expression of a series of genes. However, how 20E orderly regulates the pupa-specific expression of genes remains unclear. In this study, we report a regulatory mechanism for the pupa-specific expression of chitin synthase A 2b (CHSA-2b) in Bombyx mori. We found that Broad-Complex Z4 (BR-C Z4) was up-regulated by 20E just before pupation, while transcription factor FoxJ and CHSA-2b were up-regulated during the pupal stage. There is a Fox cis-regulatory element in the CHSA-2b promoter region, and FoxJ protein bound to this element, enhancing the CHSA-2b transcription during the pupal stage. In addition to CHSA-2b, FoxJ also up-regulated the expression of 16 out of 19 pupa-specific genes tested. However, at the prepupal stage, 20E-induced BR-C Z4 inhibited the FoxJ transcription, indirectly inhibiting the CHSA-2b transcription. These data suggest that at the pre-pupation stage, 20E-induced BR-C Z4 inhibited the expression of pupa-stage genes like CHSA-2b by inhibiting the expression of FoxJ; by the pupal stage, the expression of BR-C Z4 decreased, releasing its inhibition on FoxJ, which then up-regulated the expression of the pupa-specific genes. This study explains the elaborate regulation of the pupa-specific gene expression during metamorphosis in B. mori.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Quitina Sintase/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Animais , Quitina Sintase/metabolismo , Ecdisterona/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Insetos/metabolismo , Metamorfose Biológica , Pupa/genética , Pupa/crescimento & desenvolvimento , Regulação para Cima , Asas de Animais/crescimento & desenvolvimento
2.
Insect Biochem Mol Biol ; 116: 103255, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31654713

RESUMO

Vacuolar-type H + -adenosine triphosphatases (V-ATPases) are indispensable for lysosome acidification and participate in autophagic processes. The steroid hormone 20-hydroxyecdysone (20E) predominantly induces autophagy and regulates insect larval molting and metamorphosis; however, the specific mechanism of lysosome acidification regulation by 20E remains unclear. Here, we showed that the developmental profiles of Bombyx V-ATPases were in accordance with autophagy occurrence and lysosome acidification in the fat body during larval-pupal metamorphosis. BmV-ATPase-A and BmV-ATPase-B were required for lysosome acidification and autophagic flux. Both 20E treatment and starvation were able to induce lysosome acidification. Furthermore, BmV-ATPase transcription was induced by 20E treatment and reduced by RNAi targeting the 20E receptor BmUsp. On the one hand, 20E upregulated the transcription of BmV-ATPases through inducing Bombyx transcription factor EB (TFEB) and its nuclear translocation; on the other hand, 20E inhibited mTOR signaling to induce the transcription and assembly of BmV-ATPase subunits. Overall, 20E induces lysosome acidification by upregulating the transcription and assembly of V-ATPase subunits via activating BmTFEB and cooperating with nutrient signaling. These findings improve our understanding of the regulatory mechanisms underlying lysosome acidification and autophagic flux in Bombyx mori.


Assuntos
Adenosina Trifosfatases/metabolismo , Bombyx/fisiologia , Ecdisterona/metabolismo , Corpo Adiposo/química , Proteínas de Insetos/metabolismo , Lisossomos/química , Animais , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Metamorfose Biológica
3.
Arch Insect Biochem Physiol ; 103(2): e21627, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31701579

RESUMO

Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , MicroRNAs/genética , Sericinas/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , MicroRNAs/metabolismo , Sericinas/metabolismo
4.
Insect Biochem Mol Biol ; 116: 103258, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678582

RESUMO

The protease inhibitors found in silkworm cocoons can be divided into several families, a majority of which contain serpin, TIL, or Kunitz domains. Previously, it has been reported that TIL-type protease inhibitors have antimicrobial activity. To date, however, it has not been determined whether the Kunitz-type protease inhibitor BmSPI51, the most abundant of cocoon protease inhibitors, plays an antimicrobial role. Thus, in this study, we sought to determine the biological role of BmSPI51 in silkworm cocoons. Our results obtained from real-time quantitative reverse transcription PCR and immunofluorescence analyses indicate that BmSPI51 is expressed exclusively in the silk glands during the larval fifth instar stage and is subsequently secreted into cocoon silk. Moreover, at a molar ratio of 1:1, BmSPI51 produced via prokaryotic expression exhibited inhibitory activity against trypsin and also proved to be highly stable over wide ranges of temperature and pH values. The expression of BmSPI51 was also found to be significantly upregulated in the larval fat body after infection with three species of fungi, namely, Candida albicans, Beauveria bassiana, and Saccharomyces cerevisiae. In vitro inhibition tests revealed that BmSPI51 significantly inhibited the sporular growth of all three of these fungal species. Further, results obtained from a binding assay showed that BmSPI51 binds to ß-d-glucan and mannan on the surface of fungal cells. In this study, we, thus, revealed the antimicrobial activity of BmSPI51 and its underlying mechanism in silkworm, thereby contributing to our present understanding of defense mechanisms in silkworm cocoons.


Assuntos
Bombyx/metabolismo , Bombyx/microbiologia , Proteínas de Insetos/genética , Inibidores de Serino Proteinase/genética , Animais , Antifúngicos/metabolismo , Beauveria/fisiologia , Bombyx/crescimento & desenvolvimento , Candida albicans/fisiologia , Corpo Adiposo/química , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/microbiologia , Saccharomyces cerevisiae/fisiologia , Inibidores de Serino Proteinase/metabolismo
5.
Insect Biochem Mol Biol ; 117: 103293, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31809784

RESUMO

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction. In the hemolymph of Lepidoptera, these lipophilic sesquiterpenic hormones are transported from their site of synthesis to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). Lepidopteran JHBPs belong to a recently uncovered, yet very ancient family of proteins sharing a common lipid fold (TULIP domain) and involved in shuttling various lipid ligands. One important, but poorly understood aspect of JHs action, is the mechanism of hormone transfer to or through the plasma membranes of target cells. Since many membrane-active peptides and proteins, such as the pore-forming bacterial toxins, are activated by low pH or interaction with phospholipid membranes, we have examined the effect of these factors on JH binding by JHBPs. The affinity of Bombyx mori and Manduca sexta JHBPs for JH III was determined by the DCC assay, equilibrium dialysis, and isothermal titration calorimetry, and found to be greatly reduced at low pH, in agreement with previous observations. Loss of binding was accompanied by changes in fluorescence and near-UV CD spectra, indicating significant changes in protein structure in the environment of aromatic residues. The apparent dissociation rate constant (koff) of the JHBP-JH III complex was greater at acidic pH, suggesting that low pH favors ligand release by opening of the binding pocket. The affinity of recombinant B. mori JHBP (rBmJHBP) was also decreased in the presence of anionic phospholipid vesicles. Measurements of steady-state fluorescence anisotropy with the lipophilic probe TMA-DPH demonstrated that rBmJHBP specifically interacts with anionic membranes. These results suggest the existence of a collisional mechanism for ligand release that may be important for delivery of JHs to the target cells, and could be relevant to the function of related members of this emerging family of lipid-transport proteins.


Assuntos
Proteínas de Transporte/genética , Proteínas de Insetos/genética , Mariposas/genética , Animais , Transporte Biológico , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Insetos/metabolismo , Ligantes , Metabolismo dos Lipídeos , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo
6.
Insect Mol Biol ; 29(1): 66-76, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31301266

RESUMO

Storage proteins are haemolymph-specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune-responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real-time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co-immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP-6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two-hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Animais , Bombyx/genética , Bombyx/imunologia , Linhagem Celular , Corpo Adiposo/metabolismo , Proteínas de Fluorescência Verde , Hemolinfa/imunologia , Imunidade Inata , Proteínas de Insetos/genética , Larva/genética , Larva/imunologia , Larva/metabolismo , Proteínas Recombinantes
7.
Insect Biochem Mol Biol ; 117: 103279, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31756435

RESUMO

In the present study, we demonstrated that bombyxin, an insect insulin-like peptide, modulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) through redox signaling. Our results showed that bombyxin treatment resulted in a transient increase in intracellular reactive oxygen species (ROS) concentration, as measured using 2',7'-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant N-acetylcysteine (NAC) abolished the bombyxin-induced increase in fluorescence in Bombyx PGs. Furthermore, bombyxin-induced ROS production was inhibited by mitochondrial oxidative phosphorylation inhibitors (rotenone and antimycin A), indicating mitochondria-mediated ROS production. The stimulation of ROS production in response to bombyxin appears to undergo development-specific changes. We further investigated the action mechanism of bombyxin-stimulated ROS signaling. Results showed that in the presence of either NAC, rotenone, or antimycin A, bombyxin-stimulated phosphorylation of insulin receptor, Akt, and 4E-binding protein (4E-BP) was blocked and bombyxin-stimulated ecdysteroidogenesis in PGs was greatly inhibited. From these results, we conclude that ROS signaling appears to be involved in bombyxin-stimulated ecdysteroidogenesis of PGs in B. mori by modulating the phosphorylation of insulin receptor, Akt, and 4E-BP. To our knowledge, this is the first demonstration of redox regulation in insulin signaling in an insect system.


Assuntos
Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Bombyx/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/metabolismo
8.
Insect Sci ; 27(1): 2-13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29943911

RESUMO

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified. However, molecular mechanisms of most of these mutants remain to be explored. Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing (mw) mutation which exhibits exceedingly small wings during pupal and adult stages. Compared with the wild type silkworm, relative messenger RNA expression of Bm-app is significantly decreased in the u11 mutant strain which shows mw phenotype. A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity. Furthermore, clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant, demonstrating that Bm-app controls wing development in B. mori. Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins, indicating that it is required for cell mitosis and growth during wing development. We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B. mori.


Assuntos
Aciltransferases/genética , Bombyx/genética , Proteínas de Insetos/genética , Asas de Animais/crescimento & desenvolvimento , Aciltransferases/metabolismo , Animais , Bombyx/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento
9.
J Insect Sci ; 19(6)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31765475

RESUMO

The present study was carried out to determine the influence of 2% aqueous honey (Apis dorsata Fabricius, 1793 [Hymenoptera: Apidae]) on larval growth and silk cocoon yield of fifth-instar larvae of the silkworm, Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae). The larvae of silkworms (Chinese HUAKAND2) were divided into a control and an experimental groups (n = 20 in each group). Control group was fed with plain mulberry leaves throughout the fifth instar, whereas the experimental group was offered mulberry leaves dipped in 2% aqueous solution of honey every other day for 4 d (days 1, 3, 5, and 7). On the other days (days 2, 4, 6, and 8), plain mulberry leaves were offered to larvae. Results showed that the average weight gain in larvae of the experimental group was 348.23 and 204.54% in case of the control group. Uneaten mulberry leaves were weighed; the control group left 34.05% of their leaves and the treated group 28.54%. The cocoon formation in the honey-treated larvae was more uniform in shape than the control group. Furthermore, honey-treated larvae began to form cocoons 7.8 ± 0.23 h earlier than the control group. We also recorded an increase of 15.34% in average weight of cocoons of the experimental group when compared with the control. Average shell percentage of fresh silk cocoons of the control and experimental groups was 20.5 and 23.5%, respectively. It is concluded from the study that 2% aqueous honey has positive impact on the larval growth and cocoon yield of B. mori.


Assuntos
Bombyx/crescimento & desenvolvimento , Mel , Larva/crescimento & desenvolvimento , Seda/biossíntese , Animais , Abelhas , Bombyx/metabolismo , Larva/metabolismo
10.
Pestic Biochem Physiol ; 160: 154-162, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31519250

RESUMO

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05-0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.


Assuntos
Benzofenantridinas/toxicidade , Bombyx/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Isoquinolinas/toxicidade , Larva/efeitos dos fármacos , Animais , Bombyx/crescimento & desenvolvimento , Hemolinfa/metabolismo , Inseticidas/farmacologia , Metabolômica
11.
Mol Biotechnol ; 61(11): 852-859, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473916

RESUMO

To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Vírus da Dengue/metabolismo , Corpo Adiposo/metabolismo , Expressão Gênica , Vetores Genéticos , Hemolinfa/metabolismo , Heparina/metabolismo , Larva/metabolismo , Sinais Direcionadores de Proteínas/genética , Sorogrupo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificação , Vírion/ultraestrutura
12.
Ecotoxicology ; 28(8): 903-912, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392633

RESUMO

Silkworm (Bombyx mori) is an economic insect of the Lepidoptera. Chlorantraniliprole (CAP) exposure results in reduced growth and development of B. mori and failure in cocooning, seriously affecting the development of sericulture. To study the mechanisms underlying the damage to silkworm caused by sublethal doses of CAP, we examined the oxidative damage, the activities of digestive enzymes in midgut, and the expressions of midgut-related genes at the mRNA level. We found that CAP exposure inhibited the growth of silkworm, decreased the body mass and caused the accumulation of reactive oxygen species (ROS) [the levels of O2-, H2O2 and lipid peroxidation (MDA) were increased by 1.62-, 1.87- and 1.46-fold, respectively]. Moreover, we also found that the midgut cells were disintegrated, microvilli disappeared, the stroma became thinner, and the chromatin of nucleus became aggregated after CAP exposure by the analysis of transmission electron microscopy (TEM). In addition, the activities of digestive enzymes were dysregulated in midgut (the activities of α-amylase and trypsin were decreased 0.69- and 0.20-fold, respectively). Furthermore, digital gene expression (DGE) profiling analysis revealed that the expressions of oxidative phosphorylation pathway and antioxidant defense system related genes in midgut were decreased, indicating that it was the oxidative damage in midgut caused by CAP that mainly affected the growth of silkworm, rather than the toxicological effects of CAP. Collectively, this study provided valuable insights into the toxic effects of CAP on insects.


Assuntos
Bombyx/efeitos dos fármacos , Inseticidas/toxicidade , ortoaminobenzoatos/toxicidade , Animais , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Bombyx/fisiologia , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/fisiologia , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Estresse Oxidativo/efeitos dos fármacos
13.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370143

RESUMO

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.


Assuntos
Adenosina Quinase/genética , Bombyx/genética , Proteínas de Insetos/genética , Larva/genética , Pupa/genética , Adenosina/química , Adenosina/metabolismo , Adenosina Quinase/química , Adenosina Quinase/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bombyx/enzimologia , Bombyx/crescimento & desenvolvimento , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Cinética , Larva/enzimologia , Larva/crescimento & desenvolvimento , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pupa/enzimologia , Pupa/crescimento & desenvolvimento , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
Insect Biochem Mol Biol ; 113: 103205, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31421207

RESUMO

The number of cells in tissues is under strict genetic control, and research on the determination of cell number is of great importance to understand the growth and development of organs. Bmsage, a bHLH transcription factor, is involved in the development of the silk gland during the embryonic stage in Bombyx mori. However, the mechanism by which it influences silk gland development is unclear. In the present study, we determined via immunofluorescence staining during the embryonic stage of Bombyx mori that Bmsage is expressed in silk gland cells from the beginning of development of the silk gland until its complete formation. By comparing different silkworm strains, we found that Bmsage expression is positively correlated with the number of silk gland cells. Bmsage knockdown by RNAi resulted in shorter silk glands and lower cell numbers, especially in the posterior silk gland. The silk gland lumen also shriveled, and the silk protein content was significantly lower than that in the control. Further investigation revealed that all cyclins decreased after knock down of Bmsage, and cyclin B and cyclin 3 were significantly down-regulated. Bmsage may be involved in the regulation of the cyclin pathway to control silk gland development. Taken together, it can be concluded from our results that Bmsage is involved in the determination of cell number in silk glands. Our results help clarify the process of cell number determination in silk gland and identify a potential target for silkworm breeding.


Assuntos
Bombyx/fisiologia , Proteínas de Insetos/fisiologia , Seda , Animais , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Contagem de Células , Glândulas Exócrinas/fisiologia , Larva/genética , Larva/fisiologia , Seda/biossíntese
15.
J Insect Sci ; 19(4)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31343690

RESUMO

Bombyx mori (Lepidoptera: Bombycidae) is an important economic insect and a classic Lepidopteran model system. Although immune-related genes have been identified at a genome-wide scale in the silkworm, proteins involved in immune defense of the silkworm have not been comprehensively characterized. In this study, two types of bacteria were injected into the silkworm larvae, Gram-negative Escherichia coli (Enterobacteriales: Enterobacteriaceae), or Gram-positive Staphylococcus aureus (Bacillales: Staphylococcaceae). After injection, proteomic analyses of hemolymph were performed by liquid chromatography-tandem mass spectrometry. In total, 514 proteins were identified in the uninduced control group, 540 were identified in the E. coli-induced group, and 537 were identified in the S. aureus-induced group. Based on Uniprot annotations, 32 immunological recognition proteins, 28 immunological signaling proteins, and 21 immunological effector proteins were identified. We found that 127 proteins showed significant upregulation, including 10 immunological recognition proteins, 4 immunological signaling proteins, 11 immunological effector proteins, and 102 other proteins. Using real-time quantitative polymerase chain reaction in the fat body, we verified that immunological recognition proteins, signaling proteins, and effector proteins also showed significant increases at the transcriptional level after infection with E. coli and S. aureus. Five newly identified proteins showed upregulation at both protein and transcription levels after infection, including 30K protein, yellow-d protein, chemosensory protein, and two uncharacterized proteins. This study identified many new immune-related proteins, deepening our understanding of the immune defense system in B. mori. The data have been deposited to the iProX with identifier IPX0001337000.


Assuntos
Bombyx/genética , Bombyx/imunologia , Escherichia coli/fisiologia , Proteínas de Insetos/imunologia , Proteoma/imunologia , Staphylococcus aureus/fisiologia , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/microbiologia , Proteínas de Insetos/análise , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/microbiologia , Proteoma/análise , Proteômica
16.
Mol Genet Genomics ; 294(6): 1375-1383, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31214765

RESUMO

Forkhead box O (FoxO) is a downstream transcription factor of the insulin-signaling pathway, which plays vital roles in the growth and metabolism of organisms. In this study, BmFoxO was overexpressed in BmE cells, in which proliferation was inhibited and apoptosis was increased. The transgenic vector overexpressing BmFoxO was constructed, and the transgenic silkworm line A4FoxO was generated via embryonic microinjection. The body size of A4FoxO silkworm was smaller than that of non-transgenic silkworm (WT). The quantitative polymerase chain reaction results revealed that the insulin pathway was enhanced and the growth-related TOR pathway was suppressed. Furthermore, the translation of proteins in the fat body of A4FoxO silkworm was inhibited. The expression level of genes involved in the glucose synthesis and lipolysis pathways was increased, whereas that of genes involved in fat synthesis was decreased. Oil red O staining revealed that the amount of lipid droplets was reduced in A4FoxO silkworms compared with WT. Further analysis showed that the content of triglyceride and glycogen was significantly decreased in fat body, but the content of glucose and trehalose was increased in the hemolymph of A4FoxO silkworms. These results suggest that the enhanced expression of BmFoxO disturbs glycolipid metabolism and affects silkworm growth.


Assuntos
Bombyx/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose/biossíntese , Proteínas de Insetos/metabolismo , Lipólise , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/embriologia , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Linhagem Celular , Proliferação de Células , Corpo Adiposo/metabolismo , Fatores de Transcrição Forkhead/genética , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Metabolismo dos Lipídeos/genética , Lipólise/genética , Açúcares/metabolismo , Triglicerídeos/metabolismo
17.
Insect Biochem Mol Biol ; 111: 103175, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31150761

RESUMO

The silkworm Bombyx mori is a well-characterized model organism for studying the silk gland development and silk production process. Using positional cloning and gene sequencing, we have previously reported that a truncated fibroin heavy chain was responsible for silkworm naked pupa (Nd) mutant. However, the mechanisms by which the mutant FibH causes developmental defects and secretion-deficiency of the silk gland remain to be fully elucidated. Here, silk gland's developmental features, histomorphology, and transcriptome analyses were used to characterize changes in its structure and gene expression patterns between Nd mutant and WT/Dazao. Whole larval stage investigation showed that Nd-PSG undergoes an arrested/delayed development, which eventually resulted in a gland degeneration. By using section staining and transmission electron microscope, a blockade in intracellular vesicle transport from endoplasmic reticulum to Golgi apparatus (secretion-deficiency) and an increased number of autophagosomes and lysosomes were found in Nd-PSG's cytoplasm. Next, by using RNA sequencing and comparative transcriptomic analysis, 2178 differentially expressed genes were identified between Nd-PSG and WT-PSG, among which most of the DEGs associated with cellular stress responses (autophagy, ubiquitin-proteasome system, and heat shock response) were significantly up-regulated in Nd-PSG, suggesting that mutant FibH perturbed cellular homeostasis and resulted in an activation of adaptive responses in PSG cells. These findings reveal the molecular mechanism of the Naked pupa (Nd) mutation and provide insights into silk gland development as well as silk protein production in silkworm Bombyx mori.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Seda/metabolismo , Transcriptoma , Animais , Bombyx/metabolismo , Glândulas Exócrinas/citologia , Glândulas Exócrinas/crescimento & desenvolvimento , Fibroínas/biossíntese , Fibroínas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Larva/citologia , Larva/crescimento & desenvolvimento , Mutação , Análise de Sequência de RNA , Seda/genética
18.
BMC Genomics ; 20(1): 342, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060506

RESUMO

BACKGROUND: In our previous study, we identified four isoforms of the Bmovo gene, Bmovo-1, Bmovo-2, Bmovo-3 and Bmovo-4 from the silkworm ovary and verified that ovarian development was regulated by the BmOVO proteins. RESULTS: To understand the regulatory mechanisms of ovarian development, the regulation of four BmOVO isoforms on the B. mori ovarian tumor (Bmotu) promoter activity was investigated with luciferase reporter assays. The results showed the Bmotu promoter activity was positively regulated by BmOVO-1, BmOVO-2, BmOVO-3 and BmOVO-4 in a dose-dependent manner, of which BmOVO-2 had the highest transcriptional activation. However, the first (A1) and third acidic domains (A3) at the N-terminus of BmOVO-1 are transcriptional repression domains, while the fourth (A4) and fifth acidic domains (A5) are transcriptional activation domains. A recombinant BmOVO zinc-finger domain was found to bind to the GTACCGTTGTA sequence located at the Bmotu promoter. Furthermore, the Bmotu promoter activity was negatively regulated by 'Tal-like' peptide, which can trigger BmOVO-1 degradation at the N-terminus. CONCLUSIONS: These results will help us to further understand the regulatory mechanisms of BmOVO isoforms on Bmotu promoter activity and ovarian development in the silkworm.


Assuntos
Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Ovário/metabolismo , Regiões Promotoras Genéticas , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Ovário/crescimento & desenvolvimento , Isoformas de Proteínas , Ativação Transcricional
19.
Environ Toxicol ; 34(9): 1043-1051, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31120183

RESUMO

Acetamiprid is widely used for agricultural pest control. However, it remains poorly understood whether the environmental residues of acetamiprid have the potential effects on economic insect. In this study, we evaluated the effects of acetamiprid on silkworm growth and development. The exposure to trace amounts of acetamiprid significantly decreased body weight, viability, and spinning ability. In addition, the activity of trypsin in the midgut was decreased after exposure. DGE and KEGG pathway enrichment analysis revealed that the significantly differentially expressed genes were mainly involved in nutrient metabolism, stress responses, and inflammation pathways. These results, in combination with hematoxylin-eosin staining and transmission electron microscopy, indicated that acetamiprid could cause oxidative damage to midgut, lead to inflammatory responses, and affect the activities of midgut digestive enzymes, thus resulting in abnormal growth and development. Our findings greatly contributed to the evaluation of the effects of acetamiprid residues on other nontarget beneficial insect.


Assuntos
Bombyx/efeitos dos fármacos , Larva/efeitos dos fármacos , Neonicotinoides/toxicidade , Resíduos de Praguicidas/toxicidade , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/ultraestrutura , Relação Dose-Resposta a Droga , Dose Letal Mediana , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Tripsina/metabolismo
20.
Insect Biochem Mol Biol ; 110: 10-18, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31004794

RESUMO

The dynamic variability of transcription factors (TFs) and their binding sites makes it challenging to conduct genome-wide transcription regulation research. The silkworm Bombyx mori, which produces silk, is one of the most valuable model insects in the order Lepidoptera. The "opening" and "closing" of chromatin in different silk yield strains is associated with changes in silk production, making this insect a good model for studying the transcriptional regulation of genes. However, few studies have examined the open chromatin regions (OCRs) of silkworms, and studying OCR synergism and their function in silk production remains challenging. Here, we performed formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate OCRs from the silk glands of fifth-instar larvae of the DaZao and D872 strains. In total, 128,908 high confidence OCRs were identified and approximately 80% of OCRs were located in non-coding regions. OCRs upregulated adjacent genes and showed signal-dependent vulnerability to single-nucleotide polymorphisms. Mid- and low-signal OCRs were more likely to have single-nucleotide polymorphisms (SNP). Further, OCRs interacted with each other within a distance of 5 kb. We named the OCR interaction complex as the "cluster of related regions" (COREs). The functions of the CORE and its harbored OCRs showed some differences. Additionally, COREs enriched many silk protein synthesis-associated genes, some of which were upregulated. This study identified numerous high confidence regulation sites and synergistic regulatory modes of OCRs that affect adjacent genes. These results provide insight into silkworm transcriptional regulation and improve our understanding of cis-element cooperation.


Assuntos
Bombyx/genética , Cromatina/genética , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Seda/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Cromatina/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Seda/biossíntese
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