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1.
J Invertebr Pathol ; 170: 107323, 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31926972

RESUMO

Bombyx mori nucleopolyhedrosis virus (BmNPV) has always been a great challenge to the development and stability of the sericulture industry. LncRNAs have been reported to play important roles in gene expression regulation, development and immune response but the roles of lncRNAs in BmNPV infection and silkworm-BmNPV interaction are not clear. We used a genome-wide transcriptome analysis to identify the lncRNAs in Bombyx mori cells (BmN cells) and analyzed the differentially expressed lncRNAs, microRNAs and protein-coding genes in silkworm cells with or without BmNPV infection. A total of 13,159 candidate lncRNAs were identified in the BmN cells, among which 4450 lncRNAs were differentially expressed (DE) with 2837 up-regulated and 1613 down-regulated. In addition, 66 differentially expressed miRNAs (DEmiRNAs) and 7448 differentially expressed mRNAs (DEmRNAs) were identified, and DElncRNA-DEmiRNA-DEmRNA regulatory network was constructed. Gene expression was variable in 4973 of predicted lncRNA cis target genes in BmNPV infected cells. KEGG pathway analysis indicated that the target genes of DElncRNAs are enriched in ubiquitin mediated proteolysis, endocytosis and lysosome pathways. qRT-PCR validated the differential expression of several lncRNAs and miRNAs. Our results suggested that DElncRNAs participate in host response to BmNPV infection via interactions with their target genes and miRNAs. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmNPV infection and provide new insights into silkworm-pathogen interactions.

2.
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
3.
BMC Genomics ; 20(1): 736, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615392

RESUMO

BACKGROUND: Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the sustainability of the sericultural industry. DNA methylation is a widespread gene regulation mode in epigenetics, which plays an important role in host immune response. Until now, little has been known about epigenetic regulation on virus diseases in insects. This study aims to explore the role of DNA methylation in BmNPV proliferation. RESULTS: Inhibiting DNA methyltransferase (DNMT) activity of silkworm can suppress BmNPV replication. The integrated analysis of transcriptomes and DNA methylomes in silkworm midguts infected with or without BmNPV showed that both the expression pattern of transcriptome and DNA methylation pattern are changed significantly upon BmNPV infection. A total of 241 differentially methylated regions (DMRs) were observed in BmNPV infected midguts, among which, 126 DMRs were hyper-methylated and 115 DMRs were hypo-methylated. Significant differences in both mRNA transcript level and DNA methylated levels were found in 26 genes. BS-PCR validated the hypermethylation of BGIBMGA014008, a structural maintenance of chromosomes protein gene in the BmNPV-infected midgut. In addition, DNMT inhibition reduced the expression of inhibitor of apoptosis family genes, iap1 from BmNPV, Bmiap2, BmSurvivin1 and BmSurvivin2. CONCLUSION: Our results indicate that DNA methylation plays positive roles in BmNPV proliferation and loss of DNMT activity could induce the apoptosis of infected cells to suppress BmNPV proliferation. Our results may provide a new idea and research direction for the molecular mechanism on insect-virus interaction.


Assuntos
Bombyx/virologia , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica/veterinária , /fisiologia , Animais , Bombyx/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Análise de Sequência de RNA , Replicação Viral/efeitos dos fármacos
4.
J Invertebr Pathol ; 166: 107227, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31386830

RESUMO

Beauveria bassiana, a pathogen of the economically important silkworm (Bombyx mori), causes serious losses in the sericulture industry; however, the mechanisms underlying B. bassiana infection and the silkworm response are not fully understood. To obtain new insights into the interaction between B. bassiana and its host, hemolymph samples from fifth instar silkworm larvae infected with B. bassiana were analyzed at 36-h post-inoculation using a label-free LC-MS/MS proteomic technique. In total, 671 proteins were identified in the hemolymph, including 87 differentially expressed proteins, 42 up-regulated and 45 down-regulated in infected larvae. Six were detected only in infected larvae, and five were detected only in uninfected larvae. Based on GO annotations, 48 of the differentially expressed proteins were involved in molecular functions, 42 were involved in biological processes, and 39 were involved in cell components. A KEGG pathway analysis indicated that these differentially expressed proteins participate in 85 signal transduction pathways, including the amoebiasis, MAPK signaling, Hippo signaling, Toll and Imd signaling, and lysosome pathways. The silkworm hemolymph is the main site for B. bassiana replication. We identified differentially expressed proteins involved in the regulation of the host response to B. bassiana infection, providing important experimental data for the identification of key factors contributing to the interaction between the pathogenic fungus and its host.

5.
J Proteomics ; 203: 103379, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31102755

RESUMO

Heat shock protein 90, an essential chaperone responsible for the correct maturation of key proteins, has been confirmed to facilitate Bombyx mori nucleopolyhedrovirus (BmNPV) proliferation but the mechanism is not clear. In this study, we use quantitative proteomics analysis to investigate the mechanism of Hsp90 in BmNPV replication. In total, 195 differentially expressed proteins (DEPs) were identified with 136 up-regulated proteins and 59 down-regulated proteins. The protein expression level of small heat shock proteins, immune-related proteins, cellular DNA repair-related proteins and zinc finger proteins is significantly enhanced while that of protein kinases is declined. KEGG pathway analysis reveals that DEPs are involved in longevity regulating pathway, mTOR signaling pathway, FoxO signaling pathway and Toll and Imd signaling pathway. Based on the DEPs results, we speculate that inhibition of Hsp90 suppresses the BmNPV infection may because it could not only stimulate the host innate immune, induce small heat shock proteins expression to maintain the cellular proteostasis but activate host transcription factors to bind to virus DNA or protein and subsequently hinder virus replication. The results will help understand the roles of Hsp90 in BmNPV infection and shed light on new clue to illustrate the molecular mechanism of silkworm-virus interaction. SIGNIFICANCE: This is the first report on Hsp90 roles in BmNPV infection based on proteomic analysis. Our findings may provide new clue and research orientation to illustrate the molecular mechanism of silkworm-virus interaction and a set of BmHsp90 candidate clients, which may involve in BmNPV infection in BmN cells.

6.
Mol Immunol ; 109: 134-139, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30947109

RESUMO

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Accumulating studies in recent years suggest that insect viruses infection can change the host microRNAs (miRNAs) expression profile and both cellular and viral miRNAs play roles in host-pathogen interactions. Until now, the functional analysis of miRNA encoded by silkworm for host-virus interaction is limited. In this study, we validate the down-regulation of bmo-miR-2819 upon BmNPV infection by qRT-PCR and confirm the BmNPV immediately early 1 gene, ie-1 is one of the targets for bmo-miR-2819 based on the results of dual luciferase report assay. Overexpression of bmo-miR-2819 can significantly decline the abundance of IE-1 protein level in BmNPV-infected silkworm larvae. Further, the expression level of polyhedrin gene and VP39 protein of BmNPV in the infected larvae after applying bmo-miR-2819 mimics was significantly decreased comparing with that of larvae with mimic control. Our results suggest that overexpression of bmo-miR-2819 could suppress BmNPV replication by down-regulating the expression of BmNPV ie-1 gene, which demonstrate that cellular miRNAs could affect virus infection by regulating the expression of virus genes.


Assuntos
Bombyx/genética , Bombyx/virologia , Genes Virais , MicroRNAs/genética , /fisiologia , Replicação Viral , Animais , Sequência de Bases , Linhagem Celular , Regulação para Baixo/genética , MicroRNAs/metabolismo , Reprodutibilidade dos Testes
7.
Insect Sci ; 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30869181

RESUMO

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5' untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.

8.
J Invertebr Pathol ; 163: 34-42, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30825479

RESUMO

Storage proteins in the 30 K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30 K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30 K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311 bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4 h and the time for death of all infected larvae was postponed by 10 h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.

9.
Sci Rep ; 9(1): 898, 2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696919

RESUMO

The complete genome of Cordyceps militaris was sequenced using single-molecule real-time (SMRT) sequencing technology at a coverage over 300×. The genome size was 32.57 Mb, and 14 contigs ranging from 0.35 to 4.58 Mb with an N50 of 2.86 Mb were assembled, including 4 contigs with telomeric sequences on both ends and an additional 8 contigs with telomeric sequences on either the 5' or 3' end. A methylome database of the genome was constructed using SMRT and m4C and m6A methylated nucleotides, and many unknown modification types were identified. The major m6A methylation motif is GA and GGAG, and the major m4C methylation motif is GC or CG/GC. In the C. militaris genome DNA, there were four types of methylated nucleotides that we confirmed using high-resolution LCMS-IT-TOF. Using PacBio Iso-Seq, a total of 31,133 complete cDNA sequences were obtained in the fruiting body. The conserved domains of the nontranscribed regions of the genome include TATA boxes, which are the initial regions of genome replication. There were 406 structural variants between the HN and CM01 strains, and there were 1,114 structural variants between the HN and ATCC strains.

10.
Sci Rep ; 7(1): 16013, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29167521

RESUMO

DNA methylation is an important epigenetic modification that regulates a wide range of biological processes including immune response. However, information on the epigenetics-mediated immune mechanisms in insects is limited. Therefore, in this study, we examined transcriptomes and DNA methylomes in the fat body and midgut tissues of silkworm, Bombyx mori with or without B. mori cytoplasmic polyhedrosis virus (BmCPV) infection. The transcriptional profile and the genomic DNA methylation patterns in the midgut and fat body were tissue-specific and dynamically altered after BmCPV challenge. KEGG pathway analysis revealed that differentially methylated genes (DMGs) could be involved in pathways of RNA transport, RNA degradation, nucleotide excision repair, DNA replication, etc. 27 genes were shown to have both differential expression and differential methylation in the midgut and fat body of infected larvae, respectively, indicating that the BmCPV infection-induced expression changes of these genes could be mediated by variations in DNA methylation. BS-PCR validated the hypomethylation of G2/M phase-specific E3 ubiquitin-protein ligase-like gene in the BmCPV infected midgut. These results demonstrated that epigenetic regulation may play roles in host-virus interaction in silkworm and would be potential value for further studies on mechanism of BmCPV epithelial-specific infection and epigenetic regulation in the silkworm.


Assuntos
Bombyx/virologia , Metilação de DNA/genética , Epigênese Genética/genética , Reoviridae/genética , Reoviridae/patogenicidade , Animais , Proteínas de Insetos/genética , MicroRNAs
11.
Virus Genes ; 53(4): 643-649, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28493152

RESUMO

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major pathogens that pose a big challenge to the sericulture industry. Growing evidences have shown that microRNAs play key roles in the regulations of host-pathogen interactions in insects. MicroRNAs have been found in silkworms, whether and how they affect the silkworm-BmCPV interactions are still unknown. Here we investigate the effect of miR-274-3p on the BmCPV replication in the BmCPV-infected silkworm larvae. In our study, BmCPV Nonstructural protein 5 (NS5) was identified to be the target of miR-274-3p based on bioinformatics analysis and luciferase reporter assay. The abundance of NS5 was significantly increased in the presence of miR-274-3p inhibitor based on the qRT-PCR and Western blotting results. Further, qRT-PCR results revealed that the expression of polyhedrin gene of BmCPV in the larvae after applying miR-274-3p inhibitor was significantly increased comparing with that of larvae with negative control. Our results suggest that inhibition of miR-274-3p could facilitate BmCPV replication by up-regulating BmCPV NS5 gene expression and are insightful for further exploring the interactions between silkworm and BmCPV.


Assuntos
Bombyx/metabolismo , Bombyx/virologia , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Reoviridae/fisiologia , Proteínas não Estruturais Virais/genética , Replicação Viral , Animais , Bombyx/genética , Regulação Viral da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , MicroRNAs/metabolismo , Reoviridae/genética , Reoviridae/crescimento & desenvolvimento , Proteínas não Estruturais Virais/metabolismo
12.
Virus Res ; 233: 86-94, 2017 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-28286035

RESUMO

Viral microRNAs (miRNAs) have been demonstrated to play important roles in virus-host interactions. Some RNA virus-encoded miRNAs have been reported to promote viral replication and may be used as potential drug targets. Bombyx mori cypovirus (BmCPV), an important pathogen of silkworm, is a double-stranded RNA virus frequently causing serious damages in sericulture. Research on miRNA encoded by BmCPV may be useful to elucidate the BmCPV-host interaction and to develop new anti-viral methods. In our previous study, small RNA libraries of the midgut of BmCPV-infected silkworm have been generated by deep sequencing and several BmCPV-encoded putative miRNAs were predicted. In this study, two putative miRNAs encoded by BmCPV were identified and then validated by stem-loop qRT-PCR and northern blot. They are BmCPV-miR-3 encoded by the third genomic RNA segment of BmCPV (478-497bp) and BmCPV-miR-5 encoded by the fifth genomic RNA segment (2481-2500bp), both are 20bp and encoded by ORF regions. miRNA expression could be detected as early as 5h after BmCPV infection, and the expression level of BmCPV-miR-3 is much higher than that of BmCPV-miR-5 in the course of infection. Three potential target genes were predicted in the host genome, two for BmCPV-miR-3 and one for BmCPV-miR-5, but just one in the virus genome for BmCPV-miR-3 only, with the binding sites all in coding regions. Dual luciferase assay and qRT-PCR indicated that BmCPV-miR-3 could down-regulate the expression of both its two target genes, but no regulatory effect by BmCPV-miR-5 on its target gene was detected. In contrast, BmCPV-miR-3 could up-regulate the viral target. This is the first report that an insect double stranded RNA virus may generate miRNAs and the results obtained will benefit the future study of the functions of BmCPV-encoded miRNAs on viral replication and virus-host interaction.


Assuntos
Genoma Viral , MicroRNAs/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Reoviridae/genética , Animais , Sequência de Bases , Bombyx/virologia , Genes Reporter , Interações Hospedeiro-Patógeno , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/biossíntese , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/metabolismo , RNA Viral/biossíntese , Reoviridae/metabolismo , Reoviridae/patogenicidade , Replicação Viral
13.
Biomed Res Int ; 2017: 9390803, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28194425

RESUMO

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.


Assuntos
Beauveria/imunologia , Bombyx , Regulação da Expressão Gênica/imunologia , Proteínas de Insetos , Animais , Bombyx/genética , Bombyx/imunologia , Bombyx/metabolismo , Bombyx/microbiologia , Clonagem Molecular , Biologia Computacional , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia
14.
Gene ; 600: 55-63, 2017 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-27836665

RESUMO

Gloverin2 is a cationic and glycine-rich antimicrobial peptide whose expression can be induced in fat body of silkworm (Bombyx mori) larvae exposed to bacteria. The purpose of this study is to identify the roles of Bombyx mori gloverin2 (Bmgloverin2) during entomopathogenic fungus Beauveria bassiana infection. Fluorescent quantitative real-time PCR analysis indicated that the relative expression level of Bmgloverin2 gene was up-regulated in the silkworm larvae infected by B. bassiana. The cDNA of Bmgloverin2 was cloned from the silkworm by RT-PCR and the DNA segment of the Bmgloverin2 peptide (without signal peptide sequence) was inserted into pCzn1 expression plasmid and expressed in E. coli ArcticExpress (DE3). SDS-PAGE results revealed that soluble recombinant Bmgloverin2 was successfully expressed and purified. Polyclonal antibody against the Bmgloverin2 was successfully produced with the expressed recombinant protein. Western blot analysis indicated that Bmgloverin2 could be detected in the fat body of silkworm larvae infected with B. bassiana, suggesting that the expression of Bmgloverin2 could be induced by B. bassiana infection in silkworm. Antifungal assays indicated that the Bmgloverin2 had a synergistic antifungal activity with B. mori cecropin A (BmCecA) to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that Bmgloverin2 exhibits synergistic effect with BmCecA in antifungal activity against B. bassiana. The study demonstrates that Bmgloverin2 is an antifungal protein which plays an important role in synergistic antifungal activity with other antimicrobial peptide in silkworm.


Assuntos
Antifúngicos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Beauveria/efeitos dos fármacos , Beauveria/patogenicidade , Bombyx/genética , Proteínas de Insetos/administração & dosagem , Proteínas de Insetos/genética , Proteínas/administração & dosagem , Proteínas/genética , Animais , Peptídeos Catiônicos Antimicrobianos/fisiologia , Bombyx/microbiologia , Bombyx/fisiologia , Sinergismo Farmacológico , Genes de Insetos , Proteínas de Insetos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas/fisiologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Regulação para Cima
15.
J Proteomics ; 152: 300-311, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-27908826

RESUMO

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. SIGNIFICANCE: The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes the silkworm to death, which negatively affects the sericulture industry. Studies on insect antiviral immunity and on interactive mechanisms between host cells and BmCPV are in their infancy and remain insufficient. In order to obtain an overall view of silkworm response to BmCPV infection, we performed a proteomic analysis of the midgut of silkworm responses to BmCPV infection by iTRAQ. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection.


Assuntos
Bombyx/virologia , Sistema Digestório/química , Proteoma/análise , Proteômica/métodos , Reoviridae/patogenicidade , Animais , Bombyx/anatomia & histologia , Bombyx/química , Cromatografia Líquida , Sistema Digestório/virologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Insetos/metabolismo , RNA Mensageiro , Espectrometria de Massas em Tandem
17.
Saudi J Biol Sci ; 24(7): 1614-1619, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30294230

RESUMO

MicroRNAs (miRNAs) have emerged as key players in host-pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.

18.
PLoS One ; 11(11): e0165865, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27806111

RESUMO

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV.


Assuntos
Bombyx/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Análise de Sequência de RNA/métodos , Animais , Bombyx/genética , DNA (Citosina-5-)-Metiltransferases/genética , Regulação da Expressão Gênica , Ontologia Genética , Biblioteca Genômica , Interações Hospedeiro-Patógeno , Proteínas Virais/genética
19.
Gene ; 595(1): 69-76, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27693371

RESUMO

Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better understand the molecular mechanisms of JAK/STAT signaling pathway in antifungal immune response in silkworm.


Assuntos
Beauveria/imunologia , Bombyx , Proteínas de Insetos , Janus Quinases , Fatores de Transcrição STAT , Transdução de Sinais , Animais , Bombyx/genética , Bombyx/imunologia , Bombyx/microbiologia , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Janus Quinases/genética , Janus Quinases/imunologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia
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