RESUMO
The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.
Assuntos
Bombyx , Genoma de Inseto , Animais , Bombyx/genética , Biologia Computacional , Genômica , Metadados , MultiômicaRESUMO
Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development.
Assuntos
Bombyx , Proteínas de Insetos , Fatores de Transcrição Box Pareados , Animais , Bombyx/classificação , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Filogenia , Interferência de RNA , Seda/genética , Seda/metabolismoRESUMO
The bHLH domain transcription factor, Bombyx mori-derived dimmed (Bmdimm), is directly regulated by the JH-BmMet/BmSRC-BmKr-h1 pathway and plays a key role in regulating the expression of FibH, which codes the main component of silk protein. However, the other roles of Bmdimm in silk protein synthesis remain unclear. Here, we established a Bmdimm knockout (KO) line containing a 7-bp deletion via CRISPR/Cas9 system, which led to the absence of the bHLH domain. The expression level of silk protein genes and silk yield decreased significantly in the Bmdimm KO line. Moreover, knocking out Bmdimm led to shortened larval stages and significant weight loss in larvae and adults. Bmdimm was found to be highly expressed in the silk gland, but it was also expressed in the fat body. The expression level of Bmkr-h1 in the fat body was significantly downregulated in the Bmdimm KO line. Exogenous JHA treatment upregulated Bmkr-h1 and rescued the phenotype of larval growth in the Bmdimm KO line. In conclusion, knocking out Bmdimm led to a shortened larval stage via the inhibition of Bmkr-h1 expression, then reduced silk yield. These findings help to elucidate the regulatory mechanism of fibroin synthesis and larval development in silkworms.
Assuntos
Bombyx , Fibroínas , Animais , Seda/genética , Bombyx/genética , Bombyx/metabolismo , Larva/genética , Larva/metabolismo , Técnicas de Inativação de Genes , Fibroínas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
In Bombyx mori, there are more than 30 mutant strains exhibiting the translucent larval skin resulting from a decrease in the uric acid content in epidermal cell. Of these, the Chinese translucent (oc) mutant presents a moderately translucent larval skin. Previously, we narrowed the region linked to the oc phenotype to approximately 234 kb by positional cloning, and found that BmMCT9 was severely suppressed in the mutant. Here, we analyzed the mutation and potential molecular function of BmMCT9. Sequence analysis showed that a 2,624-bp fragment of BmMCT9 promoter region was replaced by a 22-bp sequence in the mutant. Luciferase reporter gene assay confirmed that BmMCT9 promoter activity in the mutant was significantly lower than that in the wild type. Knockdown of BmMCT9 caused a translucent phenotype in the first-instar silkworm larvae. Immunoblotting analysis showed that BmMCT9 expression was severely reduced in the mutant than in the wild type, and immunofluorescence showed that BmMCT9 existed mainly within the cytoplasm of epidermal cells. Together, our results suggest that reduced levels of BmMCT9 were responsible for the translucent phenotype of oc mutant, and that BmMCT9 might function in intracellular vesicles facing the cytoplasm including urate granules in silkworm integument.
Assuntos
Bombyx/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Úrico/metabolismo , Animais , Bombyx/metabolismo , Clonagem Molecular/métodos , Genótipo , Proteínas de Insetos/metabolismo , Larva/genética , Transportadores de Ácidos Monocarboxílicos/genética , Mutação/genética , Fenótipo , Filogenia , Pigmentação/genética , Transporte Proteico/fisiologia , Análise de Sequência de DNA/métodosRESUMO
Spodoptera litura is a polyphagous pest and can feed on more than 100 species of plants, causing great damage to agricultural production. The SNP results showed that there were gene exchanges between different regions. To explore the variations of larger segments in S. litura genome, we used genome resequencing samples from 14 regions of China, India, and Japan to study the copy number variations (CNVs). We identified 3976 CNV events and 1581 unique copy number variation regions (CNVRs) occupying the 108.5â¯Mb genome of S. litura. A total of 5527 genes that overlapped with CNVRs were detected. Selection signal analysis identified 19 shared CNVRs and 105 group-specific CNVRs, whose related genes were involved in various biological processes in S. litura. We constructed the first CNVs map in S. litura genome, and our findings will be valuable for understanding the genomic variations and population differences of S. litura.
Assuntos
Variações do Número de Cópias de DNA , Spodoptera/genética , Animais , Expressão Gênica , Genes de Insetos , Genoma de Inseto , Seleção Genética , Spodoptera/metabolismoRESUMO
Osiris is an insect-specific gene family with multiple biological roles in development, phenotypic polymorphism, and protection. In the silkworm, we have previously identified twenty-five Osiris genes with high evolutionary conservation and remarkable synteny among several insects. Bombxy mori Osiris9a (BmOsi9a) is expressed only in the silk gland, particularly in the middle silk gland (MSG). However, the biological function of BmOsi9a is still unknown. In this study, we overexpressed BmOsi9a in the silk gland by germline transgene expression. BmOsi9a was overexpressed not only in the MSG but also in the posterior silk gland (PSG). Interestingly, BmOsi9a could be secreted into the lumen in the MSG but not in the PSG. In the silk fiber, overexpressed BmOsi9a interacted with Sericin1 in the MSG, as confirmed by a co-immunoprecipitation assay. The overexpression of BmOsi9a altered the secondary structure and crystallinity of the silk fiber, thereby changing the mechanical properties. These results provide insight into the mechanisms underlying silk proteins secretion and silk fiber formation.
Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Sericinas/metabolismo , Seda/ultraestrutura , Animais , Animais Geneticamente Modificados , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Estrutura Secundária de Proteína , Sericinas/química , Seda/química , Seda/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.
Assuntos
Bacillus/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Bacillus/patogenicidade , Bombyx , Linhagem Celular , AMP Cíclico/metabolismo , Humanos , Isoquinolinas/farmacologia , Modelos Biológicos , Sulfonamidas/farmacologiaRESUMO
Bombyx mori is a poikilothermic insect and is economically important for silk production. Drastic changes in the ambient temperature have a negative impact on sericulture. However, the reason as to why high temperature is associated with the occurrence of diseases in silkworm and the response of silkworm to low temperature remain unclear and were the focus of the present study. Dazao silkworm exposed to 13 °C (DZ-13), 25 °C (DZ-25), and 37 °C (DZ-37) were used for RNA-seq analysis. There were 478 and 194 upregulated differentially expressed genes (DEGs) in DZ-13 and DZ-37 while 49 and 273 downregulated DEGs in DZ-13 and DZ-37, respectively. Eight DEGs were co-upregulated, in which seven genes were for heat shock proteins (Hsps), implying that Hsps play important roles in the tolerance of silkworm to high and low temperature. Gene ontology analysis revealed that the developmental process was downregulated in DZ-13. All the DEGs in the oxidative phosphorylation and insulin signaling pathways were upregulated in DZ-13. Several cuticular proteins and ATP synthesis-related genes were upregulated in DZ-13, suggesting that thickening of the cuticle and increase in the ATPase expression would help silkworms to protect themselves from low temperature-induced stress. Several immune-related genes, such as BmRel and BmSerpin-2, were downregulated in DZ-37, revealing that the resistance of silkworm is decreased under high temperature shock resulting in susceptibility to pathogens. Thus, the increase in the thermo-tolerance of silkworm should be related to the enhancement in the pathogen resistance.
Assuntos
Bombyx/genética , Perfilação da Expressão Gênica , Temperatura Alta , Proteínas de Insetos/genética , Animais , Replicação do DNA , Regulação da Expressão Gênica , Ontologia Genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Insetos/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Seda/metabolismo , Estresse Fisiológico/genéticaRESUMO
The genes responsible for silk biosynthesis are switched on and off at particular times in the silk glands of Bombyx mori. This switch appears to be under the control of endogenous and exogenous hormones. However, the molecular mechanisms by which silk protein synthesis is regulated by the juvenile hormone (JH) are largely unknown. Here, we report a basic helix-loop-helix transcription factor, Bmdimm, its silk gland-specific expression, and its direct involvement in the regulation of fibroin H-chain (fib-H) by binding to an E-box (CAAATG) element of the fib-H gene promoter. Far-Western blots, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays revealed that Bmdimm protein interacted with another basic helix-loop-helix transcription factor, Bmsage. Immunostaining revealed that Bmdimm and Bmsage proteins are co-localized in nuclei. Bmdimm expression was induced in larval silk glands in vivo, in silk glands cultured in vitro, and in B. mori cell lines after treatment with a JH analog. The JH effect on Bmdimm was mediated by the JH-Met-Kr-h1 signaling pathway, and Bmdimm expression did not respond to JH by RNA interference with double-stranded BmKr-h1 RNA. These data suggest that the JH regulatory pathway, the transcription factor Bmdimm, and the targeted fib-H gene contribute to the synthesis of fibroin H-chain protein in B. mori.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fibroínas/genética , Proteínas de Insetos/genética , Hormônios Juvenis/genética , Seda/biossíntese , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Bombyx/genética , Fibroínas/metabolismo , Proteínas de Insetos/biossíntese , Hormônios Juvenis/metabolismo , Larva , Regiões Promotoras Genéticas/genética , Sericinas/biossíntese , Sericinas/genéticaRESUMO
Juvenile hormone (JH) regulates the insect growth and development. JH appears in the hemolymph bound by a specific glycoprotein, juvenile hormone-binding protein (JHBP), which serves as a carrier to release the hormone to target tissues and cells. However, JHBP family candidates, expression patterns, and functional implications are still unclear. In this study, we identified 41 genes-containing conserved JHBP domains distributed across eight chromosomes of the silkworm Bombyx mori. A phylogenetic tree showed that the silkworm JHBP (BmJHBP) genes could be classified into two major branches and four subfamilies. Microarray data revealed that BmJHBP genes exhibit various expression patterns and are expressed in different tissues, periods, and sexes. The expression of BmJHBP genes was generally higher in the head, integument, midgut, fat body, testis, and ovary than in the anterior of the silk gland (ASG), median of the silk gland (MSG), posterior of the silk gland (PSG), hemocyte, and Malpighian tubule. BmJHBPd2, in particular, was investigated by Western Blotting, and immunofluorescent assay and was found to be highly expressed in the PSG cytoplasm on day 3 of the fifth instar, coinciding with silk production. Taken together, our findings will be useful in improving understanding the complexity of the JHBP family, and will lay the foundation of explaining functional characterization for further research.
Assuntos
Bombyx/genética , Proteínas de Transporte/genética , Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cromossomos de Insetos/genética , Regulação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Família Multigênica , Filogenia , Seda/biossínteseRESUMO
Several pathogenic microorganisms have been used to investigate the genome-wide transcriptional responses of Bombyx mori to infection. However, studies have so far each focused on one microorganism, and systematic genome-wide comparison of transcriptional responses to different pathogenic microorganisms has not been undertaken. Here, we surveyed transcriptional responses of B. mori to its natural bacterial, viral, and fungal pathogens, Bacillus bombyseptieus, B. mori nucleopolyhedrovirus (BmNPV), and Beauveria bassiana, respectively, and to nonpathogenic Escherichia coli, by microarray analysis. In total, the expression of 2,436, 1,804, 1,743, and 912 B. mori genes was modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, and E. coli, respectively. Notably, the expression of 620, 400, 177, or 165 of these genes was only modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, or E. coli, respectively. In contrast to the expression of genes related to juvenile hormone synthesis and metabolism, that of genes encoding juvenile hormone binding proteins was microorganism-specific. Three basal metabolic pathways were modulated by infection with any of the four microorganisms, and 3, 14, 5, and 2 metabolic pathways were specifically modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, and E. coli, respectively. Interestingly, BmNPV infection modulated the JAK/STAT signaling pathway, whereas both the Imd and Toll signaling pathways were modulated by infection with B. bombyseptieus, B. bassiana, or E. coli These results elucidate potential molecular mechanisms of the host response to different microorganisms, and provide a foundation for further work on host-pathogen interaction.
Assuntos
Bombyx/genética , Bombyx/microbiologia , Interações Hospedeiro-Patógeno/genética , Transcrição Gênica , Animais , Bacillus/fisiologia , Beauveria/fisiologia , Bombyx/crescimento & desenvolvimento , Bombyx/virologia , Escherichia coli/fisiologia , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Larva/microbiologia , Larva/virologia , Análise em Microsséries , Nucleopoliedrovírus/fisiologia , Transdução de SinaisRESUMO
The ATP-binding cassette (ABC) superfamily is a large protein family with diverse physiological functions in all kingdoms of life. One distinguished subfamily, the pleiotropic drug resistance (PDR) transporters, has only been identified in plants and fungi. Here, we identified a Nicotiana tabacum PDR gene, NtPDR6, which is a homolog of Petunia hybrida PDR1. The full-length cDNA of NtPDR6 had a 4482-bp open reading frame encoding a full-size ABC transporter with 1493 amino acids. Sequence comparison showed that NtPDR6 had high homology with plant PDR proteins. NtPDR6 was strongly induced by phosphate starvation as well as by 1-naphthalene acetic acid. Tissue expression pattern analysis showed that NtPDR6 was detected in all surveyed tissues but preferentially in roots. We cloned the 1.3-kb NtPDR6 promoter and found that there was one phosphate starvation response-related element Pho-like and several root-specific expression-related elements rootmotiftapox1 in the NtPDR6 promoter. A tissue-specific pattern of NtPDR6 promoter-ß-glucuronidase expression was dominantly observed in subepidermal cells and the elongation zone of lateral roots. RNA interference technology was used to knock down NtPDR6 expression, and there was a significantly increased branching phenotype in the NtPDR6 knockdown plants. These data suggest that NtPDR6 plays a key role in regulation of shoot branching processes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Morfogênese , Nicotiana/genética , Proteínas de Plantas/genética , Brotos de Planta/crescimento & desenvolvimento , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Especificidade de Órgãos/genética , Fenótipo , Fosfatos/deficiência , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA , Nicotiana/metabolismoRESUMO
The CRISPR/Cas9 gene-editing system is a standard technique in functional genomics, with widespread applications. However, the establishment of a CRISPR/Cas9 system is challenging. Previous studies have presented numerous methodologies for establishing a CRISPR/Cas9 system, yet detailed descriptions are limited. Additionally, the difficulties in obtaining the necessary plasmids have hindered the replication of CRISPR/Cas9 techniques in other laboratories. In this study, we share a detailed and simple CRISPR/Cas9 knockout system with optimized steps. The results of gene knockout experiments in vitro and in vivo show that this system successfully knocked out the target gene. By sharing detailed information on plasmid sequences, reagent codes, and methods, this study can assist researchers in establishing gene knockout systems.
RESUMO
Silk proteins, as natural macromolecules, have extensive applications in biomaterials and biomedicine. In the silkworm, the expression of silk protein genes is negatively associated with ecdysone during the molt stage, while it is positively correlated with juvenile hormone during the intermolt stage. In our previous study, overexpression of an isoform Z2 of Broad Complex (BmBrC-Z2), an ecdysone early response factor, significantly reduced the expression of silk protein genes. However, the underlying regulatory mechanism remains unclear. In this study, we conducted transcriptomic analysis and found that overexpressing BmBrC-Z2 significantly upregulated the expression level of multiprotein bridging factor 2 (BmMBF2), an inhibitor of fibroin heavy chain (FibH). Further investigations revealed that BmBrC-Z2 directly regulated BmMBF2 by binding to cis-regulatory elements, as demonstrated using Dual-Luciferase Reporter Gene Assay, EMSA, and ChIP-PCR assay. Additionally, when using the CRISPR/Cas9 system to knock out BmMBF2, silk protein genes were significantly upregulated during the molt stage of mutant larvae. These findings uncover the negative regulation of silk protein synthesis by the ecdysone signaling cascade, specifically through the manipulation of BmMBF2 expression during the molt stage. This study enhances to our understanding of the temporal regulatory mechanism governing silk protein synthesis and offers a potential strategy for improving silk yield.
RESUMO
The efficient synthesis of silk protein is heavily reliant on the ingestion of massive nutrients during the peak growth phase in the silkworm. However, the molecular mechanism of nutritional regulation of silk protein synthesis remains unknown. In this study, we investigated the impact of nutrient deficiency on the synthesis of silk protein. Nutritional deficiency led to a reduction in silk yield, accompanied by decreased levels of silk proteins and fibroin heavy chain (FibH)-activating transcription factors SGF1 and Dimm. Furthermore, insulin enhanced the protein levels of SGF1 and Dimm, which can be attenuated by specific inhibitors of PI3K. Co-immunoprecipitation analysis showed that the nutrient pathway factor protein kinase B (Akt) could interact with SGF1 protein. Knockdown of Akt reduced the phosphorylation level of SGF1 and impedes its nuclear translocation. Further studies revealed that SGF1 was directly bound to Fkh site in the 22-43 region upstream of ATG of Dimm gene to activate its transcription. In conclusion, during the peak growth phase, nutrition promotes the massive synthesis of silk protein through the PI3K-Akt-SGF1-Dimm pathway. This study offers valuable insights into the efficient synthesis of silk proteins and establishes a theoretical foundation for improving silk yield.
RESUMO
The midgut is an important organ for digestion and absorption of nutrients and immune defense in the silkworm Bombyx mori. In an attempt to create a tool for midgut research, we cloned the 1080 bp P2 promoter sequence (P2P) of a highly expressed midgut-specific gene in the silkworm. The transgenic line (P2) was generated via embryo microinjection, in which the expression of EGFP was driven by P2P. There was strong green fluorescence only in the midgut of P2. RT-PCR and Western blot showed that P2P was a midgut-specific promoter with activity throughout the larval stage. A transgenic truncation experiment suggested that regions -305 to -214 and +107 to +181 were very important for P2P activity. The results of this study revealed that we have identified a midgut-specific promoter with a high level of activity in the silkworm that will aid future research and application of silkworm genes.
Assuntos
Bombyx/genética , Trato Gastrointestinal/citologia , Genes de Insetos , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Fluorescência , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/genética , Larva/metabolismo , Microinjeções , Dados de Sequência Molecular , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
The mitogen-activated protein (MAP) kinase cascade is an important signaling module which is involved in biotic and abiotic stress responses as well as plant growth and development. In this study, we identified 17 tobacco MAPKs including 11 novel tobacco MAPK genes that have not been identified before. Comparative analysis with MAPK gene families from other plants, such as Athaliana thaliana, rice and poplar, suggested that tobacco MAPKs (such as NtMPK1, NtMPK3 and NtMPK8) might play similar functions in response to abiotic and biotic stresses. QRT-PCR analysis revealed that a total of 14 NtMPKs were regulated by SA and/or MeJA, suggesting their potential roles involved in plant defense response. In addition, 6 NtMPKs were induced by drought treatment, implying their roles in response to drought stress. Our results indicated that most of tobacco MAPK might be involved in plant defense response, which provides the basis for further analysis on physiological functions of tobacco MAPKs.
Assuntos
Evolução Molecular , Proteínas Quinases Ativadas por Mitógeno/genética , Nicotiana/enzimologia , Nicotiana/genética , Proteínas de Plantas/genética , Acetatos/farmacologia , Sequência de Aminoácidos , Clonagem Molecular , Sequência Consenso , Ciclopentanos/farmacologia , Indução Enzimática , Regulação da Expressão Gênica de Plantas , Anotação de Sequência Molecular , Dados de Sequência Molecular , Oxilipinas/farmacologia , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Reguladores de Crescimento de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Ácido Salicílico/farmacologia , Homologia de Sequência de Aminoácidos , Estresse FisiológicoRESUMO
Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wild-type neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30 degrees C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (sch(l)) do not hatch even at room temperature (25 degrees C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BmTh coding sequences were identical among sch, sch(l), and wild-type. However, in sch the approximately 70-kb sequence was replaced with approximately 4.6 kb of a Tc1-mariner type transposon located approximately 6 kb upstream of BmTh, and in sch(l), a large fragment of an L1Bm retrotransposon was inserted just in front of the transcription start site of BmTh. In both cases, we observed a drastic reduction of BmTh expression. Use of RNAi with BmTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, pnd(+) and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BmTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color.
Assuntos
Bombyx/enzimologia , Bombyx/genética , Genes de Insetos/genética , Mutação/genética , Pigmentação/genética , Caracteres Sexuais , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Mapeamento Cromossômico , Ligação Genética , Genoma/genética , Larva , Fenótipo , Reprodutibilidade dos TestesRESUMO
Juvenile hormone esterase (JHE) is the specific enzyme that degrades juvenile hormone (JH) and regulates the JH titer in insects. JH also regulates the development of the silk gland and the synthesis and secretion of silk proteins in Bombyx mori. Here, we identified nine possible JHE family members, Bmjhe1-9. Notably, Bmjhe6 is specifically expressed in the silk gland. Using semi-quantitative, quantitative real-time RT-PCR and Western blot, it was confirmed that Bmjhe6 was specifically expressed in the middle silk gland (MSG) with high levels in the anterior region of the MSG (A-MSG). The immunofluorescence localization analysis revealed that Bmjhe6 is produced within cells, secreted into the gland lumen, and co-transported with silk proteins into the anterior silk gland (ASG). In vitro hormone induction experiments demonstrated that Bmjhe6 responds to a JH analog, increasing its expression after 12-24 h, whereas 20-hydroxyecdysone inhibited it. In addition, Bmjhe6 knockdown using dsBmjhe6 injections accelerated larval development, resulting in increased larval body and silk gland weight. This induced disordered sericin genes (Ser2, Ser3) expression, and key genes in the JH synthesis pathway (BmKr-h1 and BmMet1) were significantly upregulated along with the transcription factors (SGF-1 and Sage). These results indicate that Bmjhe6 plays an important role in silk gland growth and silk protein synthesis by modulating JH signal.
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Transgenic technology is a powerful tool for improving disease-resistant species. Bmlipase-1, purified from the midgut juice of Bombyx mori, showed strong antiviral activity against B. mori nucleopolyhedrovirus (BmNPV). In an attempt to create an antiviral silkworm strain for sericulture, a transgenic vector overexpressing the Bmlipase-1 gene was constructed under the control of a baculoviral immediate early-1 (IE1) promoter. Transgenic lines were generated via embryo microinjection. The mRNA level of Bmlipase-1 in the midguts of the transgenic line was 27.3 % higher than that of the non-transgenic line. After feeding the silkworm with different amounts of BmNPV, the mortality of the transgenic line decreased to approximately 33 % compared with the non-transgenic line when the virus dose was 10(6) OB/larva. These results imply that overexpressing endogenous antiviral genes can enhance the antiviral resistance of silkworms.