RESUMO
Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fígado/irrigação sanguínea , Proteômica , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Nonalcoholic fatty liver (NAFL) remains relatively benign, but high-risk to end-stage liver diseases become highly prevalent when it progresses into nonalcoholic steatohepatitis (NASH). Our current understanding of the development of NAFL to NASH remains insufficient. In this study, we revealed MAP kinase (MAPK) activation as the most notable molecular signature associated with NASH progression across multiple species. Furthermore, we identified suppressor of IKKε (SIKE) as a conserved and potent negative controller of MAPK activation. Hepatocyte-specific overexpression of Sike prevented NASH progression in diet- and toxin-induced mouse NASH models. Mechanistically, SIKE directly interacted with TGF-ß-activated kinase 1 (TAK1) and TAK1-binding protein 2 (TAB2) to interrupt their binding and subsequent TAK1-MAPK signaling activation. We found that indobufen markedly up-regulated SIKE expression and effectively improved NASH features in mice and macaques. These findings identify SIKE as a MAPK suppressor that prevents NASH progression and provide proof-of-concept evidence for targeting the SIKE-TAK1 axis as a potential NASH therapy.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/metabolismo , Transdução de Sinais/fisiologia , Hepatócitos/metabolismo , Perfilação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fígado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is a prominent insect for the bioconversion of various organic wastes. As a saprotrophic insect, the BSF inhabits microbe-rich environments. However, the influences of the intestinal microorganisms on BSF growth and development are not very clear. In this study, the dynamics of the intestinal bacterial community of BSF larvae (BSFL) were analyzed using pyrosequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most prevalent bacterial phyla in the intestines of all larval instars. The dynamic changes in bacterial community compositions among different larval instars were striking at the genus level. Klebsiella, Clostridium, Providencia, and Dysgonomonas were the relatively most abundant bacteria in the 1st- to 4th-instar BSFL, respectively. Dysgonomonas and Providencia also dominated the 5th- and 6th-instar larvae, at ratios of 31.1% and 47.2%, respectively. In total, 148 bacterial strains affiliated with 20 genera were isolated on different media under aerobic and anaerobic conditions. Among them, 6 bacteria, BSF1-BSF6, were selected for further study. The inoculation of the 6 isolates independently into germ-free BSFL feeding on an artificial diet showed that all the bacteria, except BSF4, significantly promoted BSF growth and development compared with the germ-free control. Citrobacter, Dysgonomonas, Klebsiella, Ochrobactrum, and Providencia promoted BSF development significantly by increasing the weight gains of larvae and pupae, as well as increasing the prepupae and eclosion rates. In addition, Citrobacter, Klebsiella and Providencia shortened the BSF life cycle significantly. The results illustrate the promotive effects of intestinal bacteria on BSF growth and development.
Assuntos
Dípteros , Animais , Larva , Bactérias , Dieta , BacteroidetesRESUMO
Cotton plants accumulate phytotoxins, including gossypol and related sesquiterpene aldehydes, to resist insect herbivores and pathogens. To counteract these defensive plant secondary metabolites, cotton bollworms (Helicoverpa armigera) elevate their production of detoxification enzymes, including cytochrome P450 monooxygenases (P450s). Besides their tolerance to phytotoxin, cotton bollworms have quickly developed resistance to deltamethrin, a widely used pyrethroid insecticide in cotton field. However, the relationship between host plant secondary metabolites and bollworm insecticide resistance is poorly understood. Here, we show that exogenously expressed CYP6AE14, a gossypol-inducible P450 of cotton bollworm, has epoxidation activity towards aldrin, an organochlorine insecticide, indicating that gossypol-induced P450s participate in insecticide metabolism. Gossypol-ingested cotton bollworm larvae showed higher midgut P450 enzyme activities and exhibited enhanced tolerance to deltamethrin. The midgut transcripts of bollworm larvae administrated with different phytochemicals and deltamethrin were then compared by microarray analysis, which showed that gossypol and deltamethrin induced the most similar P450 expression profiles. Gossypol-induced P450s exhibited high divergence and at least five of them (CYP321A1, CYP9A12, CYP9A14, CYP6AE11 and CYP6B7) contributed to cotton bollworm tolerance to deltamethrin. Knocking down one of them, CYP9A14, by plant-mediated RNA interference (RNAi) rendered the larvae more sensitive to the insecticide. These data demonstrate that generalist insects can take advantage of secondary metabolites from their major host plants to elaborate defence systems against other toxic chemicals, and impairing this defence pathway by RNAi holds a potential for reducing the required dosages of agrochemicals in pest control.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Gossipol/farmacologia , Resistência a Inseticidas/genética , Mariposas/genética , Aldrina , Animais , Gossypium , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Mariposas/efeitos dos fármacos , Mariposas/enzimologia , Nitrilas , Análise de Sequência com Séries de Oligonucleotídeos , Piretrinas , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
Yolk proteins are the main source of nutrients during embryonic and early larval development in oviparous animals. Therefore, vitellogenesis is crucial for reproduction. The silkworm, Bombyx mori, is a model lepidopteran insect in which there are three yolk proteins: vitellin, 30-kDa protein, and egg-specific protein (Esp). In this study, we explored the gene function of Esp through transgenic clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 technology-mediated mutations in the silkworm. We found that Esp mutation resulted in female sterility but had no effect on male fertility. Female mutants could lay eggs after mating, but the eggs were smaller and lighter colored than those laid by wild-type females. The most important finding is that the eggs laid by female mutants did not hatch. Furthermore, we observed stable inheritance of female sterility caused by Esp mutation through successive generations. Thus, Esp encodes a yolk protein that is crucial for female reproductive success and is a potential target for pest control.
Assuntos
Bombyx , Infertilidade Feminina , Animais , Animais Geneticamente Modificados , Bombyx/genética , Proteínas do Ovo , Feminino , Proteínas de Insetos/genética , MasculinoRESUMO
The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bombyx/crescimento & desenvolvimento , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Insetos/genética , Tegumento Comum/fisiologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Filogenia , Conformação ProteicaRESUMO
Purpose: This study aimed to identify the potential prognostic role of HK3 and provide clues about glycolysis and the microenvironmental characteristics of ccRCC. Methods: Based on the Cancer Genome Atlas (TCGA, n = 533) and Gene expression omnibus (GEO) (n = 127) databases, real-world (n = 377) ccRCC cohorts, and approximately 15,000 cancer samples, the prognostic value and immune implications of HK3 were identified. The functional effects of HK3 in ccRCC were analyzed in silico and in vitro. Results: The large-scale findings suggested a significantly higher HK3 expression in ccRCC tissues and the predictive efficacy of HK3 for tumor progression and a poor prognosis. Next, the subgroup survival and Cox regression analyses showed that HK3 serves as a promising and independent predictive marker for the prognosis and survival of patients with ccRCC from bioinformatic databases and real-world cohorts. Subsequently, we found that HK3 could be used to modulate glycolysis and the malignant behaviors of ccRCC cells. The comprehensive results suggested that HK3 is highly correlated with the abundance of immune cells, and specifically stimulates the infiltration of monocytes/macrophages presenting surface markers, regulates the immune checkpoint molecules PD-1 and CTLA-4 of exhaustive T cells, restrains the immune escape of tumor cells, and prompts the immune-rejection microenvironment of ccRCC. Conclusion: In conclusion, the large-scale data first revealed that HK3 could affect glycolysis, promote malignant biologic processes, and predict the aggressive progression of ccRCC. HK3 may stimulate the abundance of infiltrating monocytes/macrophages presenting surface markers and regulate the key molecular subgroups of immune checkpoint molecules of exhaustive T cells, thus inducing the microenvironmental characteristics of active anti-tumor immune responses.
Assuntos
Carcinoma de Células Renais/genética , Hexoquinase/genética , Neoplasias Renais/genética , Linfócitos do Interstício Tumoral/imunologia , Evasão Tumoral/genética , Microambiente Tumoral , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/mortalidade , Feminino , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Hexoquinase/metabolismo , Humanos , Neoplasias Renais/imunologia , Neoplasias Renais/mortalidade , Linfócitos do Interstício Tumoral/metabolismo , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Prognóstico , Análise de Sobrevida , Regulação para CimaRESUMO
Myocardial ischemia-reperfusion (MIR) injury is a major cause of adverse outcomes of revascularization after myocardial infarction. To identify the fundamental regulator of reperfusion injury, we performed metabolomics profiling in plasma of individuals before and after revascularization and identified a marked accumulation of arachidonate 12-lipoxygenase (ALOX12)-dependent 12-HETE following revascularization. The potent induction of 12-HETE proceeded by reperfusion was conserved in post-MIR in mice, pigs, and monkeys. While genetic inhibition of Alox12 protected mouse hearts from reperfusion injury and remodeling, Alox12 overexpression exacerbated MIR injury. Remarkably, pharmacological inhibition of ALOX12 significantly reduced cardiac injury in mice, pigs, and monkeys. Unexpectedly, ALOX12 promotes cardiomyocyte injury beyond its enzymatic activity and production of 12-HETE but also by its suppression of AMPK activity via a direct interaction with its upstream kinase TAK1. Taken together, our study demonstrates that ALOX12 is a novel AMPK upstream regulator in the post-MIR heart and that it represents a conserved therapeutic target for the treatment of myocardial reperfusion injury.
Assuntos
Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Animais , Araquidonato 12-Lipoxigenase , Camundongos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miócitos Cardíacos , SuínosRESUMO
Lipotoxicity is a recognized pathological trigger and accelerator of nonalcoholic steatohepatitis (NASH). However, the molecular basis of lipotoxicity-induced NASH remains elusive. Here, we systematically mapped the changes in hepatic transcriptomic landscapes in response to lipotoxic insults across multiple species. Conserved and robust activation of the arachidonic acid pathway, in particular the arachidonate 12-lipoxygenase (ALOX12) gene, was closely correlated with NASH severity in humans, macaques with spontaneously developed NASH, as well as swine and mouse dietary NASH models. Using gain- and loss-of-function studies, we found that ALOX12 markedly exacerbated NASH in both mice and Bama pig models. ALOX12 was shown to induce NASH by directly targeting acetyl-CoA carboxylase 1 (ACC1) via a lysosomal degradation mechanism. Overall, our findings reveal a key molecular driver of NASH pathogenesis and suggest that ALOX12-ACC1 interaction may be a therapeutic target in NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Modelos Animais de Doenças , Fígado/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , SuínosRESUMO
Nonalcoholic steatohepatitis (NASH) is a progressive liver disease and has become a leading indication for liver transplantation in the United States. The development of effective therapies for NASH is a major unmet need. Here, we identified a small molecule, IMA-1, that can treat NASH by interrupting the arachidonate 12-lipoxygenase (ALOX12)acetyl-CoA carboxylase 1 (ACC1) interaction. IMA-1 markedly blocked diet-induced NASH progression in both male mice and Cynomolgus macaque therapeutic models. The anti-NASH efficacy of IMA-1 was comparable to ACC inhibitor in both species. Protein docking simulations and following functional experiments suggested that the anti-NASH effects of IMA-1 were largely dependent on its direct binding to a pocket in ALOX12 proximal to its ACC1-interacting surface instead of inhibiting ALOX12 lipoxygenase activity. IMA-1 treatment did not elicit hyperlipidemia, a known side effect of direct inhibition of ACC enzymatic activity, in both mice and macaques. These findings provide proof of concept across multiple species for the use of small moleculebased therapies for NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Acetil-CoA Carboxilase , Animais , Fígado/metabolismo , Macaca/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Gossipol/toxicidade , Proteínas de Insetos/antagonistas & inibidores , Lepidópteros/enzimologia , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas/parasitologia , Interferência de RNA , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Sistema Enzimático do Citocromo P-450/genética , Digestão , Resistência a Medicamentos/genética , Inativação Gênica , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Gossypium/genética , Gossypium/parasitologia , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Nicotiana/genética , Nicotiana/parasitologiaRESUMO
The non-lepis wing of silkworm (Bombyx mori) is controlled by the recessive gene, nlw. Owning to lack of crossing over in females, the reciprocal backcrossed F(1) (BC(1)) progenies were used for linkage analysis and mapping of nlw based on the SSR linkage map and STS markers using the wild type (+(nlw)/+(nlw)) silkworm strain P50 and U06 with scaleless wing (nlw/nlw). The nlw gene was linked to eight SSR markers and one STS marker. All the individuals with the wild type in the BC1F (Using F(1) as female to backcross to the recessive parent, that is (U06xP50)xU06) showed heterozygous profile of (U06xP50) F(1), and the ones with non-lepis wing in BC1F exhibited the homozygous profile of the strain U06. Using a reciprocal BC1M (Using F1 as male to backcross to the recessive parent, that is U06x(U06xP50))cross, we constructed a linkage map of 125.6 cM, and the distance between nlw and the nearest marker cash2p was 11.4 cM.
Assuntos
Bombyx/genética , Marcadores Genéticos , Proteínas de Insetos/genética , Sequências Repetitivas de Ácido Nucleico , Asas de Animais , Animais , Bombyx/crescimento & desenvolvimento , Mapeamento Cromossômico , Feminino , Humanos , Endogamia , Masculino , Asas de Animais/crescimento & desenvolvimentoRESUMO
MicroRNAs (miRNAs) are a class of short, non-coding transcripts that bind to 3'-untranslated regions to trigger messenger RNA degradation or translational inhibition. Here we explored how miRNAs regulate sex determination in Bombyx mori, a lepidopteran model insect. Genes known to be involved in sex determination, BmPSI, Bmdsx, and BmMasc, are predicted targets of the species-specific miR-2738. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-2738 suppressed transcription of BmPSI, Bmdsx, and BmMasc. The levels of BmPSI and BmMasc were significantly down-regulated in B. mori miR-2738 overexpression. In contrast, the genetic disruption of miR-2738 using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 transgenic system increased the levels of BmPSI and BmMasc transcripts, whereas splicing of Bmdsx was unaltered by miR-2738 depletion or overexpression. Taken together, this study implicates miR-2738 as a minor regulator of sex determination genes in the silkworm.
Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Processos de Determinação Sexual/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Sequência de Bases , Bombyx/metabolismo , Células HEK293 , Humanos , MicroRNAs/metabolismoRESUMO
Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.
Assuntos
Melaninas/metabolismo , Pigmentação/genética , Pupa/metabolismo , Spodoptera/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Feminino , Expressão Gênica , Genótipo , Filogenia , Spodoptera/metabolismoRESUMO
The translationally controlled tumor protein (TCTP) is a highly conserved and multifunctional protein with activities ranging from cytoskeletal regulation to transcription regulation in numerous organisms. In insects, TCTP is essential for cell growth and proliferation. Recently, TCTP has been reported to affect the innate intestinal immune pathway in the Bombyx mori silkworm, a lepidopteran model insect. However, the comprehensive physiological roles of TCTP in the silkworm remain poorly understood. Here, we performed functional analysis of BmTCTP by using a binary transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/RNA-guided CRISPER-associated protein 9 nucleases) system. Disruption of BmTCTP led to developmental arrestment and subsequent lethality in third instar larvae. Histological analysis revealed that growth impairment originated from decreased cell size, and the proliferation and differentiation of intestinal epithelial cells were also affected. RNA-seq analysis revealed that genes involved in carbohydrate metabolism, lipid metabolism and digestive system pathways were significantly affected by BmTCTP depletion. Together, the results demonstrated that BmTCTP plays a key role in controlling larval growth and development.
Assuntos
Biomarcadores Tumorais/fisiologia , Bombyx/genética , Animais , Bombyx/crescimento & desenvolvimento , Sistemas CRISPR-Cas , Larva/crescimento & desenvolvimento , Plasmídeos , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/µL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.
Assuntos
Proteínas de Insetos/genética , Spodoptera/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Proteínas de Insetos/metabolismo , Masculino , Fenótipo , Spodoptera/crescimento & desenvolvimento , Spodoptera/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmß4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.
Assuntos
Bombyx/genética , Edição de Genes , Regiões Promotoras Genéticas , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Linhagem Celular , Embrião não Mamífero , Genes Reporter , Proteínas de Fluorescência Verde , Transformação GenéticaRESUMO
Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.
Assuntos
Proteínas de Insetos/fisiologia , Mariposas/genética , Diferenciação Sexual , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Feminino , Masculino , Mutação , FenótipoRESUMO
We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.
Assuntos
Bombyx/imunologia , Densovirinae/fisiologia , Genes de Insetos , Interações Hospedeiro-Patógeno , Animais , Bombyx/genética , Bombyx/virologia , DNA Complementar , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/virologia , Expressão Gênica , Larva/imunologia , Larva/virologia , Reação em Cadeia da PolimeraseRESUMO
Microsatellites or simple sequence repeats (SSRs) are co-dominant molecular markers. When we used fluorescent SSR markers to construct a linkage map for the female heterogametic silkworm (Bombyx mori, ZW), we found that some loci did not segregate in a Mendelian ratio of 1:1 in a backcross population. These loci segregated in a 3:1 ratio of single bands compared with double bands. Further examination of band patterns indicated that three types of SSR bands were present: two homozygotes and one heterozygote. In the beginning, we considered to discard these markers. By scoring male and female F1 individuals, we confirmed that these loci were located on the Z chromosome. Using the sex-linked visible mutation sch (K05) and its wild-type (C108), we constructed an F1 male backcross (BC1M) mapping population. The combination of sch backcross and SSR data enabled us to map the SSR markers to the Z chromosome. By adjusting input parameters based on these data, we were able to use Mapmaker software to construct a linkage map. This strategy takes advantage of co-dominant markers for positional cloning of genes on the Z chromosome. We localized sch to the Z chromosome relative to six SSR markers and one PCR marker, covering a total of 76.1 cM. The sch mutation is an important sex-linked visible mutation widely used in breeding of commercial silkworms (e.g. male silkworm selection rearing). Localization of the sch gene may prove helpful in cloning the gene and developing strains for marker-assisted selection in silkworm breeding.