Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 39
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Zool Res ; 42(5): 637-649, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34472225

RESUMO

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 Proteica
2.
Cell Metab ; 33(10): 2059-2075.e10, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34536344

RESUMO

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.

3.
Int J Biol Sci ; 17(9): 2205-2222, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34239350

RESUMO

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.

4.
Insect Sci ; 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33629486

RESUMO

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.

5.
Insect Sci ; 27(4): 646-654, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31131541

RESUMO

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/metabolismo
6.
Hepatology ; 70(5): 1750-1769, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31077413

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 C57BL
7.
Insect Sci ; 26(6): 1011-1019, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30688002

RESUMO

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/metabolismo
8.
Insect Sci ; 26(6): 1037-1044, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30088858

RESUMO

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 , Fenótipo
9.
Insect Sci ; 26(6): 991-999, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30549429

RESUMO

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 Genética
10.
Insect Sci ; 26(6): 1000-1010, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29808584

RESUMO

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/metabolismo
11.
Insect Sci ; 26(6): 973-982, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29316276

RESUMO

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
12.
Insect Sci ; 25(6): 1017-1024, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30328670

RESUMO

Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.


Assuntos
Sistemas CRISPR-Cas/genética , Larva/crescimento & desenvolvimento , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/genética , Tirosina 3-Mono-Oxigenase/deficiência , Tirosina 3-Mono-Oxigenase/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Lepidópteros/enzimologia , Mutação , Controle Biológico de Vetores , Fenótipo
13.
Insect Sci ; 25(6): 1006-1016, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29808962

RESUMO

As a core member of the Hippo signaling pathway, Hpo plays a critical role in regulating growth and development. Previous studies reported that loss of function of Hpo results in increased proliferation, reduced apoptosis and induction of tissue overgrowth in Drosophila. In this study, we used CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) to study Hpo gene (BmHpo) function in the lepidopteran insect Bombyx mori, known commonly as the silkworm. Sequence analysis of BmHpo revealed an array of deletions in mutants. We found that BmHpo knockout resulted in defects in body size regulation, in developmental defects and pigment accumulation and early death. Our data show that BmHpo is essential for regulation of insect growth and development and that CRISPR/Cas9 technology can serve as a basis for functional analysis of target genes in lepidopteran insects.


Assuntos
Tamanho Corporal/genética , Bombyx/crescimento & desenvolvimento , Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Mutação , Animais , Sequência de Bases , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Insetos/deficiência , Mutagênese , Filogenia
14.
Insect Sci ; 23(4): 543-7, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27234337

RESUMO

The hindgut of lower termites harbors various symbiotic protists, which perform varied functions in lignocellulose decomposition. As termites are social insects, the species and numbers of these flagellated protists in the termite gut vary among the different castes. Juvenile hormones (JHs) can regulate caste differentiation in termites. In this study, we used the juvenile hormone analog fenoxycarb to induce termite workers (Reticulitermes flaviceps) to differentiate into pre-soldiers. A metatranscriptomic investigation of the protistan community was then performed by 454 pyrosequencing. From a thorough analysis based on 597 312 generated reads, we found that the starch and sucrose metabolism pathway was the most abundant pathway across the metatranscriptome. The current study demonstrates that the metatranscriptome of the protistan community in termites contains an abundance of lignocellulase, which plays a vital role in termite nutrition.


Assuntos
Isópteros/microbiologia , Isópteros/parasitologia , Microbiota/genética , Transcriptoma , Animais , Celulase/genética , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Isópteros/efeitos dos fármacos , Fenilcarbamatos/farmacologia , Análise de Sequência de RNA , Simbiose
15.
Insect Sci ; 23(3): 332-4, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27145185
16.
Insect Biochem Mol Biol ; 75: 1-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27192033

RESUMO

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.


Assuntos
Proteínas de Transporte/genética , Quimiotaxia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Insetos/genética , Atrativos Sexuais/metabolismo , Spodoptera/fisiologia , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Edição de Genes , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/fisiologia , Masculino , Mutação , Óvulo/crescimento & desenvolvimento , Óvulo/fisiologia , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Spodoptera/genética , Spodoptera/crescimento & desenvolvimento
17.
Insect Sci ; 23(3): 469-77, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27061764

RESUMO

Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.


Assuntos
Sistemas CRISPR-Cas , Spodoptera/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Embrião não Mamífero , Marcação de Genes , Larva/crescimento & desenvolvimento , Mutagênese , RNA Guia/genética , RNA Mensageiro/genética , Spodoptera/embriologia , Spodoptera/crescimento & desenvolvimento
18.
Mol Ecol ; 21(17): 4371-85, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22515600

RESUMO

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 , Transcriptoma
19.
Nan Fang Yi Ke Da Xue Xue Bao ; 30(10): 2323-6, 2010 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-20965836

RESUMO

OBJECTIVE: To observe the change in the amount of sialic acids on hepatocellular carcinoma (HCC) cell membrane. METHODS: Surgical specimens of HCC and liver cirrhosis tissues were obtained from 28 patients to prepare carcinoma cell and hepatocyte suspensions by collagenase digestion. For assay of α2, 3 and α2, 6-sialic acids, the cells were suspended in the staining buffer containing either fluorescein isothiocyanate-Maackia amurensis lectin (FITC-MAL) or fluorescein isothiocyanate-Sambucus nigra bark lectin (FITC-SNA) and incubated for 1 h, respectively. Flow cytometric analysis was carried out to measure the mean fluorescence intensity (MFI) on the cell surface. RESULTS: In both FITC-MAL- and FITC-SNA-incubated HCC cells, the MFI on the cell surface was greater than that of the hepatocytes. CONCLUSION: Both of α2, 3 and α2, 6- sialic acids increases significantly on the hepatocyte membrane after the carcinomatous change.


Assuntos
Carcinoma Hepatocelular/metabolismo , Membrana Celular/metabolismo , Neoplasias Hepáticas/metabolismo , Ácidos Siálicos/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia
20.
Nan Fang Yi Ke Da Xue Xue Bao ; 30(3): 529-1, 2010 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-20335128

RESUMO

OBJECTIVE: To evaluate the therapeutic effect of in vitro induced autologous bone marrow-derived liver stem cell transplantation for posthepatitic cirrhosis. METHODS: Between Jun 2008 and Mar 2009, 12 patients with posthepatitic cirrhosis and portal hypertensive underwent azygousportal disconnection and splenectomy in our department. The patients were then divided into two groups to receive autologous bone marrow-deprived liver stem cell infusion via the hepatic artery after in vitro induction for 7 days (n=6) or saline (n=6). The therapeutic effects of the operations on the liver functions and liver fibrosis index were evaluated. RESULTS: All the patients recovered uneventfully and no side effect of the operation was found. After the operation, the patients receiving bone marrow-deprived liver stem cell infusion showed better hepatic function improvement than those receiving saline infusion (P<0.05). CONCLUSION: Transplantation of in vitro induced autologous bone marrow-derived liver stem cell via the hepatic artery is safe and effective for treatment of posthepatitic cirrhosis.


Assuntos
Hepatite Viral Humana/complicações , Cirrose Hepática/etiologia , Cirrose Hepática/terapia , Transplante de Células-Tronco , Adulto , Células da Medula Óssea/citologia , Feminino , Humanos , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Transplante Autólogo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...