Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 63
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Arch Insect Biochem Physiol ; 109(1): e21854, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34783381

RESUMO

Cytorhinus lividipennis is a natural enemy of rice planthoppers and leafhoppers. Improving the fecundity of C. lividipennis will be helpful to improve its control effect on pests. However, little is known about the hormonal regulatory mechanism of reproduction in C. lividipennis. In the current study, we examined the role of 20-hydroxyecdysone (20E) biosynthesis relative gene Shadow in the reproduction of C. lividipennis. The complementary DNA sequence of ClSad is 2018 -bp in length with an open reading frame of 1398-bp encoding 465 amino acid residues. ClSad was readily detected in nymphal and adult stages, and highly expressed in the adult stage. ClSad was highly expressed in the midgut and ovaries of adult females. Moreover, RNA interference-mediated knockdown of ClSad reduced the 20E titers and ClVg transcript level, resulting in fewer fully developed eggs and a decrease in the number of eggs laid by dsSad-injected adult females within 15 days. These results suggest that ClSad plays a critical role in the reproduction of C. lividipennis. The present study provides insights into the molecular mechanism of the ClSad gene for the reproduction of C. lividipennis.


Assuntos
Ecdisterona/genética , Fertilidade/genética , Heterópteros/genética , Animais , Ecdisterona/biossíntese , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterópteros/metabolismo , Masculino , Ovário/crescimento & desenvolvimento , Interferência de RNA , Análise de Sequência de DNA
2.
J Steroid Biochem Mol Biol ; 213: 105976, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34418528

RESUMO

The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear. We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes. Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense.


Assuntos
Proteínas de Artrópodes/genética , Sistema Enzimático do Citocromo P-450/genética , Ecdisterona/biossíntese , Muda/genética , Oócitos/metabolismo , Ovário/metabolismo , Palaemonidae/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Ecdisterona/genética , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Ovário/citologia , Ovário/crescimento & desenvolvimento , Palaemonidae/classificação , Palaemonidae/crescimento & desenvolvimento , Palaemonidae/metabolismo , Filogenia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
3.
Bull Entomol Res ; 111(4): 485-498, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33745467

RESUMO

The oriental armyworm, Mythimna separata (Walker) is a serious pest of agriculture that does particular damage to Gramineae crops in Asia, Europe, and Oceania. Metamorphosis is a key developmental stage in insects, although the genes underlying the metamorphic transition in M. separata remain largely unknown. Here, we sequenced the transcriptomes of five stages; mature larvae (ML), wandering (W), and pupation (1, 5, and 10 days after pupation, designated P1, P5, and P10) to identify transition-associated genes. Four libraries were generated, with 22,884, 23,534, 26,643, and 33,238 differentially expressed genes (DEGs) for the ML-vs-W, W-vs-P1, P1-vs-P5, and P5-vs-P10, respectively. Gene ontology enrichment analysis of DEGs showed that genes regulating the biosynthesis of the membrane and integral components of the membrane, which includes the cuticular protein (CP), 20-hydroxyecdysone (20E), and juvenile hormone (JH) biosynthesis, were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs were enriched in the metabolic pathways. Of these DEGs, thirty CP, seventeen 20E, and seven JH genes were differentially expressed across the developmental stages. For transcriptome validation, ten CP, 20E, and JH-related genes were selected and verified by real-time PCR quantitative. Collectively, our results provided a basis for further studies of the molecular mechanism of metamorphosis in M. separata.


Assuntos
Genes de Insetos , Metamorfose Biológica/genética , Mariposas/fisiologia , Transcriptoma , Animais , Ecdisterona/biossíntese , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis/biossíntese , Larva/metabolismo , Pupa/metabolismo
4.
Insect Biochem Mol Biol ; 123: 103428, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32553573

RESUMO

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.


Assuntos
Ecdisteroides , Hemípteros/metabolismo , Animais , Ecdisteroides/biossíntese , Ecdisteroides/genética , Ecdisteroides/metabolismo , Ecdisterona/biossíntese , Ecdisterona/genética , Ecdisterona/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes de Insetos , Hemípteros/embriologia , Hemípteros/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Muda/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Oviposição/genética
5.
Protoplasma ; 256(3): 601-614, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30357479

RESUMO

Pfaffia glomerata is a medically important species because it produces the phytoecdysteroid 20-hydroxyecdysone (20-E). However, there has been no ready-to-use transcriptome data available in the literature for this plant. Here, we present de novo transcriptome sequencing of RNA from P. glomerata in order to investigate the 20-E production as well as to understand the biochemical pathway of secondary metabolites in this non-model species. We then analyze the effect of photoautotrophy on the production of 20-E genes phylogenetically identified followed by expression analysis. For this, total messenger RNA (mRNA) from leaves, stems, roots, and flowers was used to construct indexed mRNA libraries. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 164,439 transcripts were annotated. In addition, the effect of photoautotrophy in two genes putatively involved in the 20-E synthesis pathway was analyzed. The Phantom gene (CYP76C), a precursor of the route, showed increased expression in P. glomerata plants cultured under photoautotrophic conditions. This was accompanied by increased production of this metabolite indicating a putative involvement in 20-E synthesis. This work reveals that several genes in the P. glomerata transcriptome are related to secondary metabolism and stresses, that genes of the P450 family participate in the 20-E biosynthesis route, and that plants cultured under photoautotrophic conditions promote an upregulated Phantom gene and enhance the productivity of 20-E. The data will be used for future investigations of the 20-E synthesis pathway in P. glomerata while offering a better understanding of the metabolism of the species.


Assuntos
Amaranthaceae/genética , Processos Autotróficos , Sistema Enzimático do Citocromo P-450/genética , Ecdisterona/biossíntese , Genes de Plantas , Família Multigênica , Processos Fototróficos , Transcriptoma/genética , Processos Autotróficos/genética , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , Processos Fototróficos/genética , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA
6.
Int J Mol Sci ; 19(7)2018 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-29954065

RESUMO

Cyanotis arachnoidea contains a rich array of phytoecdysteroids, including 20-hydroxyecdysone (20E), which displays important agrochemical, medicinal, and pharmacological effects. To date, the biosynthetic pathway of 20E, especially the downstream pathway, remains largely unknown. To identify candidate genes involved in 20E biosynthesis, the comparative transcriptome of C. arachnoidea leaf and root was constructed. In total, 86.5 million clean reads were obtained and assembled into 79,835 unigenes, of which 39,425 unigenes were successfully annotated. The expression levels of 2427 unigenes were up-regualted in roots with a higher accumulation of 20E. Further assignments with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified 49 unigenes referring to the phytoecdysteroid backbone biosynthesis (including 15 mevalonate pathway genes, 15 non-mevalonate pathway genes, and 19 genes for the biosynthesis from farnesyl pyrophosphate to cholesterol). Moreover, higher expression levels of mevalonate pathway genes in roots of C. arachniodea were confirmed by real-time quantitative PCR. Twenty unigenes encoding CYP450s were identified to be new candidate genes for the bioreaction from cholesterol to 20E. In addition, 90 transcription factors highly expressed in the roots and 15,315 unigenes containing 19,158 simple sequence repeats (SSRs) were identified. The transcriptome data of our study provides a valuable resource for the understanding of 20E biosynthesis in C. arachnoidea.


Assuntos
Commelinaceae/metabolismo , Ecdisterona/biossíntese , Transcriptoma/genética , Commelinaceae/genética , Ecdisterona/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Ontologia Genética , Repetições de Microssatélites/genética , Anotação de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo
7.
Plant Physiol Biochem ; 129: 394-399, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29945075

RESUMO

The steroid 20-hydroxyecdysone (20E) is a major component of phytoecdysteroid in plants and may play a defensive role against insect pests in higher plants. In spinach, the biosynthesis and accumulation of 20E have been investigated during the vegetative stage; however, these processes have not been clearly studied during the reproductive stage, particularly in male and female individuals. In this study, we analyzed the level and distribution of 20E in individual male and female spinach plants during the reproductive stage via high performance liquid chromatography (HPLC). We found that 20E biosynthesis and accumulation were markedly different between male and female spinach during the late flowering stage. Compared with the male plant, biosynthesis of 20E in the leaves was more active and its accumulation in the floral parts was higher in female plants during the late flowering stage. These results indicate that the female reproductive organs at least in PE-positive plants could be effectively protected against harmful insects via active biosynthesis and accumulation of PE during the late flowering stage to protect floral parts from harmful insects for seed formation and store the available 20E in seeds for the next generation.


Assuntos
Ecdisterona/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Spinacia oleracea/metabolismo , Ecdisterona/biossíntese , Flores/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Folhas de Planta/metabolismo , Reprodução , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Spinacia oleracea/fisiologia
8.
Phytochemistry ; 127: 23-8, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27017303

RESUMO

20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3ß,14α-dihydroxy-5ß-cholest-7-en-6-one (5ß-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3ß-hydroxy-5ß-cholestan-6-one (5ß-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5ß-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5ß-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5ß-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.


Assuntos
Ajuga/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ecdisterona/biossíntese , Ecdisterona/metabolismo , Lamiaceae/química , Ajuga/genética , Colestanonas/metabolismo , Colesterol/química , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Desidrocolesteróis/metabolismo , Ecdisteroides/química , Lamiaceae/metabolismo , Estrutura Molecular , Raízes de Plantas/química
9.
J Insect Physiol ; 80: 31-41, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25770979

RESUMO

Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mß (E(spl)mß), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mß was 3-8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mß was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mß are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Hormônios Juvenis/metabolismo , Fatores de Transcrição/genética , Regulação para Cima , Sequência de Aminoácidos , Animais , Bombyx/química , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Ecdisteroides/biossíntese , Ecdisterona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/química , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
10.
Phytochemistry ; 111: 59-64, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25593010

RESUMO

3ß-Hydroxy-5ß-cholestan-6-one was identified in the EtOAc extract of Ajuga hairy roots by micro-analysis using LC-MS/MS in the multiple reaction mode (MRM). Furthermore, administration of (2,2,4,4,7,7-(2)H6)- and (2,2,4,4,6,7,7-(2)H7)-cholesterols to the hairy roots followed by LC-MS/MS analysis of the EtOAc extract of the hairy roots indicated that cholesterol was converted to the 5ß-ketone with hydrogen migration from the C-6 to the C-5 position. These findings, in conjunction with the previous observation that the ketone was efficiently converted to 20-hydroxyecdysone, strongly suggest that the 5ß-ketone is an intermediate immediately formed after cholesterol during 20-hydroxyecdysone biosynthesis in Ajuga sp. In addition, the mechanism of the 5ß-ketone formation from cholesterol is discussed.


Assuntos
Ajuga/química , Colestanonas/isolamento & purificação , Colesterol , Ecdisterona/biossíntese , Colestanonas/química , Colestanonas/metabolismo , Colesterol/administração & dosagem , Colesterol/análogos & derivados , Colesterol/química , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Cetonas/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Espectrometria de Massas em Tandem
11.
Arch Insect Biochem Physiol ; 88(3): 179-91, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25354095

RESUMO

Spook has essential roles in the biogenesis of the molting hormone 20-hydroxyecdysone (20-E). The function of spook in the rice striped stem borer (SSB) Chilo suppressalis remains unclear, prompting our hypothesis that it exerts actions similar to those reported for other insect species. Here we amplified the full-length transcript of spook (Cs-Spook) in SSB by 5' and 3' rapid amplification of cDNA ends. Cs-Spook has conserved P450 motifs such as Helix-C, Helix-I, Helix-K, and PERF motif (PxxFxPxRF). It was highly expressed in late instar larvae but less so in newly molted larvae. Cs-Spook was highly expressed in prothoracic glands. Cs-Spook was knocked down by dsRNA treatments. Compared with controls, the gene expression level was reduced to 9% at 24 h post injection (PI), 33% at 48 h PI, and 24% at 72 h PI. The ecdysteroid titer decreased significantly in the dsRNA-treated group (P < 0.05), resulting in delayed larval development. The delayed development in dsRNA-treatment group was rescued by treating with 20-E. Our work demonstrates that Cs-Spook participates in the biogenesis of 20-E and regulates the molt of SSB, as seen in other species.


Assuntos
Ecdisterona/biossíntese , Genes de Insetos , Muda , Mariposas/crescimento & desenvolvimento , Mariposas/genética , Animais , Sequência de Bases , DNA Complementar , Ecdisona , Regulação da Expressão Gênica no Desenvolvimento , Larva/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Interferência de RNA , RNA de Cadeia Dupla , Análise de Sequência de Proteína
12.
Insect Sci ; 22(2): 191-202, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24282064

RESUMO

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.


Assuntos
Hemípteros/genética , Proteínas de Insetos/genética , Interferência de RNA , Sequência de Aminoácidos , Animais , Ecdisterona/biossíntese , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes de Insetos , Hemípteros/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Ninfa/genética , Ninfa/crescimento & desenvolvimento , RNA de Cadeia Dupla/administração & dosagem , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Alinhamento de Sequência
13.
Insect Mol Biol ; 24(1): 13-28, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25255866

RESUMO

The specialized wasp cells teratocytes (TCs) are derived from the embryonic serosal membrane of some parasitic hymenopteran insects. As a parasitic factor, TCs are multifunctional in host regulation, such as host nutritional deprivation, immunosuppression and developmental arrest; however, little is understood about their genetic constituents. The present study provides a comprehensive view of the genes expressed by TCs through a transcriptome analysis based on RNA sequencing technology. The assembled 34 686 contigs (>200 base pairs) were annotated into different functional categories, indicating a distinct distribution in gene transcripts compared with those of haemocytes and fat body. The TC transcriptome contained components of insulin signalling and biosyntheses of juvenile hormone and 20-hydroxyecdysone. TCs also expressed various groups of digestive enzymes, indicating that they have nutritional role for the growing parasitoid larvae in parasitism. Furthermore, through this transcriptome analysis two kinds of immunosuppressive serine protease inhibitors (serpins) and Rho GTPase-activating proteins (RhoGAPs) were annotated. To determine the biological functions of these factors, we devised ex vivo RNA interference (RNAi) by conducting knockdown of gene expression in in vitro-cultured TCs followed by injection of the treated TCs to test insects. Ex vivo RNAi revealed that some serpins and RhoGAPs expressed in TCs inhibited host cellular immunity. This study reports a transcriptome of the unique TC animal cell and its immunosuppressive genetic factors using ex vivo RNAi technology.


Assuntos
Interações Hospedeiro-Parasita/genética , Mariposas/parasitologia , Vespas/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Ecdisterona/biossíntese , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita/imunologia , Proteínas de Insetos/metabolismo , Larva/imunologia , Larva/parasitologia , Mariposas/imunologia , Interferência de RNA , Análise de Sequência de RNA , Serpinas/metabolismo , Vespas/genética
14.
Mol Biol Evol ; 31(12): 3302-13, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25213334

RESUMO

Although there are many studies to show a key role of transposable elements (TEs) in adaptive evolution of higher organisms, little is known about the molecular mechanisms. In this study, we found that a partial TE (Taguchi) inserted in the cis-regulatory region of the silkworm ecdysone oxidase (EO) gene, which encodes a crucial enzyme to reduce the titer of molting hormone (20-hydroxyecdysone, 20E). The TE insertion occurred during domestication of silkworm and the frequency of the TE insertion in the domesticated silkworm (Bombyx mori) is high, 54.24%. The linkage disequilibrium in the TE inserted strains of the domesticated silkworm was elevated. Molecular population genetics analyses suggest that this TE insertion is adaptive for the domesticated silkworm. Luminescent reporter assay shows that the TE inserted in the cis-regulatory region of the EO gene functions as a 20E-induced enhancer of the gene expression. Further, phenotypic bioassay indicates that the silkworm with the TE insertion exhibited more stable developmental phenotype than the silkworm without the TE insertion when suffering from food shortage. Thus, the inserted TE in the cis-regulatory region of the EO gene increased developmental uniformity of silkworm individuals through regulating 20E metabolism, partially explaining transformation of a domestication developmental trait in the domesticated silkworm. Our results emphasize the exceptional role of gene expression regulation in developmental transition of domesticated animals.


Assuntos
3-Hidroxiesteroide Desidrogenases/genética , Bombyx/genética , Elementos de DNA Transponíveis , Proteínas de Insetos/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Região 5'-Flanqueadora , Animais , Bombyx/enzimologia , Ecdisterona/biossíntese , Evolução Molecular , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Desequilíbrio de Ligação , Mutagênese Insercional , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA
15.
J Chem Ecol ; 40(8): 913-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25139758

RESUMO

Silkworm (Bombyx mori), a model Lepidoptera insect, is economically important. Its growth and development are regulated by endogenous hormones. During the process of transition from larvae to pupae, 20-hydroxyecdysone (20E) plays an important role. The recent surge in consumer products and applications using metallic nanoparticles has increased the possibility of human or ecosystem exposure due to their unintentional release into the environment. We investigated the effects of exposure to titanium dioxide nanoparticles (TiO2 NPs) on the action of 20E in B. mori. Titanium dioxide nanoparticle treatment shortened the molting duration by 8 hr and prolonged the molting peak period by 10 %. Solexa sequencing profiled the changes in gene expression in the brain of fifth-instar B. mori in response to TiO2NPS exposure for 72 hr, to address the effects on hormone metabolism and regulation. Thirty one genes were differentially expressed. The transcriptional levels of pi3k and P70S6K, which are involved in the target of the rapamycin (TOR) signaling pathway, were up-regulated. Transcriptional levels of four cytochrome P450 genes, which are involved in 20E biosynthesis, at different developmental stages (48, 96, 144, and 192 hr) at 5th instars of all displayed trends of increasing expression. Simultaneously, the ecdysterone receptors, also displayed increasing trends. The 20E titers at four developmental stages during the 5th instar were 1.26, 1.23, 1.72, and 2.16 fold higher, respectively, than the control group. These results indicate that feeding B. mori with TiO2 NPs stimulates 20E biosynthesis, shortens the developmental progression, and reduces the duration of molting. Thus, application of TiO2 NPs is of high significance for saving the labor force in sericulture, and our research provides a reference for the ecological problems in the field of Lepidoptera exposured to titanium dioxide nanoparticles.


Assuntos
Bombyx/efeitos dos fármacos , Bombyx/fisiologia , Ecdisterona/metabolismo , Nanopartículas Metálicas/toxicidade , Transdução de Sinais , Titânio/toxicidade , Agricultura , Animais , Bombyx/crescimento & desenvolvimento , Ecdisterona/biossíntese , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Seda
16.
PLoS One ; 8(12): e79861, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24324583

RESUMO

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5ß-ketodiol, commonly referred as the "black box", remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.


Assuntos
Anopheles/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Ecdisterona/biossíntese , Ovário/enzimologia , Testículo/enzimologia , Animais , Anopheles/genética , Anopheles/crescimento & desenvolvimento , Colestenonas/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Desidrocolesteróis/metabolismo , Ecdisterona/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reprodução/genética
17.
Gene ; 531(2): 168-74, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24055487

RESUMO

Ecdysteroid hormone 20-hydroxyecdysone (20E) plays fundamental roles in insect development and reproduction, whereas the primary role of ecdysone (E) is the precursor for 20E. A cytochrome P450 monooxygenase (CYP), encoded by a Halloween gene Shade (Shd, cyp314a1), catalyzes the conversion of E into 20E in representative insect species in Diptera, Lepidoptera and Orthoptera. We describe here the cloning and characterization of LsShd in a hemipteran insect species, the small brown planthopper Laodelphax striatellus. LsSHD has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal expression pattern of LsShd was determined through the fourth-instar and the early fifth-instar stages by qPCR. LsShd showed two expression peaks in day 2 and day 5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Dietary introduction of double-stranded RNA (dsRNA) of LsShd into nymphs successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, and caused nymphal lethality and delayed development. Ingestion of 20E did not increase LsShd expression level, but almost completely rescued LsEcR mRNA level, and relieved the negative effects on the survival and development in LsShd-dsRNA-exposed nymphs. In contrast, dietary introduction of E had little rescue effects. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects, and LsSHD functions to regulate metamorphotic processes by converting E to 20E even in a hemipteran insect, L. striatellus.


Assuntos
Ecdisteroides/biossíntese , Técnicas de Silenciamento de Genes , Genes de Insetos/fisiologia , Hemípteros/genética , Hemípteros/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ecdisona/biossíntese , Ecdisona/metabolismo , Ecdisterona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica/genética , Genes de Insetos/genética , Hemípteros/enzimologia , Metamorfose Biológica/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
18.
Insect Biochem Mol Biol ; 41(5): 283-93, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21277979

RESUMO

We previously reported preferential expression of genes for ecdysteroid signaling in the mushroom bodies of honeybee workers, suggesting a role of ecdysteroid signaling in regulating honeybee behaviors. The organs that produce ecdysteroids in worker honeybees, however, remain unknown. We show here that the expression of neverland and Non-molting glossy/shroud, which are involved in early steps of ecdysteroid synthesis, was enhanced in the ovary, while the expression of CYP306A1 and CYP302A1, which are involved in later steps of ecdysone synthesis, was enhanced in the brain, and the expression of CYP314A1, which is involved in converting ecdysone into active 20-hydroxyecdysone (20E), was enhanced in the brain, fat body, and ovary. In in vitro organ culture, a significant amount of ecdysteroids was detected in the culture medium of the brain, fat body, and hypopharyngeal glands. The ecdysteroids detected in the culture medium of the fat body were identified as ecdysone and 20E. These findings suggest that, in worker honeybees, cholesterol is converted into intermediate ecdysteroids in the ovary, whereas ecdysone is synthesized and secreted mainly by the brain and converted into 20E in the brain and fat body.


Assuntos
Abelhas , Ecdisona/biossíntese , Ecdisterona/biossíntese , Proteínas de Insetos/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Abelhas/citologia , Abelhas/enzimologia , Abelhas/genética , Cromatografia Líquida de Alta Pressão , Meios de Cultivo Condicionados , Sistema Enzimático do Citocromo P-450/metabolismo , Ecdisona/genética , Ecdisterona/genética , Europa (Continente) , Corpo Adiposo/citologia , Corpo Adiposo/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Insetos/genética , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Esteroide Hidroxilases/genética
19.
Insect Biochem Mol Biol ; 41(5): 294-305, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21288489

RESUMO

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.


Assuntos
Ecdisterona/biossíntese , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Transdução de Sinais , Tribolium/fisiologia , Vitelogeninas/biossíntese , Animais , Sequência de Bases , Regulação para Baixo , Ecdisterona/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Insulina/genética , Insulina/metabolismo , Hormônios Juvenis/genética , Análise em Microsséries , Dados de Sequência Molecular , Fenômenos Fisiológicos da Nutrição , Oócitos/metabolismo , Oogênese/genética , Interferência de RNA , RNA Mensageiro/análise , Reprodução/genética , Transdução de Sinais/genética , Inanição/genética , Inanição/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima , Vitelogênese/genética , Vitelogeninas/genética
20.
Biosci Biotechnol Biochem ; 74(11): 2226-31, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21071854

RESUMO

There is increasing interest in phytoecdysteroids (PEs) because of their potential role in plant defense against insects. To understand the mechanism regulating their levels in plants, the fluctuation, distribution, and biosynthesis of PE 20-hydroxyecdysone (20E) examined in Achyranthes japonica. The total amount of 20E per individual plant initially remained at a constant level, and increased markedly after the first leaf pair (LP) stage, while the concentration of 20E in a given plant decreased rapidly during vegetative growth. In addition, the incorporation of [2-(14)C]-mevalonic acid into 20E did not differ significantly depending on plant organs and developmental stages, suggesting that biosynthesis of 20E is not restricted to particular organs or growth stages.


Assuntos
Achyranthes/metabolismo , Ecdisterona/biossíntese , Achyranthes/imunologia , Animais , Isótopos de Carbono , Insetos/imunologia , Ácido Mevalônico/metabolismo , Distribuição Tecidual
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...