Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
BMC Plant Biol ; 21(1): 340, 2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34273968

RESUMO

BACKGROUND: TLPs (Tubby-like proteins) are widespread in eukaryotes and highly conserved in plants and animals. TLP is involved in many biological processes, such as growth, development, biotic and abiotic stress responses, while the underlying molecular mechanism remains largely unknown. In this paper we characterized the biological function of cucumber (Cucumis sativus L.) Tubby-like protein 8 (CsTLP8) in Arabidopsis. RESULTS: In cucumber, the expression of the tubby-like protein CsTLP8 was induced by NaCl treatment, but reduced by PEG (Polyethylene Glycol) and ABA (Abscisic Acid) treatment. Subcellular localization and transcriptional activation activity analysis revealed that CsTLP8 possessed two characteristics of classical transcription factors: nuclear localization and trans-activation activity. Yeast two-hybrid assay revealed interactions of CsTLP8 with CsSKP1a and CsSKP1c, suggesting that CsTLP8 might function as a subunit of E3 ubiquitin ligase. The growth activity of yeast with ectopically expressed CsTLP8 was lower than the control under NaCl and mannitol treatments. Under osmotic and salt stresses, overexpression of CsTLP8 inhibited seed germination and the growth of Arabidopsis seedlings, increased the content of MDA (Malondialdehyde), and decreased the activities of SOD (Superoxide Dismutase), POD (Peroxidase) and CAT (Catalase) in Arabidopsis seedlings. Overexpression of CsTLP8 also increased the sensitivity to ABA during seed germination and ABA-mediated stomatal closure. CONCLUSION: Under osmotic stress, CsTLP8 might inhibit seed germination and seedling growth by affecting antioxidant enzymes activities. CsTLP8 acts as a negative regulator in osmotic stress and its effects may be related to ABA.


Assuntos
Ácido Abscísico/metabolismo , Cucumis sativus/metabolismo , Germinação , Pressão Osmótica , Proteínas de Plantas/metabolismo , Sementes , Transdução de Sinais , Antioxidantes/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/embriologia , Cloreto de Sódio , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
2.
Genomics ; 113(3): 1170-1179, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33705887

RESUMO

Complete chloroplast genomes of ten wild Fragaria species native to China were sequenced. Phylogenetic analysis clustered Fragaria species into two clades: The south clade (F. iinumae, F. chinensis, F. pentaphylla, F. nilgerrensis, F. daltoniana, F. corymbosa, F. moupinensis, F. tibetica, F. nipponica, F. gracilis, and F. nubicola and north clade (F. viridis, F. orientalis, F. moschata, F. mandshurica, F. vesca, F. chiloensis, F. virginiana, and F. × ananassa), while F. iinumae is the oldest extant species. Molecular clock analysis suggested present Fragaria species share a common ancestor 3.57 million years ago (Ma), F. moschata and octoploid species evolve 0.89 and 0.97 Ma, respectively, but F. moschata be not directly involved in current octoploid species formation. Drastic global temperature change since the Palaeocene-Eocene, approx. 55 Ma, especially during uplifting of the Qinghai-Tibet plateau and quaternary glaciation may have driven the formation of Fragaria, separation of two groups and polyploidization.

3.
Plant Cell Environ ; 43(11): 2666-2679, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32799324

RESUMO

Improving plant water-use efficiency (WUE) is important to plant survival and crop yield in the context of water limitation. In this study, SlTLFP8 (Tubby-like F-box protein 8) was identified as an osmotic-induced gene in tomato. Transgenic tomato with up-regulated expression of SlTLFP8 showed enhanced water-deficient resistance, whereas knockout mutants generated by CRISPR/Cas9 were more sensitive to water deficit. SlTLFP8 overexpression significantly enhanced WUE by suppressing transpiration under both water-sufficient and water-deficient conditions. Further study showed that overexpressing SlTLFP8 significantly increased leaf epidermal cell size and thereby decreased stomatal density 10-20%, conversely SlTLFP8 knockout resulted in decreased cell size and thereby increased stomatal density 20-50%. SlTLFP8 overexpression and knockout modulated ploidy levels in leaf cells. Changes in expression of cell cycle related genes also indicated that SlTLFP8 affected cell size and stomatal density through endocycle transition. Despite changes in stomata density and transpiration, altering the expression of SlTLFP8 did not change photosynthesis. Additionally, biomass was not altered and there was little difference in fruit yield for transgenic and wild type lines under water-sufficient and water-deficient conditions. Our results demonstrate the effect of SlTLFP8 on endoreduplication and the potential of SlTLFP8 for improvement of WUE. BRIEF SUMMERY: This work found a new mechanism of TLP (Tubby like protein) response to water-deficient stress. SlTLFP8, a member of TLP family, regulates water-deficient resistance by modulating water loss via affecting stomatal density. Expression of SlTLFP8 was induced by osmotic stress. Transgenic tomato lines with SlTLFP8 overexpression or SlTLFP8 knockout showed significantly differences in water-use efficiency (WUE) and water-deficient resistance. The difference of leaf water loss caused by transpiration is the main explanation of the difference in WUE and water-deficient resistance. Additionally, overexpressing SlTLFP8 significantly decreased stomatal density, while SlTLFP8 knockout resulted in increased stomatal density, and SlTLFP8 affected stomatal density through endoreduplication and altered epidermal cell size. Despite changes in stomata density, altering the expression of SlTLFP8 did not result in distinct changes in photosynthesis, biomass and yield of tomato.


Assuntos
Endorreduplicação , Proteínas F-Box/fisiologia , Proteínas de Plantas/fisiologia , Estômatos de Plantas/anatomia & histologia , Transpiração Vegetal , Água/metabolismo , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Tamanho Celular , Proteínas F-Box/metabolismo , Técnicas de Silenciamento de Genes , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiologia , Proteínas de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
4.
Biol Psychiatry ; 88(12): 922-934, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-32172944

RESUMO

BACKGROUND: Serotonin (5-HT) 1B/1D receptor (5-HT1B/1DR) agonists undergo an abstinence-induced switch in their effects on cocaine-related behaviors, which may involve changes in modulation of dopamine (DA) neurons in the ventral tegmental area (VTA). However, it is unclear how 5-HT1B/1DRs affect VTA DA neuronal function and whether modulation of these neurons mediates the abstinence-induced switch after chronic cocaine exposure. METHODS: We examined the ability of 5-HT1B/1DRs to modulate D2 autoreceptors (D2ARs) and synaptic transmission in the VTA by slice recording and single unit recording in vivo in naïve mice and in mice with chronic cocaine treatment. RESULTS: We report a bidirectional modulation of VTA DA neuronal firing through the interaction of VTA 5-HT1B/1DRs and D2ARs. In both VTA slices and the VTA of anesthetized mice, the 5-HT1B/1DR agonist CP94253 decreased DA neuronal firing rate and evoked excitatory postsynaptic currents to DA neurons in slice. Paradoxically, CP94253 decreased quinpirole-induced inhibition of DA neurons by reducing D2AR-mediated G protein-gated inwardly rectifying potassium current. This manifested decreased GABAA (gamma-aminobutyric acid A) receptor-mediated evoked inhibitory postsynaptic currents in slice, resulting in disinhibition of DA neurons, in opposition to the 5-HT1B/1DR-induced inhibition. This dual effect was verified in chronic cocaine-treated and mild stress-treated, male mice on days 1 and 20 posttreatment. CONCLUSIONS: This study revealed dual effects of CP94253 on VTA DA neurons that are dependent on D2AR sensitivity, with anti-inhibition under normal D2AR sensitivity and inhibition under low D2AR sensitivity. These dual effects may underlie the ability of CP94253 to both enhance and inhibit cocaine-induced behaviors.


Assuntos
Cocaína , Área Tegmentar Ventral , Animais , Cocaína/farmacologia , Dopamina , Neurônios Dopaminérgicos , Masculino , Camundongos , Serotonina
5.
Plant Sci ; 283: 385-395, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128709

RESUMO

High temperature is a major environmental factor affecting plant growth. Heat shock proteins (Hsps) are molecular chaperones that play important roles in improving plant thermotolerance during heat stress. Spinach (Spinacia oleracea) is very sensitive to high temperature; however, the specific function of Hsps in spinach is unclear. In this study, cytosolic heat shock 70 protein (SoHSC70), which was induced by heat stress, was cloned from spinach. Overexpressing SoHSC70 in spinach calli and Arabidopsis enhanced their thermotolerance. In contrast, spinach seedlings with silenced SoHSC70 by virus-induced gene silencing (VIGS) showed more sensitivity to heat stress. Further analysis revealed that overexpressing SoHSC70 altered relative electrical conductivity (REC), malondialdehyde (MDA) content, photosynthetic rate, reactive oxygen species (ROS) accumulation and the activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) after the heat treatment. Taken together, our results suggest that overexpressing SoHSC70 positively affects heat tolerance by reducing membrane damage and ROS accumulation and improving activities of antioxidant enzymes.


Assuntos
Antioxidantes/metabolismo , Membrana Celular/metabolismo , Proteínas de Choque Térmico/fisiologia , Proteínas de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Spinacia oleracea/metabolismo , Termotolerância , Arabidopsis , Membrana Celular/fisiologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Malondialdeído/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Spinacia oleracea/genética , Spinacia oleracea/fisiologia
6.
Plant Sci ; 280: 66-76, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824030

RESUMO

Switchgrass (Panicum virgatum L.) is a bioenergy crop; thus, it is important to improve biomass to effectively produce bioethanol, particularly under adverse stress conditions. NAC transcription factors are involved in the abiotic stress response. PvNAC1 was isolated in the nucleus of switchgrass, with its C-terminal region containing a transcriptional activation domain. PvNAC1 expression was induced by dehydration, salt, H2O2, and abscisic acid treatments. Overexpressing (OE) PvNAC1 improved growth performance, leading to significantly taller and heavier (dry weight) plants. Moreover, cellulose content was significantly higher in OE plants, indicating that PvNAC1 plays an important role regulating growth and bioethanol production. PvNAC1 RNA interference (RNAi) switchgrass plants exhibited reduced dry weight and cellulose content. OE PvNAC1 enhanced tolerance to salt stress, through higher reactive oxygen species scavenging ability and less Na+ and more K+ accumulation in roots and shoots. RNAi plants were more sensitive to salt stress. The quantitative polymerase chain reaction results revealed that some stress responsive genes, three antioxidant enzymatic genes, and an ion homeostasis-related gene were upregulated in OE plants and downregulated in RNAi plants. These results show that PvNAC1 functions as a transcriptional activator in response to salt stress and growth.


Assuntos
Panicum/metabolismo , Sódio/metabolismo , Biomassa , Celulose/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tolerância ao Sal
7.
Plant Cell Physiol ; 60(3): 562-574, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496548

RESUMO

Melatonin plays an important role in stress tolerance in plants. In this study, exogenous melatonin significantly alleviated the dwarf phenotype and inhibited the decrease of plant fresh weight induced by excess copper (Cu2+). Our results indicated that melatonin alleviated Cu2+ toxicity by improving Cu2+ sequestration, carbon metabolism and ROS (reactive oxygen species) scavenging, rather than by influencing the Cu2+ uptake under excess Cu2+ conditions. Transcriptome analysis showed that melatonin broadly altered gene expression under Cu2+ stress. Melatonin increased the levels of glutathione and phytochelatin to chelate excess Cu2+ and promoted cell wall trapping, thus keeping more Cu2+ in the cell wall and vacuole. Melatonin inhibited ROS production and enhanced antioxidant systems at the transcriptional level and enzyme activities, thus building a line of defense in response to excess Cu2+. The distribution of nutrient elements was recovered by melatonin which was disturbed by Cu2+. In addition, melatonin activated carbon metabolism, especially glycolysis and the pentose phosphate pathway, to generate more ATP, an intermediate for biosynthesis. Taken together, melatonin alleviated Cu2+ toxicity in cucumber via multiple mechanisms. These results will help to resolve the toxic effects of Cu2+ stress on plant growth and development. These results can be used for new strategies to solve problems associated with Cu2+ stress.


Assuntos
Cucumis sativus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peroxidação de Lipídeos/genética , Peroxidação de Lipídeos/fisiologia , Melatonina/metabolismo , Melatonina/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcriptoma/genética
8.
Plant Cell Physiol ; 59(5): 930-945, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29415202

RESUMO

The NAC transcription factors play vital roles in responding to drought stress in plants; however, the molecular mechanisms remain largely unknown in cucumber. Suppression of CsATAF1 via RNA interference (RNAi) weakened drought stress tolerance in cucumber due to a higher water loss rate in leaves, a higher level of hydrogen peroxide (H2O2) and superoxide radicals (O2·-), increased malondialdehyde (MDA) content, lower Fv/Fm ratios and lower antioxidant enzyme activity. The analysis of root length and stomatal apertures showed that CsATAF1-RNAi cucumber plants were less responsive to ABA. In contrast, CsATAF1-overexpression (OE) plants showed increased drought stress tolerance and sensitivity to ABA. Quantitative PCR (qPCR) analysis showed that expression of several stress-responsive genes was significantly up-regulated in CsATAF1-OE transformants and down-regulated in CsATAF1-RNAi transformants. CsABI5, CsCu-ZnSOD and CsDREB2C were verified as direct target genes of CsATAF1. Yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) further substantiated that CsATAF1 bound to the promoters of CsABI5, CsCu-ZnSOD and CsDREB2C. Transient expression in tobacco leaves and cucumber protoplasts showed that CsATAF1 directly up-regulated the expression of CsABI5, CsCu-ZnSOD and CsDREB2C. Our results demonstrated that CsATAF1 functioned as a positive regulator in response to drought stress by an ABA-dependent pathway and decreasing reactive oxygen species (ROS) accumulation in cucumber.


Assuntos
Ácido Abscísico/farmacologia , Adaptação Fisiológica , Cucumis sativus/fisiologia , Secas , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Adaptação Fisiológica/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cucumis sativus/genética , Cucumis sativus/crescimento & desenvolvimento , Sequestradores de Radicais Livres/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Germinação/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Análise de Sequência de DNA , Estresse Fisiológico/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
9.
CNS Neurosci Ther ; 23(9): 748-758, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28786172

RESUMO

INTRODUCTION: Apolipoprotein E4 (APOE4) is a major genetic risk factor for late-onset sporadic Alzheimer disease. Emerging evidence demonstrates a hippocampus-associated learning and memory deficit in aged APOE4 human carriers and also in aged mice carrying human APOE4 gene. This suggests that either exogenous APOE4 or endogenous APOE4 alters the cognitive profile and hippocampal structure and function. However, little is known regarding how Apoe4 modulates hippocampal dendritic morphology, synaptic function, and neural network activity in young mice. AIM: In this study, we compared hippocampal dendritic and spine morphology and synaptic function of young (4 months) mice with transgenic expression of the human APOE4 and APOE3 genes. METHODS: Hippocampal dendritic and spine morphology and synaptic function were assessed by neuronal imaging and electrophysiological approaches. RESULTS: Morphology results showed that shortened dendritic length and reduced spine density occurred at hippocampal CA1 neurons in Apoe4 mice compared to Apoe3 mice. Electrophysiological results demonstrated that in the hippocampal CA3-CA1 synapses of young Apoe4 mice, basic synaptic transmission, and paired-pulse facilitation were enhanced but long-term potentiation and carbachol-induced hippocampal theta oscillations were impaired compared to young Apoe3 mice. However, both Apoe genotypes responded similarly to persistent stimulations (4, 10, and 40 Hz for 4 seconds). CONCLUSION: Our results suggest significant alterations in hippocampal dendritic structure and synaptic function in Apoe4 mice, even at an early age.


Assuntos
Apolipoproteína E4/genética , Hipocampo/citologia , Rede Nervosa/patologia , Neurônios/fisiologia , Sinapses/genética , Animais , Apolipoproteína E3/genética , Fenômenos Biofísicos , Dendritos/ultraestrutura , Espinhas Dendríticas/fisiologia , Modelos Animais de Doenças , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/genética , Hipocampo/fisiologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Estatísticas não Paramétricas , Sinapses/metabolismo , Vesículas Sinápticas/genética
10.
Sci Rep ; 7: 41397, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28169290

RESUMO

Cellulose is the major component of cell wall materials. A 300 bp specific fragment from the cDNA fragment was chosen to insert into vector pFGC1008 at forward and reverse orientations to construct the recombinant RNAi vector. Knockdown of BoiCesA caused "dwarf" phenotype with smaller leaves and a loss of the content of cellulose. Moreover, RT-PCR analysis confirmed that the expression of the RNAi apparatus could repress expression of the CesA gene. Meanwhile, examination of the leaves from the T3 of RNAi transformants indicated reduction of cell expansion in vascular bundles, particularly on their abaxial surface. The proline and soluble sugar content increased contrarily. Under the salt stress, the T3 of RNAi plants showed significant higher resistance. The expression levels of some salt tolerance related genes (BoiProH, BoiPIP2;2, BoiPIP2;3) were significantly changed in T3 of RNAi plants. The results showed that the hairpin structure of CesA specific fragment inhibited the endogenous gene expression and it was proved that the cDNA fragment was relevant to the cellulose biosynthesis. Moreover, modulation cellulose synthesis probably was an important influencing factor in polysaccharide metabolism and adaptations of plants to stresses. This will provide technological possibilities for the further study of modulation of the cellulose content of crops.


Assuntos
Adaptação Fisiológica/genética , Brassica/enzimologia , Brassica/genética , Celulose/metabolismo , Técnicas de Silenciamento de Genes , Glucosiltransferases/genética , Folhas de Planta/anatomia & histologia , Cloreto de Sódio/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Sequência de Aminoácidos , Brassica/efeitos dos fármacos , Brassica/fisiologia , Parede Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Especificidade de Órgãos/genética , Pectinas/metabolismo , Fenótipo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Prolina/metabolismo , Interferência de RNA , Análise de Sequência de DNA , Solubilidade , Açúcares/metabolismo , Transcrição Genética
11.
Sci Rep ; 6: 31814, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27545827

RESUMO

The function of aquaporin (AQP) protein in transporting water is crucial for plants to survive in drought stress. With 47 homologues in tomato (Solanum lycopersicum) were reported, but the individual and integrated functions of aquaporins involved in drought response remains unclear. Here, three plasma membrane intrinsic protein genes, SlPIP2;1, SlPIP2;7 and SlPIP2;5, were identified as candidate aquaporins genes because of highly expressed in tomato roots. Assay on expression in Xenopus oocytes demonstrated that SlPIP2s protein displayed water channel activity and facilitated water transport into the cells. With real-time PCR and in situ hybridization analysis, SlPIP2s were considered to be involved in response to drought treatment. To test its function, transgenic Arabidopsis and tomato lines overexpressing SlPIP2;1, SlPIP2;7 or SlPIP2;5 were generated. Compared with wild type, the over-expression of SlPIP2;1, SlPIP2;7 or SlPIP2;5 transgenic Arabidopsis and tomato plants all showed significantly higher hydraulic conductivity levels and survival rates under both normal and drought conditions. Taken together, this study concludes that aquaporins (SlPIP2;1, SlPIP2;7 and SlPIP2;5) contribute substantially to root water uptake in tomato plants through improving plant water content and maintaining osmotic balance.


Assuntos
Aquaporinas/genética , Aquaporinas/metabolismo , Secas , Lycopersicon esculentum/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Estresse Fisiológico , Distribuição Tecidual , Xenopus/genética
12.
Front Plant Sci ; 7: 197, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27047496

RESUMO

In this work, we found, that exogenous melatonin pretreatment improved anthocyanin accumulation (1- to 2-fold) in cabbage. To verify the relationship with melatonin and anthocyanin, an Arabidopsis mutant, snat, which expresses a defective form of the melatonin biosynthesis enzyme SNAT (Serotonin N-acetyl transferase), was employed. Under cold conditions, the foliage of wild-type Arabidopsis exhibited a deeper red color than the snat mutant. This finding further proved, that exogenous melatonin treatment was able to affect anthocyanin accumulation. To gain a better understanding of how exogenous melatonin upregulates anthocyanin, we measured gene expression in cabbage samples treated with melatonin and untreated controls. We found that the transcript levels of anthocyanin biosynthetic genes were upregulated by melatonin treatment. Moreover, melatonin treatment increased the expression levels of the transcription factors MYB, bHLH, and WD40, which constitute the transcriptional activation complex responsible for coordinative regulation of anthocyanin biosynthetic genes. We found, that free radical generation was downregulated, whereas the osmotic adjustment and antioxidant capacities were upregulated in exogenous melatonin-treated cabbage plants. We concluded, that melatonin increases anthocyanin production and benefits cabbage growth.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...