RESUMEN
γ-Aminobutyric acid (GABA) is a very important inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. GABA receptors (GABARs) are known to be the molecular targets of a class of insecticides. Members of the GABAR gene family of the silkworm, Bombyx mori, a model insect of Lepidoptera, have been identified and characterized in this study. All putative silkworm GABAR cDNAs were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Bombyx mori appears to have the largest insect GABAR gene family known to date, including three RDL, one LCCH3, and one GRD subunit. The silkworm RDL1 gene has RNA-editing sites, and the RDL1 and RDL3 genes possess alternative splicing. These mRNA modifications enhance the diversity of the silkworm's GABAR gene family. In addition, truncated transcripts were found for the RDL1 and LCCH3 genes. In particular, the three RDL subunits may have arisen from two duplication events.
Asunto(s)
Bombyx/genética , Genes de Insecto , Proteínas de Insectos/genética , Receptores de GABA/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Bombyx/metabolismo , Evolución Molecular , Amplificación de Genes , Regulación de la Expresión Génica , Orden Génico , Genes Duplicados , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Edición de ARN , Receptores de GABA/química , Receptores de GABA/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ácido gamma-Aminobutírico/metabolismoRESUMEN
A BmNPV Bacmid with the Bmvp80 gene disrupted was constructed using the ET-recombination system in Escherichia coli to investigate the role of Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in single cell infection phenotype, whereas a rescue BmBacmid restored budded virus titers to wild type levels; however, the homologous gene Ac104 (Acvp80) from AcMNPV could not complement the BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cells transfected with BmNPV lacking functional Bmvp80 revealed that the number of nucleocapsids was markedly lower. These results suggest that Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species.
Asunto(s)
Silenciador del Gen , Nucleopoliedrovirus/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Animales , Bombyx , Línea Celular , Nucleopoliedrovirus/fisiología , Especificidad de la Especie , Ensamble de Virus , Replicación ViralRESUMEN
Acetylcholinesterase (AChE; EC 3.1.1.7) is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). Because AChE is expressed in all invertebrate and vertebrate animals as a key enzyme of the cholinergic system, the toxicity of anticholinesterase insecticides to mammals and non-target species such as beneficial insects has been a great concern. In addition, the intensive use of OP and CB insecticides has resulted in the development of resistance in many insect pests, which has limited the use of anticholinesterase insecticides. Many aces encoding AChEs have been sequenced from a variety of vertebrates, insects and other invertebrates, and crystal structures of four AChEs have been determined in the past 20 years. Although the primary motifs and the three dimensional (3D) structures of different AChEs are similar, differences among AChEs are obvious. The catalytic properties and inhibition kinetics of AChEs from different groups of insects and mammals may be quite different, and two AChEs from a single insect may also show distinct differences. These differences may provide new opportunities for designing more selective insecticides for pest management.
Asunto(s)
Acetilcolinesterasa/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Insecticidas/farmacología , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Carbamatos/farmacología , Humanos , Resistencia a los Insecticidas , Compuestos Organofosforados/farmacología , Conformación Proteica , Especificidad de la EspecieRESUMEN
Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus- insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I(50) values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori.