RESUMO
Three genes encoding proteins showing sequence similarity and features typical of insect APNs were characterized in C. tremulae and designed as CtAPN1, CtAPN2 and CtAPN3. Expression analysis of the three C. tremulae APN genes showed that CtAPN2 transcript is more abundant in the fat body, whereas both CtAPN1 and CtAPN3 are specifically expressed in the midgut. Despite a similar genomic organization, lepidopteran and coleopteran APNs are phylogenetically distant, suggesting that APN gene duplication events occurred after these two insect orders split. Sequence and expression comparisons of CtAPN1, CtAPN2 and CtAPN3 cDNAs in a C. tremulae Bacillus thuringiensis (Bt)-susceptible and in a Bt-resistant strain did not show any polymorphism at the amino acid level or difference at the transcription level.
Assuntos
Proteínas de Bactérias , Antígenos CD13/genética , Besouros/enzimologia , Besouros/genética , Endotoxinas , Proteínas Hemolisinas , Controle Biológico de Vetores , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Sistema Digestório/enzimologia , Corpo Adiposo/enzimologia , Feminino , Perfilação da Expressão Gênica , Estágios do Ciclo de Vida , Filogenia , Plantas Geneticamente Modificadas/genética , Populus/genética , Alinhamento de Sequência , Tribolium/genéticaRESUMO
In order to understand how lepidopteran insects react physiologically to Bacillus thuringiensis crystal toxin ingestion, transcriptional profiling of Choristoneura fumiferana larvae exposed to sublethal doses of Cry1Ab protoxin were monitored using a C. fumiferana-specific cDNA microarray derived from a protoxin-specific subtractive library. Differential gene expression occurred primarily between 2 and 5 h postingestion. Metabolic enzymes such as lipases and proteases were generally repressed, whereas genes involved in detoxification, immune system regulation or general stress response were upregulated. A similar protoxin-specific transcriptional pattern was also observed with Manduca sexta larvae, using three upregulated genes (serpin, cytochrome P450 and carboxyl/cholinesterase) and one downregulated gene (beta-glucosidase), suggesting that a susceptible larval response to Cry toxin exposure might be universal among lepidopterous insects.
Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Endotoxinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hemolisinas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/genética , Animais , Toxinas de Bacillus thuringiensis , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/genéticaRESUMO
Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after protoxin exposure.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Lepidópteros/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Toxinas de Bacillus thuringiensis , Sequência de Bases , Primers do DNA , Biblioteca Gênica , Larva/efeitos dos fármacos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Análise de Sequência de DNARESUMO
The complete nucleotide sequence of the genomic RNA of an aphid-infecting virus, Aphid lethal paralysis virus (ALPV), has been determined. The genome is 9812 nt in length and contains two long open reading frames (ORFs), which are separated by an intergenic region of 163 nt. The first ORF (5' ORF) is preceded by an untranslated leader sequence of 506 nt, while an untranslated region of 571 nt follows the second ORF (3' ORF). The deduced amino acid sequences of the 5' ORF and 3' ORF products respectively showed similarity to the non-structural and structural proteins of members of the newly recognized genus Cripavirus (family Dicistroviridae). On the basis of the observed sequence similarities and identical genome organization, it is proposed that ALPV belongs to this genus. Phylogenetic analysis showed that ALPV is most closely related to Rhopalosiphum padi virus, and groups in a cluster with Drosophila C virus and Cricket paralysis virus, while the other members of this genus are more distantly related. Infectivity experiments showed that ALPV can not only infect aphid species but is also able to infect the whitefly Trialeurodes vaporariorum, extending its host range to another family of the order Hemiptera.
Assuntos
Afídeos/virologia , Genoma Viral , Vírus de RNA/classificação , Vírus de RNA/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Afídeos/patogenicidade , Sequência de Bases , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Hemípteros/virologia , Dados de Sequência Molecular , Filogenia , Vírus de RNA/química , Proteínas não Estruturais Virais/genéticaRESUMO
The genomic sequence of a new icosahedral DNA virus infecting Myzus persicae has been determined. Analysis of 5499 nt of the viral genome revealed five open reading frames (ORFs) evenly distributed in the 5' half of both DNA strands. Three ORFs (ORF1-3) share the same strand, while two other ORFs (ORF4 and ORF5) are detected in the complementary sequence. The overall genomic organization is similar to that of species from the genus DENSOVIRUS: ORFs 1-3 most likely encode the non-structural proteins, since their putative products contain conserved replication motifs, NTP-binding domains and helicase domains similar to those found in the NS-1 protein of parvoviruses. The deduced amino acid sequences from ORFs 4 and 5 show sequence similarities with the structural proteins of the members of the genus DENSOVIRUS: These data indicate that this virus is a new species of the genus Densovirus in the family PARVOVIRIDAE: The virus was tentatively named Myzus persicae densovirus.
Assuntos
Afídeos/virologia , Vírus de DNA/classificação , Densovirus/classificação , Genoma Viral , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Vírus de DNA/química , Vírus de DNA/genética , Densovirus/química , Densovirus/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Regiões Promotoras Genéticas , Sítios de Splice de RNA , Proteínas ViraisRESUMO
A new icosahedral DNA virus was isolated from aphids (Myzus persicae) that showed abnormal growth and development. The purified virus particles have a diameter of 20 nm and contain a single-stranded DNA molecule of approximately 5.7 kb. The viral particles are composed of five structural proteins (92, 85, 68, 64, and 57 kDa). As the main biophysical properties of this virus are similar to those of the members of the genus Densovirus it was tentatively named Myzus persicae densovirus (MpDNV). A PCR-based detection method and a polyclonal antiserum raised against MpDNV allowed the detection of the virus in a single-infected aphid. MpDNV is immunologically related to Junonia coenia densovirus, but not to other members of the subfamily Densovirinae. Biological assays showed that MpDNV could be both transmitted transovarially and horizontally via honeydew and saliva. MpDNV was able to infect whiteflies but not other aphid species tested.