RESUMO
Two cDNAs encoding novel Rel proteins were cloned from the silkworm, Bombyx mori. These cDNA clones (BmRelA and BmRelB) showed identical nucleotide sequences except for the 5'-region. BmRelB cDNA derived probably from an alternatively spliced mRNA lacked 241 bp nucleotides at the 5'-region of the BmRelA cDNA, resulting in a loss of the first 52 amino acids. Expression of antibacterial peptide genes was strongly inhibited upon infection with Micrococcus luteus in transgenic silkworms in which BmRel gene expression was knocked down, suggesting that these two Rel proteins are involved in activation of antibacterial peptide genes. Co-transfection experiments indicated that BmRelB activated the Attacin gene strongly and other genes to a lesser extent, whereas BmRelA activated Lebocin 4 gene strongly and Attacin and Lebocin 3 genes very weakly. The Rel homology domain of BmRelA and BmRelB was shown to bind specifically to kappaB sites of antibacterial peptide genes. Proline-rich domains of the BmRels were necessary for activation of antibacterial peptide genes. These results illustrate that a minor structural change in Rel proteins can provoke a dramatic differential activation of antibacterial peptide genes, suggesting a novel regulatory mechanism for insect antibacterial peptide gene expression.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bombyx/genética , Regulação da Expressão Gênica/imunologia , Proteínas de Insetos/genética , Família Multigênica/genética , Filogenia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Bases , Bombyx/imunologia , Clonagem Molecular , Análise por Conglomerados , Primers do DNA , DNA Complementar/genética , Proteínas de Drosophila/genética , Ensaio de Desvio de Mobilidade Eletroforética , Componentes do Gene , Vetores Genéticos , Glutationa Transferase , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Família Multigênica/imunologia , NF-kappa B/metabolismo , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , TransfecçãoRESUMO
We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.
