RESUMEN
Bombyx mori cathepsin B (BmCatB) is involved in the programmed cell death of the fat body during B. mori metamorphosis. For a better understanding of the functional regulatory mechanism, the promoter region of BmCatB in the transcriptional regulation has been identified and analyzed in the present study. BmCatB promoter region performed by the 5' truncation or mutagenesis of EcREs was inserted in the pFA3Luc-A3RL double fluorescence expression vector to activate the fireflies luciferase (FLuc) gene. The results indicated that the dual-luciferase activity of BmCatB gene in the silkworm larval fat body is regulated by the length of promoter. Site-directed mutagenesis of EcRE experiment has shown that the EcREs are up-regulated significantly in the regulation of the BmCatB promoter. A 142bp region (-1165 to -1023) and EcREs are the mainly fat-body tissue-specificity related region and could function as a core promoter element.
Asunto(s)
Bombyx/genética , Catepsina B/genética , Cuerpo Adiposo/fisiología , Proteínas de Insectos/genética , Regiones Promotoras Genéticas , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Bombyx/fisiología , Catepsina B/metabolismo , Regulación de la Expresión Génica , Proteínas de Insectos/metabolismo , Luciferasas/genética , Metamorfosis Biológica/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Especificidad de ÓrganosRESUMEN
The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.
