RESUMEN
The promoter of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene from Aspergillus nidulans (PgpdA) is widely used to direct expression of target genes constitutively in fungi. However, in some species, a heterogeneous promoter is found to be of low efficiency. To obtain a high-efficiency promoter for transformation of Beauveria bassiana, an entomopathogenic fungus widely used as an mycoinsecticide, a glyceraldehyde-3-phosphate dehydrogenase gene (Bbgpd) promoter, was cloned and characterized. Four deletion constructs (-2118, -1153, -726, and -354) of the 5'-upstream sequence of Bbgpd linked to a bar::gus fusion gene (phosphinothricin-resistance::beta-glucuronidase fused gene), which were used as selected marker gene and report gene, respectively, were generated. GUS activities of transgenic strains harboring -726, -1153, and -2118 deletion constructs were much stronger than that of the promoter of Aspergillus nidulans gpdA (PgpdA), with a twofold to threefold increase over that in the PgpdA construct. The -726 fragment was necessary to direct GUS expression in B. bassiana. No -354 transgenic progenies were obtained, possibly because it failed to initiate the transcription of bar::gus fusion gene. A remarkable increase of GUS activity was found between the -1153 and -726 constructs, indicating that some active transcriptional elements were located in this region. With a high expression level and relatively short sequence, PBbgpd can be used to drive target genes in B. bassiana transgenic research.
Asunto(s)
Beauveria/genética , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Regiones Promotoras Genéticas , Región de Flanqueo 5' , Secuencia de Aminoácidos , Secuencia de Bases , Beauveria/metabolismo , Proteínas Fúngicas/genética , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , Datos de Secuencia Molecular , Eliminación de SecuenciaRESUMEN
Based on the flanking sequence of T-DNA of a T-DNA insertion mutant of Beauveria bassiana, T12, the full length cDNA of carboxylic transport protein, designated MaJen1, was cloned from Metarhizium anisopliae. MaJen1 is 1,695 bp long and contained a 1,524 bp ORF which predicted a protein of 508 amino acid. The amino acid sequence of the gene showed 69% and 31% identity to the carboxylic transport protein of Neurospore crassa and Saccharomyces cerevisiae, respectively. The genome sequence, GMaJen1, was amplified by PCR, indicating that there were two introns in GMaJen1. Southern analysis indicated that GMaJen1 was present as a singl copy in Metarhizium anisopliae. The result of RT-PCR showed that expression of MaJen1 was induced by the cuticle of cockroach and repressed by glucose. A 1,626 bp upstream sequence of GMaJen1 was amplified by YADE method, which contained several putative binding domains of glucose repressor.
Asunto(s)
Ácidos Carboxílicos/metabolismo , Hypocreales/genética , Transportadores de Ácidos Monocarboxílicos/genética , Regiones Promotoras Genéticas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , ADN Complementario/química , ADN Complementario/genética , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Hypocreales/metabolismo , Datos de Secuencia Molecular , Transportadores de Ácidos Monocarboxílicos/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA library was constructed using mRNA from mycelia of Beauveria bassiana grown on cuticle/chitin cultures. A cDNA clone of the protease, designated CDEP-1, was isolated from cDNA library. CDEP-1 contained an 1,134 bp ORF that predicted a protein of 377 amino acids with M(r) = 38,616 and PI = 8.302. The amino acid sequence of the gene shows 57.9%, 83.3% and 54.7% identity to Metarhizium nisopliae Pr1, Beauveria bassiana Pr1 and proteinase K, respectively. Southern analysis indicated that CDEP-1 was present as singly copy in Beauveria bassiana.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas de Insectos/metabolismo , Hongos Mitospóricos/enzimología , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Serina Endopeptidasas/químicaRESUMEN
A Y-shaped adaptor dependent extension (YADE) method was developed to amplify the unknown genomic sequences adjacent to a known sequence, with the single-primer amplification completely suppressed. Using this method, two fragments, F027S and F027A, adjacent to a cotton ovule cDNA fragment (F027), were amplified using genomic DNA. It was demonstrated that F027S and F027A had 104 bp and 175 bp overlapped to F027, and contained 1 and 3 putative introns, respectively, all of which included conserved border sequence GT-AG and a putative branch site. The possible improvements and applications of YADE method are further discussed.
