RESUMO
Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.
Assuntos
Poligalacturonase/genética , Poligalacturonase/metabolismo , Nicotiana , Cloroplastos/genética , Peroxidase/genética , Peroxidase/metabolismo , Temperatura , Bactérias/enzimologia , Transformação Genética , Parede Celular , Southern Blotting , Reação em Cadeia da Polimerase , Fungos/enzimologia , Concentração de Íons de Hidrogênio , HidrolasesRESUMO
Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.
Assuntos
Fator de Ligação a CCAAT , Penicillium/genética , Poligalacturonase/genética , Ensaio de Desvio de Mobilidade Eletroforética , Genes Fúngicos , Regiões Promotoras Genéticas , Fatores Estimuladores UpstreamRESUMO
Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.
Assuntos
DNA Fúngico/genética , Genoma Fúngico , Proteínas Luminescentes/genética , Mutação , Penicillium/genética , Transformação Genética/genética , Proteínas Luminescentes/análise , Microscopia de Fluorescência , Penicillium/enzimologia , Plasmídeos/genética , Poligalacturonase/genética , Protoplastos/enzimologiaRESUMO
Com o objetivo de obter-se linhagens com maior produçäo de pectinases, Penicillium expansum foi mutagenizado com UV and NTG. Os mutantes selecionados foram analisados quanto à produçäo de enzimas pectinolíticas e comparados com a linhagem parental. Foram obtidos mutantes com aumento de até duas vezes na atividade de Pectina liase ou de Poligalacturonase. O mutante P462 apresenta aumento de 2 vezes na atividade de ambas enzimas