Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation.
Penicillium griseoroseum has been studied because of its efficient pectinases production. In this work, the Penicillium griseoroseum nitrate reductase gene was characterized, transcriptionally analyzed in different nitrogen sources, and used to create a phylogenetic tree and to develop a homologous transformation system. The regulatory region contained consensus signals involved in nitrogen metabolism and the structural region was possibly interrupted by 6 introns coding for a deduced protein with 864 amino acids. RT-PCR analysis revealed high amounts of niaD transcript in the presence of nitrate. Transcription was repressed by ammonium, urea, and glutamine showing an efficient turnover of the niaD mRNA. Phylogenetics analysis showed distinct groups clearly separated in accordance with the classical taxonomy. A mutant with a 122-bp deletion was used in homologous transformation experiments and showed a transformation frequency of 14 transformants/microg DNA. All analyzed transformants showed that both single- and double-crossover recombination occurred at the niaD locus. The establishment of this homologous transformation system is an essential step for the improvement of pectinase production in Penicillium griseoroseum.
Pereira
Jorge Fernando
JF
Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, 36.570-000 Viçosa-MG, Brazil.
de Queiroz
Marisa Vieira
MV
Lopes
Francis Júlio Fagundes
FJ
Rocha
Rodrigo Barros
RB
Daboussi
Marie-Josée
MJ
de Araújo
Elza Fernandes
EF
D000595:Amino Acid Sequence; D004271:DNA, Fungal / Q000032:analysis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005656:Fungal Proteins; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D009584:Nitrogen