RESUMO
The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticilliumfungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5' flanking sequence and 731 bp of 3' flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5' flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V.fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.
Assuntos
Agaricales , DNA Espaçador Ribossômico/genética , Proteínas Fúngicas/genética , Nitrato Redutases/genética , Verticillium/classificação , Verticillium/genética , Sequência de Aminoácidos , DNA Fúngico/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Verticillium/enzimologia , Verticillium/patogenicidadeRESUMO
The 3' regions of the gene encoding the cap binding protein eIF4E were successfully isolated from Agaricus bisporus and Verticillium fungicola using a degenerate primer within the eIF4E gene and an anchored oligo d(T) primer. The deduced amino acid sequences contained 173 residues for A. bisporus and 171 residues V. fungicola. Analysis of these sequences shows that despite conserved regions of homology, centering around tryptophan residues, A. bisporus and V. fungicola are very diverse at the amino acid and DNA level. Percentage homology between the two fungi is low at the nucleotide, 35%, and amino acid level, 29%. The highest degree of similarity between the A. bisporus sequence and other published sequences is with the Homo sapiens eIF4E sequence (32%). V. fungicola exhibited highest homology with the eIF4E sequence from Caenorhabditis elegans (34%). Southern analysis of genomic DNA indicated a single copy of the gene within the A. bisporus genome.