RESUMO
Lentinus tuber-regium is a sclerotium-forming basidiomycetous mushroom. It has increasingly aroused people's attention for its medicinal effects. In this study, we investigated the applicability of the Agrobacterium tume-faciens-mediated transformation (ATMT) method in L. tuber-regium. A. tumefaciens strain GV 3101 harboring the vector pPEH was used to transform the mycelium of L. tuber-regium strain ACCC50657. The genes for hygromycin B phosphotransferase (hph) and enhanced green fluorescent protein (egfp) under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter of Pleurotus ostreatus were employed as the selection marker and reporter gene, respectively. The optimal cocultivation temperature and time for transformation were 3 days and 4 days at 25°C and 20°C, respectively. Southern blot analysis showed the variation in the copy number and position of hph, which indicated random integration of hph. Polymerase chain reaction and fluorescence microscopy indicated that the P. ostreatus gpd promoter can drive the fused hph-egfp gene expression in L. tuber-regium. This is the first report that the ATMT method was successfully applied to L. tuber-regium. This reliable and efficient transformation method could be a powerful tool for strain genetic improvement and gene function study in L. tuber-regium.
Assuntos
Agrobacterium tumefaciens/genética , Lentinula/genética , Regulação Fúngica da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Lentinula/fisiologia , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
Polyporoid and lentinoid fungi contain the important producers of substances having immunomodulatory, antitumoral, antiviral, and antihyperlipidemic effects. The discovery of several phylogenetic lines within the lentinoid-polyporoid continuum will help with target metabolomic analysis of species still not studied in pharmacological respects. The purpose of the present work was to increase a resolution in the lentinoid-polyporoid phylogenetic zone by means of selection of both the main representatives of Lentinus-related genera and poorly known/intermediate taxa such as Lentinus suavissimus, Neofavolus spp., and the resupinate part of Polyporus (genera Perenniporia and Pachykytospora) in the context of the basic structure of the Polyporales tree. The molecular phylogeny of highlighting all the polyporoid and lentinoid nodes was reconstructed using nLSU ITS rDNA and TEF datasets. The data obtained from ITS, TEF, and LSU coincide in support of core Polyporaceae of 10 clades corresponded to the generic level and 7 of these (Cerioporus, Cladomeris, Favolus, Lentinus, Neofavolus, Picipes, and Polyporus s.str.) contain generic units characterized by polyporoid or lentinoid morphotypes. The other 2 clades containing lentinoid taxa are outside the core Polyporaceae, namely Panus (Meruliaceae, Polyporales) and Neolentinus (Gloeophyllaceae, Gloeophyllales). A new genus, Picipes, is described and 25 new combinations are proposed.
Assuntos
Lentinula/classificação , Polyporaceae/classificação , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Proteínas Fúngicas/genética , Lentinula/química , Lentinula/genética , Filogenia , Polyporaceae/química , Polyporaceae/genética , Polyporus/química , Polyporus/classificação , Polyporus/genética , Análise de Sequência de DNARESUMO
In order to isolate the genes expressed specifically and abundantly in the mature fruit body of Lentinula edodes, the cDNAs derived from the gill of the fruit body were compared with the cDNAs from the mycelia by differential screening. Consequently, six clones were identified as fruit-body-specific genes (fbg03, 08, 13, 14, 16, and 21). The deduced amino acid sequence of fbg14 (Le.cypfb) had significant homology with the cytochrome P450 protein. The transcriptional level of fbg16, which showed 29.9% identity with the riboflavin aldehyde-forming enzyme of Agaricus bisporus, was highest among all of the fbg clones. This result indicates that the promoter region of fbg16 may become a powerful candidate for the expression signal of the vector for the gene manipulation in the mature fruit body.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos/genética , Lentinula/genética , Sequência de Aminoácidos , Northern Blotting , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , DNA Fúngico/biossíntese , DNA Fúngico/genética , Biblioteca Gênica , Dados de Sequência Molecular , Transformação GenéticaRESUMO
The information that the deduced expression product of Lentinus edodes priA gene consists of N-terminal hydrophobic sequence, putative zinc-binding motifs and C-terminal membrane-binding-promoting unique sequence led us to analyze its function in L. edodes. Here L. edodes monokaryotic cells over-expressing priA gene were found to exhibit a remarkably decreased accumulation of zinc ion, indicating the involvement of the priA gene in regulation of the intracellular zinc concentration.