This paper reports the purification and localization of a Tuber borchii Vittad. fruitbody protein (TBF-1) and the cloning of the encoding gene. TBF-1 is detectable by SDS-PAGE analyses only in this white truffle species and presents a molecular mass of 11,994 Da. TBF-1 was purified by one-step Reversed-Phase HPLC and its complete amino acid sequence was determined after digestion with trypsin and N-Asp endoproteinase. Polyclonal antibodies were produced and tested in immunofluorescence and immunogold experiments, providing information about the protein localization. It was detected mostly on the hyphal walls, where it was colocalized with b-1,3-glucans and chitin. The sporal wall was not labeled. The encoding gene (tbf-1) was cloned using several techniques involving PCR. The coding region consists of a 360-bp open reading frame interrupted by an intron, with another intron following the stop codon. A putative signal peptide of 12 amino acids was found at the N-terminal. Northern blot analysis revealed that tbf-1 is highly expressed in unripe and ripe fruitbodies and was not detectable in culture mycelium or ectomycorrhizal roots.
The tbf-1 gene from the white truffle Tuber borchii codes for a structural cell wall protein specifically expressed in fruitbody.
BALESTRINI R;
1998
Abstract
This paper reports the purification and localization of a Tuber borchii Vittad. fruitbody protein (TBF-1) and the cloning of the encoding gene. TBF-1 is detectable by SDS-PAGE analyses only in this white truffle species and presents a molecular mass of 11,994 Da. TBF-1 was purified by one-step Reversed-Phase HPLC and its complete amino acid sequence was determined after digestion with trypsin and N-Asp endoproteinase. Polyclonal antibodies were produced and tested in immunofluorescence and immunogold experiments, providing information about the protein localization. It was detected mostly on the hyphal walls, where it was colocalized with b-1,3-glucans and chitin. The sporal wall was not labeled. The encoding gene (tbf-1) was cloned using several techniques involving PCR. The coding region consists of a 360-bp open reading frame interrupted by an intron, with another intron following the stop codon. A putative signal peptide of 12 amino acids was found at the N-terminal. Northern blot analysis revealed that tbf-1 is highly expressed in unripe and ripe fruitbodies and was not detectable in culture mycelium or ectomycorrhizal roots.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
