Open Access

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

  • Katherinne Navarrete1,
  • Amanda Roa1,
  • Inmaculada Vaca2,
  • Yeison Espinosa1,
  • Claudio Navarro1 and
  • Renato Chávez1Email author
Cellular & Molecular Biology LettersAn International Journal200914:28

https://doi.org/10.2478/s11658-009-0028-y

Received: 10 March 2009

Accepted: 23 June 2009

Published: 27 June 2009

Abstract

Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5′-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.

Key words

Penicillium camemberti niaD gene pyrG geneTransformation Aspergillus nidulans

Advertisement