Author	 FLORENCIO, C.S., University of Brasilia, UnB - Brazil, Brasília, Brazil (Presenting author)
Co-author(s)	 Pimentel, N.V.G, University of Brasilia, UnB - Brazil, Brasília, Brazil
Felipe, M.S.S, Universidade Católica de Brasília-UCB, Brasília, Brazil
Bocca, A.L, University of Brasilia, UnB - Brazil, Brasília, Brazil
Fernandes-Matos, L., University of Brasilia, UnB - Brazil, Brasília, Brazil
Abstract title	Molecular tools for gene deletion in Fonsecaea pedrosoi (Poster)
Internet ID 	 1307
Objectives:
Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis (CBM), a disease with a worldwide distribution but prevalent in tropical and subtropical countries. It is a filamentous fungus with hyphae and conidia pigmented due to the production of melanin. The biology, virulence factors and mechanisms of pathogenesis of F. pedrosoi are not explained, and therefore studies involving molecular tools for genetic deletion are essential to improve knowledge about the disease and potentially to develop new antifungal therapies. One of the most used techniques in fungi for genetic transformation in other fungus is biolistics, a technique widely used to deliver genetic material directly to cells and intact tissues by bombarding particles, which requires optimization of physical parameters. In this study the main objective was to use strains of F. pedrosoi (CBS 271.37) to optimize the particle bombardment and the construction of deletion cassettes using Double Joint PCR.



Methods:

For the construction of the gene deletion cassettes the double joint PCR technique was used and these were genetically transformed by biobalistc into conidia of F. pedrosoi (strain CBS 271.37 - ATCC 18658). To obtain a better result and also standardization of transformation parameters, purified and plated conidia and conidia germinated in 24 hours were used in the transformation. After transformation the plates were incubated for 24 hours and then the cells were plated in medium containing antibiotics. The transformants were selected by hygromycin B, the stability was verified mitotically and the gene deletion was confirmed by PCR and then Southern blot.



Results:

This work describes the construction and obtaining of an F. pedrosoi mutant by homologous recombination and deletion of genes using Biobalistica and DJ PCR . After transformation the mutants that were able to grow and remain stable were selected for phenotype tests and also for confirmation by PCR and southern blot, as well as functional characterization of the deleted gene is underway.



Conclusion:

Deletion of genes using biobalistic and PCR double joint is well characterized in other fungi, such as Aspergillus sp. and Cryptococcus sp. The experiments demonstrated that the use of biobalistic for the genetic transformation of F.pedrosoi through conidia is efficient. Mutants are able to grow in media containing antibiotics and maintain mitotic stability. Particle bombardment is a fast and advantageous method and with this study it is possible to study several genes of Fonsecaea to better understand the pathobiology, since the adjustment of protocols and use of molecular tools for genetic deletion is being described for the first time. Some tests are under way to better describe these deletions.