Blastocladiella emersonii is a saprobic aquatic fungus, belonging to the class of Chytridiomycetes (Alexopoulus et al., 1996), whose life cycle suffers dramatic biochemical and morphological changes during two stages of cell differentiation: the germination and the sporulation.
The life cycle begins with the zoospore, a motile uninucleated non-growing cell, which germinates rapidly and synchronously upon exposure to nutrient medium or an inorganic salt solution containing certain monovalent cations (Soll and Sonneborn, 1972), cyclic AMP (Gomes et al., 1980), or other inducers (Gottschalk and Sonneborn, 1982). During the first 20 min of germination at 27oC, the zoospore retracts its flagellum and forms a cell wall of chitin. The resulting round cell converts into a germling cell, with the formation of a germ tube that elongates and begins to branch at approximately 60 min, giving rise to a rhizoidal system through which the nutrients are absorbed (Lovett, 1975). During vegetative growth, the cells go through intense nuclear division without cytokinesis, generating single-celled coenocytes. Nutrient starvation at any time during growth induces the other transitional stage, the sporulation, that after 3.5-4h at 27oC culminates with the intracellular formation of the zoospores, which are then released to the medium through an opening in the cell wall denominated discharge papilla (Lovett, 1975).
B. emersonii is a primitive fungus, which has diverged early in the fungal lineage (Van der Auwera and De Wachter, 1996; Heckman et al., 2001). Despite the particular taxonomic position and the significance as an important ecological group that involves saprobes as well as plant, animal and fungal pathogens (Powell, 1993), the chytrids remain poorly characterized. Although B. emersonii has become one member of the group that has been extensively studied at different levels, current knowledge about its expressed genes is limited to the rRNA genes and eight protein coding sequences (Van der Auwera and De Wachter, 1996; Forster et al., 1990; Marques and Gomes, 1992; de Oliveira et al., 1994; Stefani and Gomes, 1995; Rocha and Gomes, 1998, 1999; de Souza and Gomes, 1998; Simão and Gomes, 2001; Fietto et al., 2002).
An efficient way to obtain information about gene expression and coding sequences of uncharacterized genomes is to sequence a large number of expressed sequence tags (ESTs). If obtained from non-normalized libraries, the EST sequencing analysis (also known as digital Northern) can represent the expression profile, including complexity and abundance levels of transcripts of different tissues, cell types and developmental stages (Adams et al., 1991; Porcel et al., 2000; Kamoun et al. 1999; Lee et al., 2002; Felipe et al., 2003).
We report here a high throughput cDNA sequencing program which is the first approach to the understanding of gene complexity in B. emersonii. We have produced 16,984 high-quality sequences corresponding to the 5’ ends of cDNAs from ten different libraries constructed using mRNA isolated from synchronized cells, collected at different stages along B. emersonii life cycle. This first large-scale sequencing project of a chytridiomycete transcriptome represents an important set of expressed sequences for studies of phylogeny as well as growth and differentiation in lower fungi.
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