EST-based identification of genes expressed in the liver of adult seabass (Dicentrarchus labrax, L.)
Introduction
The decline of ocean fisheries stocks has provided a rapid growth in fish farming (Naylor et al., 2000) that, thanks to modern technologies, has improved production and quality and, at the same time, reduced the environmental impact with benefits on the public perception of the aquaculture industry (Huntingford, 2002, Kristiansen and Juell, 2002). The increasing pressure in the field of aquaculture has determined the necessity to adopt the most recent techniques of molecular biology that, integrated with the classic methodologies, could augment the overall efficiency of the industry. Functional genomic, for instance, can offer precise information on the actual genes responsible for growth, disease resistance, flesh quality, health conditions, welfare and other attributes (Gornati et al., 2005). We are, therefore, certain that the new molecular techniques will find their place in the everyday management of fish farming, but we are also aware that the scarcity of the genomic resources for some fish species, in spite of their commercial interest, will retard the positive effects that modern biotechnology can offer to aquaculture industry. We are convinced that an effort should be made to reduce, as far as it concerns genomic resources, the gap that separates farming species from “model organisms”. For this reason in this paper, we present an EST project on sea bass, a fish species of great economical interest in the Mediterranean area. EST cataloguing and profiling of sea bass will set the basis for functional genomic research in this species, for comparative and environmental genomics, for the identification of polymorphic markers, for the discovery of new molecular markers of exposure and for the production of micro- and macro-arrays.
Section snippets
cDNA library and mass excision protocol
We have used a cDNA library from liver of Dicentrarchus labrax that we had previously constructed using ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene, CA, USA) following the manufacturer's instructions (Gornati et al., 2004). Briefly, we isolated total RNA with TRIzol and mRNA through oligo(dT)-cellulose columns (SIGMA). We synthesized the cDNAs using oligo(dT) primers containing Xho I restriction site and Stratascript reverse transcriptase (Stratagene, CA, USA). After the second strand
Sequence analysis
We performed single pass sequencing on 1229 randomly selected clones from a sea bass cDNA library targeting the 5′-terminus of each insert. For each sequence, we eliminated the vector portion at the 5′-terminus and the 3′-terminus portions when rich in undetermined bases. The length distribution of the trimmed sequences is reported in Fig. 1 and their average length resulted of about 700 bp. The sequences were deposited in the NCBI Expressed Sequence Tags database (//www.ncbi.nlm.nih.gov/projects/dbEST
Discussion
We are convinced that the beneficial effects that modern biotechnology can bring to aquaculture industry might be reduced by the scarcity of the genomic resources for fish species of commercial interest and the availability of nucleotide sequences is a fundamental tool to pursue molecular research. We have, therefore, started an EST project on sea bass by sequencing 1229 randomly selected clones from a liver cDNA library.
We have used an expression (non-normalized) library for ESTs production
Acknowledgement
This research was supported by grant 6-C-129 and 6-D-39 of MiPAF.
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