cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure.
The aim of this work was to identify in Eisenia fetida genes whose expression are regulated following exposure to a complex mixture of Metallic Trace Elements (MTE) representative of a highly polluted smelter soil. Suppression Subtractive Hybridization (SSH) was used to construct cDNA libraries enriched in upor downregulated transcripts in the immune circulating cells of the cœlomic cavities, namely cœlomocytes, from worms exposed to metallic pollution. Among 1536 SSH derived cDNA clones sequenced we identified 764 unique ESTs of which we selected 18 candidates on the basis of their redundancy. These selected candidates were subjected to a two-step validation procedure based on the study of their expression level by real-time PCR. The first step consisted in measuring the expression of the 18 candidates in worms exposed to artificial contaminated soil. The second step consisted in measuring the expression in animals exposed to a "naturally" contaminated soil sampled close to a smelter. Both steps allowed us to highlight 3 candidates that are strongly induced in worms exposed to a smelter polluted soil. These candidates are, the well-known MTE induced Cd-metallothionein and 2 original biomarkers, lysenin and a transcript which cloning of the complete coding sequence identified as the Coactosin-Like Protein.