|Other Abstract||As a unicellular eukaryotic ciliated protozoan, Tetrahymena showed the special feature in its genome: the expansion of large gene families associated with the sensing of and responding to environmental changes. Thus it is a good model organism to study the evolution of these gene families. Metallothioneins (MTs) is a well-conserved gene superfamily which is closely associated with the cellular physiological process under environmental stress. Based on the cloning, identification and evolutionary analysis of cadmium-inducible MT genes from six Tetrahymena species, this study showed:
1．A novel Cd-inducible metallothionein (MT) gene (Tpig-MT1) was cloned and sequenced from the ciliate Tetrahymena pigmentosa. The deduced polypeptide possesses CCC and CC clusters with characteristics of typical Tetrahymena Cd-MTs. The structure of Tpig-MT1 is different from the known Cd-MTs in T. pyriformis, T. thermophila and T. pigmentosa. Tpig-MT1 contains two intragenic tandem repeats with 72.9% identity， which were designated as Tpig-MT1 (repeat A1) and Tpig-MT1 (repeat A2). The transcriptional response of Tpig-MT1 gene to different heavy metals (Cd, Cu, Zn, Hg, Pb) and oxidative stress (H2O2) was measured using real-time quantitative PCR. The results showed that the gene was quickly induced (1 h) by the five heavy metals and the order of expression level was Hg> Pb> Cd> Cu>Zn. The induction effect of H2O2 was 5-fold after about 15 min, but soon decreased to a non-significant level (30 min). Based on the genetic diversity of Tetrahymena Cd-MT isoforms, We hypothesize that the module A and B of Tetrahymena subfamily 7a MTs genes may already exist respectively as the ancestral subfamily 7a MT component, and Tetrahymena subfamily 7a MT gene composed of only module B could exist.
2．Several novel Cd-MTs （Tell-MT1, Tvor-MT1, Tmob-MT1, Tmal-MT1, Tmal-MT2）and MT ESTs（Tmal-MT3, Theg-MT1和 Theg-MT3）and a cDNA sequence with full 3’terminus（Theg-MT2）were obtained from T. elliotti, T. vorax, T. mobilis, T. malaccensis and T. hegewischi. All of them showed typical characteristic of Tetrahymena MTS 7a subfamily (Cd-MTs), and composed of module “A” or “B” by intragenic tandem repeats: Tell-MT1 (AA), Tvor-MT1 (AAB), Tmob-MT1 (AAA), Tmal-MT1 (AA), Tmal-MT2 (BBBB), Tmal-MT3 (AAB), Theg-MT1 (AA) and Theg-MT3 (BBBB). The analysis of gene structures and evolution of these Cd-MT isoforms and known Tetrahymena Cd-MT isoforms revealed that, Tetrahymena Cd-MT modules A and B may already exist respectively before the divergence of two major clades (T. borealis and T. australis) within the Tetrahymena genus.|