IDENTIFICATION AND INDUCED EXPRESSION OF THE METALLOTHIONEIN GENE OF A HIGH CD-TOLERANCE COLPODA INFLATA POPULATION

Cadmium ranks first among the heavy metal pollutants under limited controll in water. Metallothionein is one of the key proteins that can bind to cadmium and regulate the oxidative stress response of organisms. The protozoan MT has been reported in two ciliates, Tetrahymena and Paramecium. In this study, we obtained a northeast population of Colpoda inflata with high cadmium tolerance. Metallothionein content of C. inflata showed a positive correlation with cadmium concentration and growth rate of population in five gradient concentration of cadmium stress experimental groups from 24—96h. The highest Cd-tolerance concentration was 10 mg/L in 96h. Metallothionein gene of Colpoda inflata was cloned and named Col-MT1. The gene sequence and amino-acid sequence were analyzed. The results showed that Col-MT1 had high homology with the amino acid sequences of other ciliates, and contains two conserved sites XXCXX and XCCX. It was a new member of subtype 7a of metallothionein gene family. TASSER protein model predicted that the secondary structure of Col-MT1 protein was composed of α-helix, β-folding and random crimp, accounting for 67.90%, 11.11% and 20.98%, respectively. SWISS-MODEL predicted that the 3D structure of Col-MT1 protein was the most similar to that of cyanobacteria metallothionein. qRT-PCR experiment confirmed that at 60h, 84h and 108h, the expression of Col-MT1 gene was up-regulated to different concentration cadmium and showed a dose-response relationship with cadmium concentration. The molecular mechanism of gene expression still needs further study. Above results supplemented the MT gene database of protozoa and lay a foundation for revealing the mechanism of C. inflata MT gene. As well as provide reference for monitoring and remediation of cadmium pollution.