The metallothionein-2 (MT-2) gene was cloned from the mandarin fish Siniperca chuatsi (Basilewsky, 1855) by using the method of rapid amplification of cDNA ends (RACE). The spliced cDNA sequence extended 388bp. The 5′ untranslated region (UTR) and 3′ UTR are 49 and 156 nt respectively. The polyadenylation signal (AATAAA) is found starting at 18 nt upstream from the polyA tail. The putative MT is predicted to be a peptide of 60 amino acids including highly conserved 20 cysteine residues. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothioneins. The amplified MT-2 genomic sequence is 865 bp in length, which consists of 3 exons and 2 introns. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The promoter region extended 1466 bp and contained 10 putative metal response elements (MREs) composing 3 clusters and TATA box, AP1, etc.
The MT-2 genome from the spotted mandarin fish Siniperca scherzeri (Steindachner, 1892) was also amplified utilizing primers designed according to the mandarin fish MT-2 cDNA sequence. It extended 865bp consisting of 3 exons and 2 introns. The promoter region extended 1167bp including 6 MREs composing 3 clusters and GREs, AP1, etc.
Real-time PCR analysis revealed that mandarin fish MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine. It was weakly detected in the brain and muscle. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm of hepatocytes in the liver, and the cytoplasm of the proximal and distal tubular epithelium in the trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution.