Abstract
Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification in living organisms. In the present study, we cloned two MT genes (OcMT1 and OcMT2) from Oxya chinensis, analyzed the expression patterns of the OcMT transcripts in different tissues and at varying developmental stages using real-time quantitative PCR (RT-qPCR), evaluated the functions of these two MTs using RNAi and recombinant proteins in an E. coli expression system. The full-length cDNAs of OcMT1 and OcMT2 encoded 40 and 64 amino acid residues, respectively. We found Cys-Cys, Cys-X-Cys and Cys-X-Y-Z-Cys motifs in OcMT1 and OcMT2. These motifs might serve as primary chelating sites, as in other organisms. These characteristics suggest that OcMT1 and OcMT2 may be involved in heavy metal detoxification by capturing the metals. Two OcMT were expressed at all developmental stages, and the highest levels were found in the eggs. Both transcripts were expressed in all eleven tissues examined, with the highest levels observed in the brain and optic lobes, followed by the fat body. The expression of OcMT2 was also relatively high in the ovaries. The functions of OcMT1 and OcMT2 were explored using RNA interference (RNAi) and different concentrations and treatment times for the three heavy metals. Our results indicated that mortality increased significantly from 8.5% to 16.7%, and this increase was both time- and dose-dependent. To evaluate the abilities of these two MT proteins to confer heavy metal tolerance to E. coli, the bacterial cells were transformed with pET-28a plasmids containing the OcMT genes. The optical densities of both the MT-expressing and control cells decreased with increasing concentrations of CdCl2. Nevertheless, the survival rates of the MT-overexpressing cells were higher than those of the controls. Our results suggest that these two genes play important roles in heavy metal detoxification in O. chinensis.
Highlights
Metallothioneins (MTs) were first discovered as cadmiumbinding proteins and isolated from horse kidneys in 1957 [1]
Our results suggest that these two genes play important roles in heavy metal detoxification in O. chinensis
Two full-length cDNA sequences were obtained from the O. chinensis transcriptome database putatively encoding two different OcMTs, which were named OcMT1 and OcMT2 (GenBank accession numbers KJ153014 and KJ153015) (Fig. 1)
Summary
Metallothioneins (MTs) were first discovered as cadmiumbinding proteins and isolated from horse kidneys in 1957 [1]. MTs are low-molecular-mass (,10 kDa), cysteine-rich proteins (15– 30% of their amino acid contents) that lack aromatic residues, resulting in their optimal capacities for metal ion coordination. These high cysteine levels are necessary for the coordination of metal ions through the thiolate cluster as facilitated by the Cys-XCys and Cys-X-Y-Cys motifs, in which X can be any amino acid other than cysteine [2]. These types of metal-binding proteins have been widely found in all organisms, including bacteria, plants, invertebrates and vertebrates [3,4,5]. Studies involving MTs have been performed in various fields, including toxicology, physiology and molecular and developmental biology [10]
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