Cloning and functional characterization of a novel metallothionein gene in Antarctic sea-ice yeast (Rhodotorula mucilaginosa)

Abstract

Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. The RmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu²⁺ , Pb²⁺ , Zn²⁺ , Cd²⁺ , and Ag⁺ . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals.

Keywords: functional analysis; heavy metal; metallothionein; polar yeast.