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Cloning of MT2 from Tartary Buckwheat (Fagopyrum tataricum) and Its Response to Cu2+ Stress |
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Abstract Abstract Metallothioneins, which are a kind of widely distributed and low molecular weight cysteine-rich proteins, were found to play an important role in heavy metal tolerance. In this study, a cDNA fragment corresponding to the type Ⅱ metallothionein (MT2) coding region (GenBank No. KY643823) was isolated from tartary buckwheat (Fagopyrum tataricum) leaf. FtMT2 encoded a deduced peptide of 78 aa residues with a calculated molecular mass of 7.87 kD and contained the cysteine-rich domains with the presence of C-C, C-X-C, and C-X-X-C (X was other amino acids other than cysteine) motifs at amino-terminus and C-X-C motifs at carboxy-terminus. Sequence and homology analysis showed that the FtMT2 protein sequence shared high homology with other plant type 2 MT-like proteins. In order to clarify the regulatory mechanism of FtMT2 in response to Cu2+ stress, 2.1 kb upstream sequence of FtMT2 was isolated by genome walking. The 2.1 kb fragment and its 5′ flanking deletions were tested for promoter activity using the dual luciferase reporter system and transient transformation in mesophyll protoplasts of F. tararicum. The promoter activities of all the fragments were up-regulated, and the full length promoter showed strongest inducibility in response to Cu2+ treatment. The role of FtMT2 protein in protecting against Cu2+ toxicity was demonstrated in pGEX4T-1-FtMT2-beared Escherichia coli, which could obviously increase the host E. coli cell's tolerance. Functional characterization of upstream region of FtMT2 provides the regulatory mechanism for establishing the method for copper contamination monitoring and offers an underlying basis for further revealing the molecular mechanisms of heavy metal resistance of Tartary buckwheat.
Keywords Tartary buckwheat, Metallothioneins (MTs), Promoter, Heavy metal tolerance
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Received: 17 November 2016
Published: 01 June 2017
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