Cloning characterization and expression of a novel metallothionein gene (mt-d) from triticum durum
Öztürk, Hasan Ümit (2003) Cloning characterization and expression of a novel metallothionein gene (mt-d) from triticum durum. [Thesis]
Two different metallothionein genes, labelled as mt-d and mt-a were identified in wheat (Triticum durum and Triticum aestivum) genomic DNA sequences were characterized. mt-d and mta, were found to contain 416 and 399 nucleotides, respectively. Nucleic acid sequence alignment showed 95 % similarity between the two. Sequencing results showed that the difference resulted from two extra TTTTTA repeats in the intron regions. cDNAs encoding mt-genes were identifed by RT-PCR. Gene alignment algorithms strongly suggested that both of these cDNAs (mt-a and mt-d) encoded an open reading frame of 75 amino acids with two cysteine-rich domains featuring Cys-XCys motifs at the amino- and carboxy termininus. The deduced amino acid sequences of mt-a and mt-d genes show striking similarity to the MT-like proteins described within the Class II as Type 1 MTs and showed 100 % similarity with each other as deduced from cDNA sequencing results. These results indicate that mt-d from T.durum forms a “novel type 1” MT. For further studies of mt-d expression, localization of the durum metallothionein protein (dMT) and its interactions with other proteins mt-d gene was inserted into the 5’ MCS of pGFPuv vector. Verification was based on sequence data and restriction enzyme analysis. However, expression could not be validated by neither by visual detection of GFP expression nor by SDS-PAGE analysis. A more detailed sequence analysis indicated that the problem was due to a point mutation within the coding sequence of the GFPuv, resulting in a stop codon and premature termination of the fusion protein. Results presented here show the presence of metallothionein gene in the wheat Triticum durum. Although our attempts to express the gene as a fusion protein together with GFP to facilitate its localization in different systems was not successful it will be important in future studies to pursue this goal and achieve expression of labelled protein in plant systems to gain insights into its exact function in plants.
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