MOLECULAR CLONING AND FUNCTIONAL ANALYSIS OF THE OSGLYOXALASE II GENE UNDER SALT STRESS TOLERANCE USING Saccharomyces cerevisiae AS A EUKARYOTIC MODEL SYSTEM
Abstract
Salt stress is known for its relation with increasing cellular methylglyoxal, which is detoxified through the glyoxalase pathway. The Osglyoxalase II gene enhances the GLYII step of this pathway, thereby improving MG clearance and contributing to greater stress tolerance. In this study, The Osglyoxalase II gene was isolated from salt-stressed Oryza sativa cv. Gz 178 seedlings by total RNA extraction, cDNA synthesis and RT-PCR amplification. The resulting 1,011 bp amplicon was cloned into pGEM-T, verified by restriction mapping and sequencing, then excised with SacII/NotI and directionally subcloned into the yeast expression vector pYES2. Furthermore, the directional subcloning was verified by restriction digestion showing the expected 5.9 kbp vector and 1011 bp insert, with sequencing confirming an intact structure. Recombinant plasmids were used to transform Saccharomyces cerevisiae via the Li-acetate method, transformants were selected on SC–Ura medium. Functional evaluation comprised: growth assays (OD₆₀₀) across a NaCl gradient, RT-PCR confirmation of transgene expression in yeast, spectrophotometric quantification of intracellular proline, and statistical analysis of replicate experiments. Transgenic yeast expressing Osgly II displayed consistently higher OD₆₀₀ values across all NaCl concentrations, maintaining substantial growth even at 2.3 M salt. RT-PCR detection of a 1,011 bp transcript exclusively in transformed yeast confirmed active gene expression. Intracellular proline levels increased progressively with salinity in the Osgly II strain, while both controls showed minimal or declining accumulation.
Data Availability Statement
The article's Material section highlights the study's original contributions; you may get in touch with the study's corresponding author for further details or clarifications.
Conflict of Interest Statement
During the study, the authors confirmed that there were no corporate or financial relations that may be believed to pose a potential conflict of interest.
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