Attempt to express the Xerophyta viscose stress-responsive gene, Xvcor, in yeast with view to functional analysis
Master Thesis
2002
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University of Cape Town
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Abstract
Low temperature is one of the environmental factors that cause substantial crop losses in the world. Recent advances in the study of plants native from temperate regions, have established that cold acclimation is regulated at the gene expression leveL To study the function ofaXerophyta viscosa cold responsive protein that accumulates in plant cells under various environmental stresses, we have cloned the Xerophyta viscosa cold-responsive cDNA gene designed Xvcor, into a less complex host, Saccharomyces cerevisiae with the purpose of carrying out in vivo functional analysis. Sequence analysis showed that Xvcor encodes a 264 amino acid residue protein (Garwe et al 2002). The Hydropathy plot indicated that the XVCOR protein is highly hydrophobic and contains 6 transmembrane domains (Garwe et al 2002). In order to achieve high-level expression of Xvcor, the gene was placed under the control of a strong promoter (Phosphoglycerate kinase). Reverse-transcription PCR amplification revealed that the Xvcor transcript accumulated in yeast cells. However, SDS-PAGE analysis could not detect the predicted 29.6 kDa recombinant protein, suggesting that the translation might be hampered. Tests designed to measure the enhancement of stress tolerance between the recombinant and the control showed similar growth performance, confirming the absence of recombinant protein. A computer search for codon usage showed that the codon usage bias in Xvcor was low compared with that of highly expressed genes of S. cerevisiae. Together, these results suggest that the codon usage in Xvcor could influence its expression in yeast.
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Bibliography: leaves 24-29.
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Tawouokam, J. 2002. Attempt to express the Xerophyta viscose stress-responsive gene, Xvcor, in yeast with view to functional analysis. University of Cape Town.