Cloning and expression of two genes (NHAS2 AND NHAS4) isolated from SYNECHOCYSTIS SP. PCC 6803 in yeast

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International Journal of Development Research

Cloning and expression of two genes (NHAS2 AND NHAS4) isolated from SYNECHOCYSTIS SP. PCC 6803 in yeast

Abstract: 

Two genes encoding Na+/H+ antiporter isolated from Cyanobacterial strain Synechocystis sp. PCC 6803 were cloned and sequenced. These genes, namely, nhaS2 and nhaS4 were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. Transgenic yeast was examined for their ability to survive under salt- stress conditions and their growth response to increasing levels of NaCl (0, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2 and 2.3M) for 7days, then the yeast growth was measured at OD600nm in spectrophotometer. The results revealed significant differences in growth between the transgenic yeast and control. The salt concentration of 1.8M NaCl was critical in determining the ability of yeast to tolerate salt stress. At this concentration, the mean growth values for the transgenic yeast harboring the genes nhaS2 or nhaS4 were 1.2250cA and 1.2180cA, respectively, compared to 0.2620cB for the control. Moreover, Increasing the salt concentration to 1.9 M NaCl, inhibited the growth of the control cells significantly. While, the transgenic yeast cells for the 2 genes maintained good growth and the growth continued until the 2.3 M NaCl, but with a lower level. RT–PCR confirmed the expression of the two genes in response to salt stress in transgenic yeast. The specific nhaS2 and nhaS4 primers successfully amplified the genes at 1623 bp and 1233bp, respectively in this transgenic yeast. In addition to the growth response of the transgenic yeast to salt stress, the results revealed that the intracellular proline accumulation in the transgenic yeast was significantly increased with raising salinization level.

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