Cloning and functional characterization of two abiotic stress tolerance genes, AtLTP1 and LeCBF1, in Saccharomyces cerevisiae

Background
Salinity is one of the most damaging abiotic stress factors in agriculture, it has a negative impact on crop growth, production, and development. It is predicted that salinity will become much more severe due to global climate change. Moreover, soil salinization affects three hectares of agricultural land every minute, increasing the salinity-affected area by 10% annually. The improvement of abiotic stress tolerance in plants was made possible by recent developments involving transgenes and the isolation of some abiotic stress tolerance genes.
Objective
The current study aimed to synthesize, clone and characterize two abiotic stress tolerance genes Lipid transfer protein (AtLTP1) of Arabidopsis thaliana and Stress-inducible transcription factor C-repeating binding factor (LeCBF1) of Solanum lycopersicum in Saccharomyces cerevisiae.
Materials and methods
The above-mentioned genes were synthesized, cloned into the pYES2 vector then transformed into Saccharomyces cerevisiae as a model eukaryotic system. The yeast growth was measured at (ODnm) in a spectrophotometer, RT-PCR expression analysis and estimation of intracellular proline content after exposure to different salt concentration were performed to characterize and evaluate the physiological roles of the selected genes in the yeast.
Results and conclusion
The AtLTP1 and LeCBF1 genes were cloned into the pYES2 vector for Saccharomyces cerevisiae expression. After being exposed to increasing concentrations of sodium chloride (0, 1.7, 1.8, 1.9, 2.0, 2., 2.2, and 2.3 M) for 7 days, transgenic yeast cells were tested for their ability to survive under increasing salt-stress conditions and their growth response. A spectrophotometer was used to measure yeast growth at ODnm. The growth of the control cells was dramatically hindered when the salt content was increased to 1.9 M NaCl. However, two transgenic yeast lines continued to grow well, at a slower rate, up to 2.3 M NaCl. The two genes’ expression in transgenic yeast in response to salt stress was verified by RT-PCR. In this transgenic yeast, the precise primers of LeCBF1 and AtLTP1 amplified the genes successfully at 633 base pairs and 368 base pairs, respectively. The findings showed that increasing salinization level considerably boosted the transgenic yeast’s intracellular proline accumulation. It was suggested that the possibility of utilizing these genes to produce salt tolerant transgenic plants, consequently, increase the amount of land that can be exploited for agriculture.