Deletion And Analysis Several Important Genes In Saccharomyces Cerevisiae Mating Pheromone Transduction Pathway

G-protein signal transduction pathways (GPSTPs) widely exist in eukaryotic organisms. GPSTPs transduce the stimulation provided by extracellular environment into the cell, resulting in the change of the cell physiological state. Yeast pheromone GPSTP mediates the mating between the different mating type haploid yeast cells. There are two kinds of mating types:mating a and mating α. Mating a haploid yeast cells secrete a pheromone (a factor) and express α pheromone receptor, while mating a haploid yeast cells secrete a pheromone (a factor) and express a pheromone receptor.Because the yeast pheromone GPSTP has been clearly studied, and in which the component and signal transduction mechanisms are similar to that in human GPSTPs, the pheromone GPCR has been replaced by human GPCR successfully. Yeast strains which expressed human GPCRs can be used for drug screening. The drugs are for the diseases caused by abnormal GPCRs. Our research is based on this research idea. We want to build a yeast pheromone GPCR mutant library, which can be used to analyse and detect target substances in food safety, medicine and environmental fields.To construct the yeast pheromone GPCR mutant library, the yeast pheromone GPSTP must be optimized firstly in order to short the detection time, improve the detection sensitivity. In this research, the starting strain was Saccharomyces cerevisiae BY4741 in which the green fluorescent protein (GFP) reporter gene had been fused to the downstream of Fusl gene (The BY4741 strain is mating a type haploid yeast which can secrete a pheromone and respond to a pheromone). The modification of the yeast pheromone GPSTP included three aspects, as following:(1) Deletion Far1 gene in order to short the detection time, a pheromone is an active factor of the yeast pheromone GPSTP, which can cause the yeast cell cycle arrest in the G1 phase (the first interval of the cell cycle, the main chromosomal proteins and DNA helicase are synthetized) through Far1 protein (a cyclin-dependent kinase inhibitor), resulting in that cell growth is inhibited, which inhibits the active signal fast amplification. The Far1 gene was deleted in order to accelerate the active signal amplification and short the detection time. (2) Deletion Sst2 gene in order to improve the detection sensitivity. Sst2 protein is an important negative regulatory factor of the pheromone GPSTP. The sensitivity of the yeast pheromone GPSTP could be improved greatly, if the Sst2 gene was deleted. In order to improve the detection sensitivity, the Sst2 gene was knocked out. (3) Deletion Ste2 gene in order to avoid the signal interference. The GPCR of yeast pheromone GPSTP is encoded by Ste2 gene, the presence of endogenous Ste2 protein can cause signal interference when we select the yeast strain in which the pheromone GPCR was mutated. The Ste2 gene was knocked out in order to remove Ste2 protein, releasing endogenous receptor interference, which provided a blank background which can be used to screen yeast pheromone GPCR mutant strains. These modified strains can be used to construct the yeast GPCR mutant library. The main results of our research were as following:1. Checking of the GFP reporter gene in BY4741 strain and analysis of the smallest response dose of BY4741 cells to a factorGFP reporter gene and mating type of the BY4741 strain were verified firstly to ensure that it could be used for detection analysis later. The GFP gene had been proved to exist in BY4741 strain completely using PCR, restriction enzyme digestion and fluorescence microscopy techniques and methods. The GFP fluorescence intensity of yeast cells was related to a factor concentration observed by fluorescence microscopy, which indicated that GFP gene had been coupled to a factor GPSTP successfully. Further analysis showed that in YPD (Yeast extract/peptone/dextrose) liquid medium, the minimum response concentration of BY4741 strain to a factor was 1μmol/L; the minimum response dose of BY4741 strain to a factor was 1μg on YPD plates.2. Deletion Far1 gene in BY4741 strainAccording to the principle of homologous recombination, geneticin (G418) as selection marker, the Far1 gene knocked-out DNA box was constructed by fusion PCR technology. The Far1 gene knocked-out DNA box was transformed into BY4741 competent cells by the LiAc/ssDNA/PEG (Lithium acetate/single-stranded DNA/polyethylene glycol) method. The results of PCR and a factor inhibition halo experiment showed that Far1 gene had been knocked-out successfully, △Far1 strains had been obtained. The results of a factor inhibition halo experiment showed that when the dose of a factor was 100μg, there was no inhibition halo formed by △Far1 strain on YPD plates, while the diameter of the inhibition halo formed by BY4741 strain on YPD plates was 24mm. It suggested that the inhibition of a factor to yeast cells growth was released, because Farl gene had been knocked-out successfully.3. Deletion Sst2 gene in BY4741 strainSst2 gene was knocked-out by a method similar to which had been used to knock-out Farl gene. Hygromycin (Hyg) was the selection marker. △Sst2 and △Sst2△Farl strains were obtained successfully. Compared with BY4741 strain, the sensitivity (100pg) of △ Sst2 strain to a factor increased 104 times, But when the dose of a factor was 10μg, there was no inhibition halo formed by △Sst2△Farl strain on YPD plates, while the diameter of the inhibition halo formed by BY4741 strain on YPD plates was 18mm, because Farl gene had been deleted in △Sst2△Farl strains. It indicated that the sensitivity of yeast strains to a factor was greatly improved because of the Sst2 gene deletion.4. Deletion Ste2 gene in BY4741 strainSte2 gene was knocked-out by a method similar to which had been used to knock-out Farl gene. Leucine (Leu) was the selection marker. △Ste2 and △Ste2△Sst2 strains were obtained successfully. When the dose of a factor was 10μg, there was no inhibition halo formed by △Ste2 and △Ste2△Sst2 strains on YPD plates, while the diameter of the inhibition halo formed by BY4741 strain on YPD plates was 19mm. It showed that the yeast pheromone GPSTP could not be activated by a factor, because the yeast endogenous a factor’ receptor had been removed successfully.