Physiological Mechanism Of Transcription Factor Crz1p In Acid Stress Response Of Candida Glabrata

This dissertation chose the mutant strain C. glabrata ATCC 55 as a model system to identify the function of transcription factor Cg Crz1 p under acidic conditions. The regulation mechanism of Cg Crz1 p and the effect of gene Cg CRZ1 deletion on cell growth, transcription level, the plasma membrane composition and performance in response to acid stress were studied in detail with the theory and method of molecular biotechnology, transcriptomics and GC-MS. The main results were described as follows.1. The mutant strains Cgcrz1Δ, Cgrlm1Δ, Cghal6Δ, Cgusv1Δ, Cgrds2Δ, Cgcst6Δ, Cgrtg1Δ and the revertant strain Cgcrz1Δ/Cg CRZ1 were constructed. Compared with the parent strain, it was found that changes were showed as follows: the final biomass of the mutant strain Cgcrz1Δ at p H2.0 decreased by 60%; the mutant strain Cgcrz1Δ exhibited a 50% reduction of growth viability after incubation in the medium at p H2.0 for 12 h; however, the final biomass of the revertant strain Cgcrz1Δ/Cg CRZ1 at p H6.0 and at p H2.0 increased by 1 and 1.25-fold, respectively; the revertant strain Cgcrz1Δ/Cg CRZ1 exhibited an 7-fold increase in growth viability after incubation in the medium at p H2.0 for 12 h.2. RNAseq was used to reveal why the mutant strain Cgcrz1Δ could not grow normally at p H 2.0. Compared with the parent strain, it was found that changes were showed as follows: the transcription level of many genes involved in membrane lipid biosynthesis and metabolism pathway was differentially regulated; the expression of genes implicated in the fatty acid synthesis and elongation pathway were downregulated while the expression of the sterol-specific enzyme coding genes in the ergosterol synthesis pathway was differentially upregulated. Genes coding for key enzymes in phospholipid biosynthesis and metabolism pathway had a downregulated expression. In addition, there was good agreement between the RNAseq profiles and the q RT-PCR data.3. Compred to the parent strain at p H2.0, it was found that changes were showed as follows: a 23% drop of β-glucan content in the cell wall was obtained in the mutant strain Cgcrz1Δ; an 10% and 30% reduction in the percentage of C16:1 and C18:1 mono-unsaturated fatty acid were observed in the plasma membrane in the mutant strain Cgcrz1Δ, and the UFA/SFA ratio lowered by 4.47, while the proportion of C16:1 and C18:1 increased by-15% and 16%, respectively, this resulted in a 2.07 higher UFA/SFA ratio in the revertant strain Cgcrz1Δ/Cg CRZ1; there were reductions of 70%, 84% and 30% in squalene, fecosterol and ergosterol content, respectively, in the mutant strain Cgcrz1Δ, while the squalene, fecosterol and ergosterol content increased by-29.7%, 50% and 40% in the revertant strain Cgcrz1Δ/Cg CRZ1; in addition, in the mutant strain Cgcrz1Δ, the content of total phospholipid content decreased, the content of phosphatidyl acid(Ptd OH) and phosphatidylinositol(Ptd Ins) reduced by 25% and 23%, respectively, and less phospholipids with unsaturated and longer fatty acid chains were observed, while the content of total phospholipid content increased, the content of phosphatidyl acid(Ptd OH) and phosphatidylinositol(Ptd Ins) increased by 48-folds and 1.8-folds, respectively, in the revertant strain Cgcrz1Δ/Cg CRZ1, and more phospholipids with unsaturated and longer fatty acid chains were noticed.4. The change of membrane lipid composition affected the membrane performance. Compared to the parent strain at p H2.0, it was found that changes were showed as follows: the plasma membrane integrity, fluidity and proton pump: H+-ATPase activity in the mutant strain Cgcrz1Δ was diminished by 45%, 9% and 52%, respectively; the plasma membrane integrity, fluidity and proton pump: H+-ATPase activity in the revertant strain Cgcrz1Δ/Cg CRZ1 increased by 30%, 6% and 17%.