Physiological Mechanism Of Transcription Factor CgRds2 In Response To Environmental Stress In Candida Glabrata

Microorganisms are often subjected to stress conditions in the process of natural growth and fermentation,which inhibit the cell growth and reduce the yield of fermentation products.It has been an effective strategy to resist the stress conditions through regulating the expression level of transcription factors.This dissertation chose Candida glabrata ATCC 55as model system to analyze the physiological mechanism of Candida glabrata in response to salt stress and acid stress.To this end,the regulation mechanisms of transcription factor CgRds2 in response to salt stress and acid stress were studied in detail with the method of molecular biology and biochemistry.The main results were described as follows:1.The knockout strain Cgrds2?and revertant strain Cgrds2?/CgRDS2 were constructed.At 1.5 M NaCl,the final biomass and cell viability of Cgrds2?strain decreased by 23.4%and39.6%,respectively,compared to that of the wild-type strain,wherease in the Cgrds2?/CgRDS2 strain,the final biomass and cell viability were 10.2%and 6.3%higher than that of the wild-type strain.At pH 2.0,the final biomass and cell viability of the Cgrds2?strain were 32.6%and 56.9%lower than that of the wild-type strain,respectively,but in the Cgrds2?/CgRDS2 strain,the final biomass was similar to that of the wild-type strain,while the cell viability incerased by 17.6%.2.Transcriptome sequencing analysis revealed that under salt stress,in the Cgrds2?strain,the expression levels of genes involved in glycolysis,ribosome biogenesis and rRNA transcription process were up-regulated;the expression levels of genes involved in lipid metabolism,MAPK signaling pathway and cell cycle were down-regulated.Among them,the expression levels of glycerophospholipid genes were the most significant differently.Futher study on the regulation mechanism of CgRds2 under salt stress found that the key kinase CgHog1 in the MAPK signaling pathway directly interacted with CgRds2 and then activated CgRds2 through phosphorylating CgRds2 at Ser64 and Thr97.Finally,CgHog1 mediated CgRds2 to regulate the expression levels of glycerophospholipid genes,glycerophospholipid composition and membrane integrity.At 1.5 M NaCl,the transcription levels of glycerophospholipid genes in the Cgrds2?strain were decreased significantly compared to that of the wild-type strain,meanwhile the total glycerophospholipid content and membrane integrity were decreased by 30.3%and 47.6%,respectively,whereas in the Cgrds2?/CgRDS2strain,the total glycerophospholipid content and membrane integrity were increased by 27.2%and 12.1%.In the Cgrds2^2A strain,the binding levels of CgRds2 to the glycerophospholipid genes were significantly decreased,and the total glycerophospholipid content and membrane integrity were decreased by 29.4%and 21.8%.3.Transcriptome sequencing analysis revealed that under acid stress,in the Cgrds2?strain,the expression levels of genes involved in meiosis,protein transport and transcription process were up-regulated;the expression levels of genes involved in translation process,membrane biosynthesis,amino acid metabolism and energy metabolism were down-regulated.Futher study on the regulation mechanism of CgRds2 under acid stress found that the calmodulin-dependent protein kinase CgCmk1 in the Ca²⁺response pathway directly interacted with the PAS domain of CgRds2,which activating CgRds2 and enabling it translocate to cell nucleus from cytoplasm.Finally,CgCmk1 mediated CgRds2 to regulate the expression levels of genes involved in energy metabolism,ATP content and membrane permeability.At pH 2.0,the transcription levels of genes involved in energy metabolism in the Cgrds2?strain were decreased significantly compared to that of the wild-type strain,meanwhile the ATP content and membrane permeability were decreased by 33.5%and 23.6%,respectively,whereas in the Cgrds2?/CgRDS2 strain,the ATP content and membrane permeability were increased by 41.5%and 18.8%.In the Cgrds2-pas strain,the binding levels of CgRds2 to the genes involved in energy metabolism were significantly decreased,and the ATP content and membrane permeability were decreased by 30.7%and 19.1%.