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Analysis Of The Physiological Mechanisms And Endogenous And Exogenous Strategies Of High Salinity Stress-induced Apoptosis In Torulopsis Glabrata

Posted on:2016-12-01Degree:MasterType:Thesis
Country:ChinaCandidate:X X YuFull Text:PDF
GTID:2180330464463614Subject:Microbiology
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In industrial microbial-manufacturing engineering, several recent reports described an apoptosis-like programmed cell death(PCD) process in yeast in response to different environmental challenges.This thesis chose the pyruvate production Torulopsis glabrata CCTCC M202019 as a model to study the effects of hyperosmotic stress caused by high Na Cl concentration in culture medium on the apoptosis. The main results were described as follows:(1) The effect of Na Cl concentration on cell growth showed that high salinitystress could significantly inhibit cell growth, and Propidium iodide(PI) and Rhodamine-123/PI(Rh123/PI) dual staining with flow cytometry showed that high salinity decreased intact cells by 16.5%, increased necrotic cells by nearly 2-fold, and the DCW(3.1 g·L-1), pyruvate production(2.5 g·L-1) and glucose consumption(17.9 g·L-1) were decreased by 79.7%, 93.1% and 77.6%, respectively, compared with unstressed control cells.(2) The physiologicalcharacteristics of T. glabrata were investigated to resist high salinity stress. These results suggested that high Na Clstress could significantly decrease or enhance mitochondrial membrane potential(?Ψm); increase the translocation of phosphatidylserine(PS) from the inner to the outer side of the plasma membrane;improvecellular reactive oxygen species(ROS) level, it was the highest when at 48 h, which was 6.45-fold as the control; improve caspase-3 activity, it was 529.8 μM·μg-1, increased by 3.2-fold, compared with the control; improve the carbony level, 6 h、12 h、24 h、36 h、48 h increased by 148%、71%、99%、70%、58%,respectively; decrease the generation of cellular ATP, it was decreased ATP/ADP by 60.5%. Furthermore, the RT-PCR was applied to detect the expression changes of some apoptosis-related genes, which showed that when under high salinity stress, FIS1, SOD1 and SOD2 genes expression were decreased by 65.7%, 70.6% and 66.7%, compared with control cells. These results showed that the decreased expression of FIS1, SOD1 and SOD2 genes may be related to the decreased of viability of the high-salinity-stressed cells, and provided an alternative strategy for rationally improving the growth performance of eukaryotes under high salinity stress.(3) Exogenous and endogenous strategies could be used to regulate the apoptosis of yeast. Addition of proline or glutathione alone(GSH-/Pro+ and GSH+/Pro-, respectively), and both additives(GSH+/Pro+) increased the percentage of intact cells by 21.1%, 22.2%,23.9%. Additionally, the strategy could enhance the capability of fermentation. Furthermore, exogenous strategy could significantly enhance the capability of?Ψm, decrease the translocation of PS from the inner to the outer side of the plasma membrane, cellular ROS level and caspase-3. Endogenous strategy was FIS1, HOG1, GPD2 and YCA1 were overexpressed. And the FIS1, HOG1 and GPD2 increased the intact cells. Additionally, the strategy could enhance the capability of fermentation. However, the YCA1 decreased the intact cells, DCW, and glucose consumption by 15%, 57% and 71.7%. Additionally, the FIS1, HOG1 and GPD2 could also enhance the capability of ?Ψm, decrease the translocation of PS from the inner to the outer side of the plasma membrane,improve cellular ROS and caspase-3 level, however, the YCA1 played an opposite role.
Keywords/Search Tags:Torulopsis glabrata, hyperosmotic stress, apoptosis, exogenous/endogenous strategy
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