酿酒酵母基因组中与邻苯二甲酸单乙基己基酯敏感性相关的基因筛选
发布时间：2018-10-29 19:52
【摘要】：邻苯二甲酸单乙基己酯(MEHP)是公认的动物内分泌干扰物,目前MEHP的研究主要集中于动物及临床相关的领域,越来越多的流行病学研究显示MEHP和广泛疾病的联系,但是在MEHP胁迫下细胞生存的机制还不是很清楚。近年来酿酒酵母基因缺失株文库被广泛应用于筛选和鉴定不同药物在细胞的作用靶点并探究药物发挥作用的通路。对酿酒酵母基因组水平上的MEHP适应性研究将有助于哺乳动物相关基因及信号途径的研究。为了研究真核细胞适应MEHP胁迫的分子机制,本课题利用真核模式生物酿酒酵母的二倍体非必需基因缺失株文库,筛选与MEHP胁迫相关的基因。本研究鉴定了96个对MEHP敏感的单基因缺失株,这些基因涉及麦角固醇生物合成途径,液泡蛋白分选途径以及液泡ATP酶相关的功能。用高效液相色谱测定了96个MEHP敏感缺失株在MEHP胁迫下胞内的MEHP含量,发现49株的胞内MEHP含量显著高于野生型菌株,21株显著低于野生型的菌株。在培养基中外源添加适量的麦角固醇或胆固醇可以部分抑制麦角固醇生物合成途径中的erg2和erg4缺失株对MEHP的敏感表型,而且对96株MEHP敏感缺失株的细胞麦角固醇含量测定发现,麦角固醇生物合成途径的erg缺失株细胞没有检测到麦角固醇,这表明酿酒酵母中麦角固醇在MEHP耐受过程中发挥重要的作用。此外,通过对钙离子/钙调磷酸酯酶信号途径有关基因的转录水平的测定,结果表明MEHP可以显著降低PMC1的转录水平,这说明酿酒酵母细胞中钙离子/钙调磷酸酯酶途径可能与细胞对MEHP胁迫应答相关。本论文的研究结果为进一步了解MEHP在真核生物细胞内的作用靶点和作用机制奠定了基础。
[Abstract]:Monoethyl hexyl phthalate (MEHP) is recognized as an endocrine disruptor in animals. At present, the study of MEHP is mainly focused on animal and clinical related fields. More and more epidemiological studies show that MEHP is associated with a wide range of diseases. However, the mechanism of cell survival under MEHP stress is not well understood. In recent years, the gene deletion library of Saccharomyces cerevisiae has been widely used to screen and identify the action targets of different drugs in cells and to explore the pathways through which drugs play a role. The study of MEHP adaptability on the genomic level of Saccharomyces cerevisiae will be helpful to the study of mammalian related genes and signaling pathways. In order to study the molecular mechanism of eukaryotic cells adapting to MEHP stress, the diploid non-essential gene deletion library of eukaryotic model Saccharomyces cerevisiae was used to screen the genes related to MEHP stress. In this study, 96 single gene deletion strains sensitive to MEHP were identified. These genes were involved in ergosterol biosynthesis pathway, vacuolar protein sorting pathway and vacuolar ATP enzyme related functions. The intracellular MEHP content of 96 MEHP sensitive deletion strains under MEHP stress was determined by high performance liquid chromatography. The results showed that the intracellular MEHP content of 49 strains was significantly higher than that of wild type strains, and 21 strains were significantly lower than wild type strains. Addition of appropriate amount of ergosterol or cholesterol to the culture medium partially inhibited the sensitive phenotypes of erg2 and erg4 deletions to MEHP in the ergosterol biosynthesis pathway, and determined the cell ergosterol content of 96 MEHP sensitive deletion strains. Ergosterol was not detected in erg deletion cells of ergosterol biosynthesis pathway, suggesting that ergosterol plays an important role in MEHP tolerance in Saccharomyces cerevisiae. In addition, the transcriptional level of genes related to calcium ion / calmodulin phosphatase signaling pathway was determined. The results showed that MEHP could significantly reduce the transcription level of PMC1. These results suggest that the Ca ~ (2 +) / Ca ~ (2 +) phosphatase pathway in Saccharomyces cerevisiae cells may be related to the response of cells to MEHP stress. The results of this paper lay a foundation for further understanding the target and mechanism of MEHP in eukaryotic cells.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
本文编号：2298647
[Abstract]:Monoethyl hexyl phthalate (MEHP) is recognized as an endocrine disruptor in animals. At present, the study of MEHP is mainly focused on animal and clinical related fields. More and more epidemiological studies show that MEHP is associated with a wide range of diseases. However, the mechanism of cell survival under MEHP stress is not well understood. In recent years, the gene deletion library of Saccharomyces cerevisiae has been widely used to screen and identify the action targets of different drugs in cells and to explore the pathways through which drugs play a role. The study of MEHP adaptability on the genomic level of Saccharomyces cerevisiae will be helpful to the study of mammalian related genes and signaling pathways. In order to study the molecular mechanism of eukaryotic cells adapting to MEHP stress, the diploid non-essential gene deletion library of eukaryotic model Saccharomyces cerevisiae was used to screen the genes related to MEHP stress. In this study, 96 single gene deletion strains sensitive to MEHP were identified. These genes were involved in ergosterol biosynthesis pathway, vacuolar protein sorting pathway and vacuolar ATP enzyme related functions. The intracellular MEHP content of 96 MEHP sensitive deletion strains under MEHP stress was determined by high performance liquid chromatography. The results showed that the intracellular MEHP content of 49 strains was significantly higher than that of wild type strains, and 21 strains were significantly lower than wild type strains. Addition of appropriate amount of ergosterol or cholesterol to the culture medium partially inhibited the sensitive phenotypes of erg2 and erg4 deletions to MEHP in the ergosterol biosynthesis pathway, and determined the cell ergosterol content of 96 MEHP sensitive deletion strains. Ergosterol was not detected in erg deletion cells of ergosterol biosynthesis pathway, suggesting that ergosterol plays an important role in MEHP tolerance in Saccharomyces cerevisiae. In addition, the transcriptional level of genes related to calcium ion / calmodulin phosphatase signaling pathway was determined. The results showed that MEHP could significantly reduce the transcription level of PMC1. These results suggest that the Ca ~ (2 +) / Ca ~ (2 +) phosphatase pathway in Saccharomyces cerevisiae cells may be related to the response of cells to MEHP stress. The results of this paper lay a foundation for further understanding the target and mechanism of MEHP in eukaryotic cells.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
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