【摘要】：鄰苯二甲酸單乙基己酯(MEHP)是公認(rèn)的動(dòng)物內(nèi)分泌干擾物,目前MEHP的研究主要集中于動(dòng)物及臨床相關(guān)的領(lǐng)域,越來(lái)越多的流行病學(xué)研究顯示MEHP和廣泛疾病的聯(lián)系,但是在MEHP脅迫下細(xì)胞生存的機(jī)制還不是很清楚。近年來(lái)釀酒酵母基因缺失株文庫(kù)被廣泛應(yīng)用于篩選和鑒定不同藥物在細(xì)胞的作用靶點(diǎn)并探究藥物發(fā)揮作用的通路。對(duì)釀酒酵母基因組水平上的MEHP適應(yīng)性研究將有助于哺乳動(dòng)物相關(guān)基因及信號(hào)途徑的研究。為了研究真核細(xì)胞適應(yīng)MEHP脅迫的分子機(jī)制,本課題利用真核模式生物釀酒酵母的二倍體非必需基因缺失株文庫(kù),篩選與MEHP脅迫相關(guān)的基因。本研究鑒定了96個(gè)對(duì)MEHP敏感的單基因缺失株,這些基因涉及麥角固醇生物合成途徑,液泡蛋白分選途徑以及液泡ATP酶相關(guān)的功能。用高效液相色譜測(cè)定了96個(gè)MEHP敏感缺失株在MEHP脅迫下胞內(nèi)的MEHP含量,發(fā)現(xiàn)49株的胞內(nèi)MEHP含量顯著高于野生型菌株,21株顯著低于野生型的菌株。在培養(yǎng)基中外源添加適量的麥角固醇或膽固醇可以部分抑制麥角固醇生物合成途徑中的erg2和erg4缺失株對(duì)MEHP的敏感表型,而且對(duì)96株MEHP敏感缺失株的細(xì)胞麥角固醇含量測(cè)定發(fā)現(xiàn),麥角固醇生物合成途徑的erg缺失株細(xì)胞沒(méi)有檢測(cè)到麥角固醇,這表明釀酒酵母中麥角固醇在MEHP耐受過(guò)程中發(fā)揮重要的作用。此外,通過(guò)對(duì)鈣離子/鈣調(diào)磷酸酯酶信號(hào)途徑有關(guān)基因的轉(zhuǎn)錄水平的測(cè)定,結(jié)果表明MEHP可以顯著降低PMC1的轉(zhuǎn)錄水平,這說(shuō)明釀酒酵母細(xì)胞中鈣離子/鈣調(diào)磷酸酯酶途徑可能與細(xì)胞對(duì)MEHP脅迫應(yīng)答相關(guān)。本論文的研究結(jié)果為進(jìn)一步了解MEHP在真核生物細(xì)胞內(nèi)的作用靶點(diǎn)和作用機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 姚廣印;張雙鳳;謝宗良;;酵母細(xì)胞麥角固醇的提取條件[J];河北大學(xué)學(xué)報(bào)(自然科學(xué)版);2007年02期

相關(guān)博士學(xué)位論文 前1條

1 張麗琳;釀酒酵母應(yīng)對(duì)鎘脅迫的分子機(jī)理研究[D];天津大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 趙剛;與鈣離子調(diào)控因子ScRch1定位相關(guān)的酵母基因篩選[D];江南大學(xué);2013年

，

本文編號(hào)：2298647