廖秀娟臺灣大學：生物環境系統工程學研究所游展維Yu, Chan-WeiChan-WeiYu2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/56140Gamma-glutamylcysteine synthetase (γ-GCS) 是glutathione (GSH) 合成的速率決定步驟酵素，證明GSH對於C. elegans抵抗砷所造成的氧化壓力扮演重要的角色，另外亦探討在C. elegans中所預測出GCS (h)的同源基因, gcs-1,對於暴露砷時所造成的反應。當C. elegans暴露於三價砷以及五價砷時，gcs-1突變種對於抵抗砷所造成的毒性比野生種線蟲更為敏感。另外，gcs-1突變種C. elegans亦比野生種C. elegans對於砷所造成的毒性反應較快。將gcs-1突變種C. elegans預先暴露於GSH，可明顯提高gcs-1突變種暴露於三價砷與五價砷時的存活率。我們亦探討C. elegans體內的GSH含量與不同砷劑量間的關係。並且利用RNA干擾技術，mRNA表達和基因轉殖C. elegans探討GCS-1的功能。我們亦探討砷對於C. elegans中其他可能和氧化壓力有關基因的表達的關係。研究的結果顯示在C. elegans中GCS-1是合成GSH所必須的，並且GSH的含量對於抵抗砷所造成的氧化壓力扮演著重要的角色，以及在C. elegans中砷會誘導化壓力相關基因mRNA的表達。Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. The intracellular GSH level increases in C. elegans exposed to As(III) and gcs-1 expression level is induced by As(III). The functional importantance of GCS-1 in C. elegans exposed to As(III) is investigated by analysis of gcs-1 transcription in transgenic C. elegans and RNAi mediated GCS-1 knock-down worms. The level of mRNA expression of several potential oxidative response gene is response to As(III) exposure was also investigated. Our results show that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress. Furthermore, As(III) is capable of inducing mRNA expression of oxidative stress-related genes.致謝 I 中文摘要 II ABSTRACT III TABLE OF CONTENTS IV LIST OF FIGURES VI LIST OF TABLE VII ABBREVIATION VIII CHAPTER 1 INTRODUCTION 1 1.1 Arsenic 1 1.2 Oxidative stress 3 1.3 Glutathione and Glutamate Cysteine Synthetase 5 1.4 Caenorhabditis. elegans 8 1.5 GCS homolog in C. elegans 9 1.6. Purpose of study 10 CHAPTER 2 MATERIALS AND METHODS 12 2.1 Chemicals 12 2.2 Strains, clones, and culture condition 12 2.3 Arsenic toxicity analyses 12 2.4 Intracellular GSH measurement 13 2.5 GSH rescue assay 14 2.6 Culture and isolation of C. elegans exposed to various stressors 14 2.7 Real-time RT-PCR 15 2.8 RNA Interference (RNAi) 16 2.9 Effect of transgenic C. elegans 19 2.10 Data analysis 20 CHAPTER 3 RESULT 21 3.1 Structure and organization of gcs-1 gene structure 21 3.2 Intracellular GSH measurement 21 3.3 Lethality tests of As(III) and As(V) and establishment of LC50 value 25 3.4 Time-dependence of As(III) and As(V) toxicity in C. elegans 28 3.5 GSH rescues gcs-1 mutant 30 3.7 Analysis of GCS-1 mRNA level affected by As(III) 32 3.8 Effects of As(III) on oxidative stress responsive genes 35 3.10 Effect of various stressors on gcs-1 transcription in transgenic C. elegans 41 CHAPTER 4 DISCUSSION 46 CHAPTER 5 CONCLUSION 53 REFERENCE 54 APPENDIX 592245988 bytesapplication/pdfen-USGamma-glutamylcysteine synthetaseglutathioneCaenorhabditis elegans砷氧化壓力arsenicthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56140/1/ntu-96-R93622018-1.pdf