廖秀娟臺灣大學：生物環境系統工程學研究所鄭權Cheng, ChuanChuanCheng2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/56000Glutathione synthetase (GS)為催化γ-glutamylcysteine以及glycine兩者生合成為glutathione (GSH)的重要酵素。GS-1是在C. elegans 中被預測出來與GS同源的基因。本篇研究目的旨在探討GS-1於抵抗砷及鎘的暴露所誘發之氧化壓力中所扮演的角色。本研究利用RNA干擾技術、基因轉殖的C. elegans 、以及mRNA表達之分析方法探討GS-1在C. elegans 體內的功能及表現。研究結果顯示，經由GS-1 RNA干擾的C. elegans，即使在正常的生長環境下也會產生許多不同的效應，其中包括其生命週期被大幅的縮短、存活率的降低、成蟲率的降低、以及形體外型的改變，而當此C. elegans生長於含有砷及鎘的環境中時，上述的效應則更加的顯著。這些結果證明了GS-1為C. elegans之生長、存活、及生命週期的必要基因，而且對於抵抗砷及鎘所誘導的氧化壓力為不可或缺的重要角色。本研究結果進一步發現C. elegans於不同的生長階段之GS-1 mRNA會有不同程度的表現，而且在L4- stage具有最高的表現量。另外，於基因轉殖的C. elegans的觀察之中也發現當C. elegans受到熱擊 (heat shock)、 巴拉刈 (paraquat)以及鎘暴露等不同來源的壓力之下，基因轉殖的C. elegans會被激發出明顯的螢光訊號，而對於其他的金屬則闕如。本論文的研究結果證明了GS-1為C. elegans生長發育和生命週期的必須基因，更顯示GS-1於抵抗砷及鎘所誘導的氧化壓力中扮演了十分重要的角色。Glutathione synthetase (GS) catalyzes the ATP-dependent synthesis of GSH from γ-glutamylcysteine (γ-Glu-Cys) and glycine. It is predicted that GS-1 is the C. elegans ortholog of GS. To investigate the protective role of cellular GSH against arsenic- and cadmium-induced oxidative stress in C. elegans, we examined the effect of GS-1 in response to arsenic and cadmium exposure. The functional importance of GS-1 in C. elegans is investigated by RNA interference (RNAi) mediated (GS-1-dificent) worms. Under normal growth conditions, pleiotropic phenotypes of GS-1 (RNAi) worms were observed, including shorten life span, decreased survival rate and adult percentage, and morphological changes, suggesting that GS-1 is essential for development and viability. These observed effects were more profound while GS-1 (RNAi) worms were grown in the presence of As (III) and Cd (II), indicating that GS-1 is required C. elegans' defense against As (III) and Cd (II) toxicity. GS-1 mRNA expression regulated by different metals in vivo and in vitro were examined by transgenic C. elegans and real-time RT-PCR analysis. GS-1 mRNA varied in different developmental stages of worms and the highest level of GS-1 mRNA was observed at L4-stage. Heat, paraquat, and Cd (II) significantly induced GS-1 expression with the highest level GFP signal, whereas other metals induced less extent. In this study, our results indicated that GS-1 plays an important role in C. elegans’ development and life span, and it is required for the protection of C. elegans against arsenic- and cadmium-induced oxidative stress.TABLE OF CONTENTS 口試委員會審定書 I The approval Sheet II 致謝 III 中文摘要 V ABSTRACT VI TABLE OF CONTENTS VIII LIST OF FIGURES X LIST OF TABLES XI Abbreviations XII CHAPTER 1 INTRODUCTION 1 1.1 Overview 1 1.2 Arsenic 1 1.3 Cadmium 3 1.4 Oxidative Stress 4 1.5 Glutathione and Glutathione Synthetase 8 1.6 Caenorhabditis elegans 13 1.7 Glutathione Synthetase Homolog in C. elegans 14 1.8 Purpose of Study 14 CHAPTER 2 MATERIALS & METHODS 16 2.1 Chemicals 16 2.2 Strains, Clones, and Worm Culture 16 2.3 RNA Interference 16 2.4 Culture and Isolation of C. elegans Exposed to Various Stressors 17 2.5 Synchronization of C. elegans 18 2.6 RNA Isolation and real-time RT-PCR 18 2.7 Effect of Transgenic C. elegans 21 2.8 Life Span Assay 21 2.9 Intracellular GSH Measurement 22 2.10 Statistical Analysis 23 CHAPTER 3 RESULTS 24 3.1 Structure and Organization of gs-1 24 3.2 RNA Interference Analysis 28 3.2.1 Phenotypic analysis of wild type worms with GS-1-deficient 28 3.2.2 As(III) and Cd(II) hypersensitivity of GS-1(RNAi) worms 35 3.2.3 Screening of putative transporter gene using GS-1(RNAi) 40 3.2.4 Down-regulation of GS-1 suppresses longevity in C. elegans 42 3.3 Analysis of GS-1 mRNA Expression level Affected by Stressors 45 3.3.1 Regulation of GS-1 mRNA expression level by As(III), As(V), and Cd(II) 45 3.3.2 Regulation of GS-1 mRNA expression by As(III) associated with development 46 3.4 Effect of Various Stressors on gs-1 Transcription in Transgenic C.elegans 47 CHAPTER 4 DISCUSSION 55 4.1 Characterization of C. elegans GS-1 55 4.2 Down-regulation of GS-1 Reduces C. elegans Life Span 57 4.3 GS-1 mRNA expression affected by various metals Exposure 58 4.4 How As(III) and Cd(II) are exported in C. elegans 61 CHAPTER 5 CONCLUSION 64 REFERENCES 66 LIST OF FIGURES Figure 1-1 Pathway for the biosynthesis of glutathione 9 Figure 1-2 The roles of GSH were played in oxidative stress managerments 12 Figure 3-1 Schematic diagram and exon/intron organization of gs-1 25 Figure 3-2 Multiple alignment of the predicted amino acid sequence of C. elegans glutathione synthetase with other known GS amino acid sequence. 26 Figure 3-3 Hydropathy profile of the GS-1 amino acid sequence. 29 Figure 3-4 Putative upstream regulatory elements in gs-1 30 Figure 3-5 Effect of GS-1 (RNAi) on C. elegans 32 Figure 3-6 GS-1 (RNAi) affects reproduction of worms 33 Figure 3-7 Morphologic change associated with GS-1 RNAi 34 Figure 3-8 RNAi effect in growth delayed of C. elegans 36 Figure 3-9 RNAi effect in C. elegans of As (III) 38 Figure 3-10 RNAi effect in C. elegans of Cd (II) 39 Figure 3-11 RNAi effect of N2 and VC287 (hmt-1 mutant) in C. elegans 41 Figure 3-12 Lifespan reduction by GS-1-dicifent and mediated with As (III) 43 Figure 3-13 Lifespan reduction by GS-1-dicifent and mediated with Cd (II) 44 Figure 3-14 GS-1 mRNA level by various heavy metals 48 Figure 3-15 The expression of GS-1 during C. elegans development with As (III)..…………………………………………………………………...50 Figure 3-16 Expression pattern of transgenic C. elegans 52 Figure 3-17 Expression pattern of transgenic C. elegans of Cd (II) exposure 53 Figure 3-18 Expression pattern of transgenic C. elegans 54 LIST OF TABLES Table 2-1 Primers used for real-time PCR 201555223 bytesapplication/pdfen-USGlutathione synthetaseCaenorhabditis elegansRNA干擾技術砷鎘氧化壓力RNA interferencearseniccadmiumoxidative stressthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56000/1/ntu-96-R94622014-1.pdf