Caenorhabditis elegans之glutathione synthetase 基因 受重金屬調控之機制之探討

Regulation of glutathione synthetase gene in Caenorhabditis elegans by heavy metals

鄭權(Chuan Cheng)

指導教授 ： 廖秀娟

國立臺灣大學/生物資源暨農學院/生物環境系統工程學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10424

摘要

Glutathione 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。

關鍵字

Glutathione synthetase ； Caenorhabditis elegans ； RNA干擾技術 ； 砷 ； 鎘 ； 氧化壓力

並列摘要

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.

並列關鍵字

Glutathione synthetase ； Caenorhabditis elegans ； RNA interference ； arsenic ； cadmium ； oxidative stress

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