廖秀娟Liao, Vivian Hsiu-Chuan臺灣大學:生物環境系統工程學研究所劉瑞桐Liu, Jui-TungJui-TungLiu2010-05-052018-06-292010-05-052018-06-292009U0001-2101200923310100http://ntur.lib.ntu.edu.tw//handle/246246/181145砷是一種自然存在的毒性物質，當暴露於砷環境的情況下會造成各種器官的病變，目前被認定為已知的人類致癌物。不論是在真核生物或原核生物，各個生物系統中都可以發現對於砷的毒性解毒機制。在Caenorhabditis elegans (C. elegans) 中,已經找到了在細菌中扮演砷解毒機制基因ArsA的同源基因asna-1。然而目前與asna-1交互作用並扮演排除三價砷的傳輸蛋白仍然未被找出。因此，本研究運用了生物資訊的方法預測出了多個可能與asna-1交互作用的基因，並且利用RNA interference (RNAi)的方法進一步篩選出此傳輸蛋白。同時，本研究也繼續探討了asna-1在C. elegans中的於砷存在時的調控機制。研究利用RNAi 篩選方法，找出了陰離子傳輸蛋白ABTS-1可能為排出砷的傳輸蛋白。我們更進一步地利用將abts-1基因移除的突變線蟲做砷的毒性測試，研究結果顯示了abts-1 缺失的線蟲對於砷的抗性有明顯的降低，證實了ABTS-1對於砷的傳輸扮演了重要的角色。研究結果亦顯示, asna-1缺失的線蟲對砷的抗性有明顯的降低。另一方面，大量表達ASNA-1蛋白質的C. elegans則可以增加對砷的抗性，更進一步證實了ASNA-1為C. elegans抗砷機制的重要蛋白。除此之外，我們也利用transgenic C. elegans以及 Real-time RT-PCR的方法對於asna-1於不同幼蟲時期的基因表達以及其暴露於砷情況下的基因調控作進一步的瞭解。我們發現了在L1幼蟲時期以及成蟲時期asna-1對於砷的誘導情況最為明顯。同時本研究也探討了asna-1以及abts-1對於C. elegans壽命的影響。結果顯示了缺乏abts-1的功能會導致C. elegans壽命的延長。本研究結果可推測出基因abts-1所轉譯的ABTS-1在對於細胞體內砷的排出扮演了重要角色並且可能為與ASNA-1作用的傳輸蛋白。同時也可推斷ASNA-1於C. elegans的幼蟲時期以及成蟲時期扮演重要的角色。Arsenic is a known human carcinogen and a potent toxin. It is a major environmental pollutant. Nearly in every organism, from prokaryotes to eukaryotes, have mechanisms for arsenic detoxification. In Caenorhabditis elegans, the gene asna-1 has been identified as the homolog of ArsA in Escherichia coli (E. coli) and involved in arsenic detoxification. To identify genes interacting with asna-1, the candidate genes were identified by gene interaction predicted program. RNA interference (RNAi) analysis was performed to screen for asna-1 interacted gene in the presence of As(III) and we found the anion transporter ABTS-1. Toxicity tests showed that both asna-1 mutant and abts-1 mutant were hypersensitive to As(III), indicating that both asna-1 and abts-1 are required for As(III) detoxification. ASNA-1 developmental mRNA expression induced by As(III) in vivo and in vitro were examined by transgenic C. elegans and real-time RT-PCR analysis, respectively. The results showed that ASNA-1 mRNA expression varied in different developmental stages of worms and adulthood had higher expression level. In addition, ASNA-1 mRNA expression was induced by As(III). Life span assay showed overexpression of ASNA-1 protein did not affect the longevity of nematodes but the deletion of abts-1 extended the life span of C. elegans. In this study, our results showed that ASNA-1 was involved in As(III) resistance and ABTS-1 might act as the corresponding transporter to As(III) detoxification. ABTS-1 might be involved in insulin-like signaling (IIS) pathway because of the extension of life span in abts-1 mutant.TABLE OF CONTENTS謝.................... .........................I文摘要...............................................IIIBSTRACT................................................VABLE OF CONTENTS................................VIIIST OF TABLES....................................IXIST OF FIGURES...........................................XIST OF APPENDIX.........................................XIBBREVIATIONS...........................................XIIHAPTER 1 INTRODUCTION..........................................1.1 Arsenic (As)....................................1.2 Detoxification system of arsenic in organisms........3.2.1 Bacteria....................................3.2.2 Saccharomyces cerevisiae.......................................4.2.3 Mammalian systems............................6.2.4 Caenorhabditis elegans (C. elegans)...........7.3 C. elegans as an animal model to explore toxicology...8.4 ArsA in organisms.............................11.4.1 ArsA in Prokaryotes..........................12.4.2 ArsA in Eukaryotes.................................12.5 Purpose of study.............................15hapter 2 MATERIALS AND METHODS.........................17.1 Chemicals................................................17.2 Strains, clone, and culture condition...............17.3 Culture and isolation of C. elegans exposed to As(III)................................................18.4 RNA Interference ( RNAi )..........................18.5 Toxicity analysis....................................19.6 Real-time RT-PCR....................................20.7 Expression of transgenic C. elegans................22.8 Arsenic exposure assay.........................22.9 Life span assay................................23.10 Statistical analysis................................25HAPTER 3 RESULTS..................................................26.1 Prediction of asna-1 interacted genes...........26.2 Identification of asna-1 interacted genes via RNA interference analysis............................27.2.1 asna-1 (RNAi)............................27.2.2 Screening of asna-1 interacted genes by RNA interference...30.3 Lethality tests of metalloids for C. elegans.........31.3.1 Effect of As(III) and Sb(III) on asna-1........31.3.2 Effect of As(III), Sb(III), and other heavy metals on abts-1...36.4 Life span assay.................................37.5 Expression of transgenic C. elegans..................41.6 Analysis of ASNA-1 mRNA expression level affected by As(III).......43hapter 4 DISCUSSION AND CONLUSION.....................49.1 Characterization of asna-1 in C. elegans.............49.2 ASNA-1 mRNA expression affected by As(III) exposure..50.3 Identification of ASNA-1 interacted genes.........52.4 Regulation of asna-1 and abts-1.................54eferences ......55application/pdf6327213 bytesapplication/pdfen-US砷ASNA-1ABTS-1Caenorhabditis elegansRNAi解毒傳輸蛋白Arsenicdetoxificationtransporter[SDGs]SDG11thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181145/1/ntu-98-R95622008-1.pdf