English  |  正體中文  |  简体中文  |  Items with full text/Total items : 27454/39300
Visitors : 2533632      Online Users : 27
RC Version 4.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
Scope Adv. Search
LoginUploadHelpAboutAdminister

Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17540

Title: 利用農桿菌轉殖小鼠砷甲基轉移酶基因至小球藻與此基因於周氏扁藻之檢測
Agrobacterium-mediated transformation of a mouse arsenic methyltransferase gene into Chlorella pyrenoidosa and the detection of arsenic methyltransferase gene in Tetraselmis chuii
Authors: Shi-Hua Gi
紀熙華
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 砷甲基轉移酶;農桿菌
green algae;arsenic methyltransferase;Agrobacterium;cyt19
Date: 2004
Issue Date: 2011-07-04
Abstract: 五價砷與三價砷之化合物乃存在於環境中之有毒物質,而將砷甲基化為生物體內一解毒機制。先前實驗發現,海水藻周氏扁藻(Tetraselmis chuii)在添加0.133mM五價無機砷培養七天後,以HG-GCT-AAS(Hydride Generation-Gamma Cold Trap-Atomic Absorption Spectrophotometer)分析後發現有單甲基砷與雙甲基砷的產生,而同樣添加無機砷培養的淡水藻小球藻(Chlorella pyrenoidosa)則無此現象。為了使藻類能將淡水水體中之砷甲基化以達到生物復育之目的,本論文將小鼠砷甲基轉移酶基因(cyt19)之開放讀取區域(1011bp)接入含Pnos 啟動子啟動外源基因的pBIN19載體後以農桿菌轉殖方式送入小球藻內,由PCR結果確認cyt19基因開放讀取區域DNA序列存在某些轉殖小球藻中,而不存在任何未轉殖小球藻體內,然而此轉殖小球藻其砷甲基化活性仍待進一步之研究。而本論文也以小鼠cyt19之cDNA(1011bp)序列製造探針,對周氏扁藻之總 RNA進行北方氏墨點法(Northern blot),發現有雜交訊號產生,其位置約1200bp-1500bp。而此雜交訊號對含五價砷條件下培養四天之周氏扁藻總 RNA會有增強之現象,培養六天此訊號則似乎減弱。根據此一訊息及網路上資料庫搜尋得到此類cyt19蛋白之資訊,觀察加砷與未加砷培養後周氏扁藻之蛋白二維電泳圖,雖然發現砷可誘發三個蛋白質合成,但在符合cyt19蛋白質特徵(等電點與分子量)範圍內,並無差異之蛋白質點,表示砷可能無法影響周氏扁藻砷甲基轉移酶之表現。
Pentavalence arsenic and trivalence arsenic chemical compounds are toxicant in the environment and methylation of arsenic species is a detoxification pathway in some organisms. Experimental analysis by HG-GCT-AAS(Hydride Generation-Gamma Cold Trap-Atomic Absorption Spectrophotometer)showed that marine alga Tetraselmis chuii cultrued with 0.133mM arsenate for 7 days generated monomethyl arsenic species (MMA) and dimethyl arsenic species (DMA), but freshwater alga Chlorella pyrenoidosa showed no methylating activity under the same condition. For producing an alga species capable of methylating arsenic in freshwater, a 1011-bp cDNA carrying the complete open reading frame of mouse arsenic methyltransferase(cyt 19) was cloned into the expression vector pBIN19 under the control of a Pnos promotor and cyt19 ORF was transformed into Chlorella via infection with Agrobacterium tumefaciens. The cyt19 cDNA was detected by PCR in several colonies of transformed Chlorella after 2-day infection and no cyt19 cDNA could be found in all nontransformed Chlorella colonies. Whether arsenic methylating activity can be produced in transformed Chlorella awaits further investigation. Northern blot analysis of total RNA of T. chuii using cyt19 ORF as the probe detected a hybridization signal and the signal intensity increased to 142% of control after culturing T. chuii in the medium containing arsenate for 4 days and returned to normal levels 6 days later. Although three arsenate-induced polypeptides were detected by 2-D gel electrophoresis of crude extract proteins of T. chuii cultured in the presence or absence of arsenate, no induction of cyt19-like proteins was found, suggesting that the expression of arsenic methyltrnsferase in T. chuii was not regulated by arsenate.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M92360011
http://ntour.ntou.edu.tw/ir/handle/987654321/17540
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

Files in This Item:

File Description SizeFormat
index.html0KbHTML149View/Open


All items in NTOUR are protected by copyright, with all rights reserved.

 


著作權政策宣告: 本網站之內容為國立臺灣海洋大學所收錄之機構典藏,無償提供學術研究與公眾教育等公益性使用,請合理使用本網站之內容,以尊重著作權人之權益。
網站維護: 海大圖資處 圖書系統組
DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - Feedback