以功能性基因體學研究細菌異化辛基苯酚  聚氧乙基醇及抗逆境之基因

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陳啟祥(Chi-Hsiang Chen) 查詢紙本館藏

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以功能性基因體學研究細菌異化辛基苯酚聚氧乙基醇及抗逆境之基因
(Functional genomic study of the genes involved in the bacteria catabolism and stress response to octylphenol polyethoxylates)

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摘要(中)

辛基苯酚聚氧乙基醇 (octylphenol polyethoxylates，OPEOn)為一常用於農業、工業以及一般家庭活動之難分解的非離子性界面活性劑。一旦排放至自然環境時，經常形成辛基苯酸，在結構上類似雌激素，具環境荷爾蒙效力。然而目前對於異化辛基苯酚聚氧乙基醇之酵素的瞭解仍非常有限，本實驗室已分離出可利用辛基苯酚聚氧乙基醇為唯一生長碳源之菌株Pseudomonas nitroreducens TX1，並利用一維及二維膠體電泳分析菌株P. nitroreducens TX1生長於以OPEOn為唯一碳源時所增加或減少表現的蛋白質，為了進一步證明上述發現之增加或減少表現蛋白質的合理性，本研究續以基因體學為研究方法參考全基因體已定序之Pseudomonas sp.，設計引子選殖參與OPEOn代謝及抗逆境反應的相關基因，並以反轉錄聚合酶鏈反應(RT-PCR)觀察其mRNA的表現量是否與一維及二維膠體電泳分析上所發現的蛋白質表現情形相符。本研究計成功地選殖出一個完整序列的基因及七個部份序列的基因，分別包括Arginine / Ornithine transport system substrate-binding proetin、periplasmic binding protein of ATP binding cassette transporter及TolB protein等三個運輸蛋白基因，dihydrolipoamide dehydrogenase所具有之三個與TCA cycle相關的基因群組，一個抗逆境蛋白質superoxide dismutase的基因及一個脂肪酸合成酵素Acetyl-CoA carboxylase carboxyl transferase的基因，另外，反轉錄聚合酶鏈反應的結果顯示，除了Arginine / Ornithine transport system substrate-binding proetin及periplasmic binding protein of ATP binding cassette transporter二個運輸蛋白外，其他蛋白質的基因表現皆與先前在一維及二維膠體電泳上所觀察到的蛋白質表現情形一致，其中，與高分子化合物輸送有關的TolB，其基因在mRNA表現量為選殖運輸蛋白中唯一有表現量增加的蛋白質，至於dihydrolipoamide dehydrogenase基因群組亦全部觀察到有增加表現的現象，初步推測此基因群組除在OPEOn為唯一碳源的表現，除了能增加能量的產生外，dihydrolipoamide dehydrogenase亦能夠利用似Fenton反應產生氫氧自由基，進而藉由氫氧自由基切斷OPEOn的聚氧乙基酸鏈，此外，自由基存在量增加時，本研究進一步發現superoxide dismutase基因的表現同時被誘發，推測除了能抵抗逆境外，文獻彙整顯示尚具有切斷由醇基轉化為羧酸的功能，故其增生表現可能同時會參與OPEOn的代謝。

摘要(英)

The octylphenol polyethoxylates (OPEOn) are recalcitrant non-ionic
surfactants. The release of OPEOn has resulted in concern due to their
metabolites in the environment showing estrogenic activity. However,
little is known about the enzymes involved in the degradation of OPEOn.
A Gram-negative rod, Pseudomonas nitroreducens TX1, was shown to be
able to grow on 0.05-20% (v/v) of OPEOn as sole carbon source. Previous
study also found several proteins to be up-regulated and down-regulated
in response to OPEOn catabolism by 1D- and 2D-electrophoresis analysis.
Consequently, this genomics study was aimed to confirm the expression
of up-regulated and down-regulated proteins from functional proteomics
as mentioned above. The genes in response to OPEOn catabolism and
stress responses were cloned by specific primers, which were designed
from reference Pseudomonas species with whole genome sequencing.
The gene expression was also investigated by RT-PCR to understand if
the expression of mRNA from these genes were consistent to that of
proteins from 1D- and 2D- electrophoresis analysis. One gene with full
length sequence and seven genes with partial sequence have been
successfully cloned in this study. These genes included three transporters
genes (arginine / ornithine transport system substrate-binding protein,
periplasmic binding protein of ATP binding cassette transporter and TolB
protein ), three genes involved in TCA cycle (2-oxoglutarate
dehydrogenase, dihydrolipoamide succinyltransferase and
dihydrolipoamide dehydrogenase), one gene for stress response and one
gene related to fatty acid synthesis. The RT-PCR analysis indicated that
the expression of these genes was consistent to that of proteins by 1D-
and 2D- electrophoresis analysis except that two transporter genes,
arginine / ornithine transport system substrate-binding protein and
periplasmic binding protein of ATP binding cassette transporter. The
expression of transporter genes found to be up-regulated only for
TolB, which is related the transportation of biopolymers. The expression
of dihydrolipoamide dehydrogenase gene clusters was also observed to be
up-regulated. These were suggested to increase the synthesis of energy in
response to OPEOn catabolism. The E3 component of these gene clusters
was further proposed to generate OH radicals by Fenton-like reaction,
which was demonstrated to be able to cleave the polyethoxylates in
OPEOn. Moreover, the expression of superoxide dismutase was also
found to be induced in response to the production of OH radicals. Since
superoxide dismutase has been found to have another function for ether
bond-splitting of carboxylated polyethoxylates from literature survey, the
expression of this enzyme was also suggested to be involved in OPEOn
catabolism.

關鍵字(中)

★ RT-PCR
★ 環境荷爾蒙
★ 辛基苯酚聚氧乙基醇

關鍵字(英)

★ RT-PCR
★ estrogenic activity
★ OPEOn

論文目次

中文摘要…..……………………………………………………………Ⅰ
英文摘要………………………………………………………………..Ⅲ
目錄……………………………………………..………………………Ⅳ
表目錄…………………………………………………………………Ⅸ
圖目錄…………………………………………………………………..Ⅹ
名詞縮寫對照表…………………………………………………..…ⅩⅡ
壹、緒論………………………………………………………………….1
一、界面活性劑的環境荷爾蒙活性…………………………............1
二、菌株Pseudomonas nitroreducens TX1篩選、鑑定與背景.....2
三、P. nitroreducens TX1耗氧酵素的純化與鑑定..............................4
四、P. nitroreducens TX1的功能性蛋白質體學分析..........................4
五、P. nitroreducens TX1的基因體學分析..........................................5
六、研究目的與動機…………………………………………………9
貳、材料及方法…………………………………………………………10
一、培養基與細菌品系………………………………………………10
二、細菌核酸之製備…………………………………………………12
三、利用聚合酶鏈反應(Polymerase Chain Reaction, PCR)做基因選殖…………..……………………………………………………14
四、利用反轉錄聚合酶鏈反應( Reverse Transcription Polymerase Chain Reaction, RT-PCR)觀察………………………………….17
五、生物資訊學的方法分析………………………………………...18
六、實驗儀器與化學藥品…………………………………………...20
叄、結果…………………………………………………………………19
一、P. nitroreducens TX1染色體中的dihydrolipoamide dehydrogenase基因群組........................................................................................22
二、以反轉錄聚合酶鏈反應觀察P. nitroreducens TX1在OPEOn下基因表現........................................................................................27
肆、討論...................................................................................................32
一、菌株P. nitroreducens TX1的dihydrolipoamide dehydrogenase基因族群選殖與功能探討..........................................................34
二、菌株P. nitroreducens TX1的dihydrolipoamide dehydrogenase基因族群啟動探討......................................................................38
三、菌株P. nitroreducens TX1在OPEOn存在下，代謝及抗逆境基因的表現......................................................................................39
伍、結論與建議........................................................................................44
陸、參考文獻............................................................................................46
表..............................................................................................................54
圖..............................................................................................................60
附錄..........................................................................................................89

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2005-7-28

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