以轉位子突變法篩選 Pseudomonas nitroreducens TX1 降解非離子性界面活性劑辛基苯酚聚氧乙基醇之基因與功能預測

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蔡博鈞(Po-Chun Tsai) 查詢紙本館藏

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論文名稱

以轉位子突變法篩選 Pseudomonas nitroreducens TX1 降解非離子性界面活性劑辛基苯酚聚氧乙基醇之基因與功能預測
(Screening of genes involved in the catabolism and stress response to octylphenol polyethoxylates (Triton X-100) in Pseudomonas nitroreducens TX1 by transposon mutagenesis)

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摘要(中)

烷基苯酚聚氧乙基醇 (Alkylphenol polyethoxylate, APEOn) 為非離子界面活性劑，並廣泛用於工業用途。一般家庭、河川底泥與都市廢水系統中皆發現此類化合物之累積。其代謝產物，如壬基苯酚 (Nonylphenol) 或辛基苯酚 (Octylphenol) ，對生物有慢性毒性及潛在的內分泌干擾性。Pseudomonas nitroreducens TX1 為從灌溉渠道底泥中分離出的細菌，能以高濃度之 APEOn 為唯一碳源生長。 P.nitroreducens TX1 的基因體序列草圖已於2014年1月發表，為 APEOn 的降解基因定位與分析提供基本資訊。本研究使用轉位子突變法 (Transposon mutagenesis) 針對辛基苯酚聚氧乙基醇 (Octylphenol polyethoxylates, Triton X-100) 分解菌株Pseudomonas nitroreducens TX1 進行突變，本研究目的在於使用此法篩選無法正常生長於 Triton X-100 培養基的突變菌株。P. nitroreducens TX1使用三親配對法 (Triparental mating) 進行隨機突變後，以生長測試篩選無法正常生長於 Triton X-100的突變菌株。基因分析方面，使用限制酶剪切染色體、DNA 連接酶處理、轉殖試驗、質體萃取、定序與序列比對等方法，進行突變基因定位及功能預測，探討各類突變基因對 P. nitroreducens TX1 分解或抵抗 Triton X-100 的影響。目前自約30000株突變菌株中篩選出145株以 Triton X-100 培養生長緩慢或無法生長的突變菌株。其中130株突變菌株已完成突變基因鑑定。已發現的突變基因依功能可分為六大類，分別為代謝酵素 (15個基因)、膜蛋白 (7個基因)、細胞膜/壁結構組成 (環境壓力反應) (11個基因) 、趨化性 (Chemotaxis) (4個基因) 、非轉錄區域序列 (non-coding sequences) (4個轉位子插入點) 以及未知功能序列 (12個基因)。另外，野生型菌株與部分代謝基因突變菌株已進行 Triton X-100 之代謝產物萃取，並以 HPLC 分析產物。雖然目前透過HPLC之結果尚未找出關鍵的降解基因，但本研究透過轉位子突變篩選的多個基因資訊，可供後續進一步系統性地探討細菌降解難以利用之碳源之代謝途徑或抵抗環境壓力之機制。

摘要(英)

Alkylphenol polyethoxylates (APEOn) are often found as pollutants in natural aquatic environments and in raw municipal wastewater. Some of the APEOn metabolites, such as nonylphenol and octylphenol, have chronic ecotoxicity and they are potential endocrine disrupters to aquatic organisms, wildlife, and humans. Pseudomonas nitroreducens TX1 is capable to use APEOn at high concentrations as a sole carbon source. In addition, the draft genome sequence of TX1 was already published in Jan., 2014 and provides an opportunity for investigating genes that play significant roles in alkylphenol polyethoxylate degradation. Transposon mutagenesis was used to create mutants of P. nitroreducens TX1for the purpose of screening the growth deficient mutants on OPEOn. The genome of mutants were treated by digestion, ligation , transformation, plasposon extraction and sequencing to predict the function of mutated genes. Over 30,000 mutants have been isolated after mating. 145 mutants grew slowly or did not grow on OPEOn medium, and the mutated genes in 130 mutants have been identified. These genes were classified to 6 group by their functions: metabolic enzyme (15 genes), membrane proteins (7 genes), stress response (11 genes), chemotaxis (4 genes), non-coding sequence (4 transposon insertion sites) and genes with unknown function (12 genes). The identifying of OPEOn metabolites in wild type TX1 and some of the mutants were performed by HPLC. Despite the fact the result of HPLC is difficult to approach the positive results. This study provides lots of candidate genes to be essential for strain TX1 to utilize and resist Triton X-100 provide the whole-picture of the mechanistic study of bacteria in the catabolism of recalcitrant carbon source and resistant strategies to environmental stresses.

關鍵字(中)

★ 烷基苯酚聚氧乙基醇
★ 采酮 X-100
★ 轉位子隨機突變

關鍵字(英)

★ Alkylphenol polyethoxylates
★ Triton X-100
★ Pseudomonas nitroreducens TX1
★ Transposon mutagenesis

論文目次

Table of contents

INTRODUCTION 1

1.1 ALKYLPHENOL ETHOXYLATES 1

1.2 ESTROGENIC ACTIVITY OF APEON 1

1.3 DEGRADATION OF ALKYLPHENOL ETHOXYLATES 2

1.4 TRANSPOSON MUTAGENESIS 2

1.5 RESEARCH AIMS 3

1.6 STUDY OUTLINE 4

MATERIAL AND METHODS 5

2.1 CULTURE MEDIUM 5

2.1.1 Antibiotics 5

2.1.2 Minimal salts basal medium 5

2.1.3 Luria- Bertani medium 6

2.2 BACTERIAL STRAINS AND CULTURE CONDITION 6

2.3 TRANSPOSON MUTAGENESIS 6

2.4 SCREENING OF OPEON GROWING MUTANTS OF TX1 7

2.4.1 Screening on plates 7

2.4.2 Growth curve monitoring 7

2.5 MUTANT GENE IDENTIFYING 8

2.5.1 Mutant genome extraction 8

2.5.2 Checking the transposon sequence in mutants 8

2.5.3 Digestion and ligation of mutant genome 8

2.5.4 Transformation of plasposon with flanking gene 8

2.5.5 Plasposon extraction and digestion 9

2.5.6 Sequencing of flanking genes 9

2.5.7 Prediction of gene function in mutants 9

2.6 IDENTIFICATION OF TRITON X-100 METABOLITES IN WILD TYPE/MUTANTS OF TX1 10

2.6.1 Reactivation of bacterial strains 10

2.6.2 Induction of degrading genes of Triton X-100 10

2.6.3 Culturing and Sampling 11

2.6.4 Extraction and Identification of metabolites 11

2.7 CHEMICALS AND INSTRUMENTS 12

2.7.1 Chemicals and enzymes 12

2.7.2 Instruments 12

RESULTS 13

3.1 SCREENING OF TRITON X-100 GROWING DEFICIENT MUTANTS 13

3.2 GROWTH CURVES OF WILD TYPE TX1 AND MUTANTS 13

3.3 IDENTIFICATION OF TX1 MUTANTS BY PCR 14

3.4 IDENTIFICATION OF THE FUNCTIONS OF MUTANT GENES 14

3.4.1 Metabolic enzymes 15

3.4.2 Membrane proteins 15

3.4.3 Stress response proteins 15

3.4.4 Chemotaxis proteins 16

3.4.5 Non-coding sequence 16

3.4.6 Function unknown proteins 16

3.5 IDENTIFICATION OF TRITON X-100 METABOLITES IN TX1 AND MUTANTS 17

DISCUSSION 18

4.1DEFENITION OF SLOW GROWER AND NON GROWER 18

4.2 FUNCTIONS OF MUTANT GENES 19

4.2.1 Metabolic enzymes 19

4.2.2 Membrane protein 23

4.2.3 Stress response 25

4.2.4 Chemotaxis proteins 25

4.2.5 Mutattions in function unlnown genes 26

4.3 HPLC RESULTS 26

REFERENCES 28

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2015-8-27

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