臺灣博碩士論文加值系統

本研究是探討環境荷爾蒙(endocrine disrupter)中，用量最多、污染情形最嚴重的鄰苯二甲酸二丁酯(Di-n-butyl phthalate, DBP)對葉菜類作物的影響。基於台灣河川及沿岸日益嚴重的環境污染，本研究選擇了小白菜(Brassica rapa subsp. chinensis)與青江菜(Brassica rapa var. chinensis)此二種，在台灣種植面積最廣、生產期最長、食用人口最多、且須引用河川水或地下水灌溉來的葉菜類作物做為供試植物。
研究結果發現，經過濃度為30 mg L-1的DBP處理下，在第三十六天時，小白菜的葉片會開始產生白化的現象，而到第四十二天時，白化的區域已擴散至全葉面。經穿透式電子顯微鏡來觀察葉片細胞的結果發現，DBP會造成小白菜葉片細胞內葉綠體結構的改變，並造成葉綠體膜的不規則外翻。此外，在不同濃度的DBP處理下，皆會造成小白菜生質量與葉綠素濃度的降低，並同時可發現DBP於植體內的累積。
在小白菜蛋白質體(proteomic analysis)與質譜儀(mass spectrometry)的分析結果中發現，DBP會造成小白菜體內兩種自由基分解酵素的升高，此兩種酵素分別是：superoxide dismutase與peroxidase 21 precursor。由此結果，我們可以推論，DBP的處理，會直接或間接的造成小白菜體內產生自由基，因此迫使面對逆境下的小白菜，會產生自由基分解酵素來因應。此外，DBP亦會造成小白菜體內四種生長酵素之異常降低，分別是：protein disulfide isomerase precursor、apocytochrome f precursor、RNA polymerase beta subunit與heat shock cognate protein 80，此四種酵素在細胞體內分別是負責蛋白質結構的折疊、電子傳遞鏈、與DNA的轉錄作用。因此，我們可以由此推論，小白菜經DBA處理後，所降低的這四種酵素，可能就是影響小白菜葉片白化及生長受阻的主要原因。
在青江菜的部分，研究結果發現，經過濃度為50 mg L-1的DBP處理下，在第三十六天時，青江菜的葉片會開始產生黃化的現象，而到第四十二天時，黃化的區域已擴散至全葉面。但由穿透式電子顯微鏡的觀察中，則並無發現葉綠體結構的明顯改變。此外，如同小白菜的測試結果，在DBP的處理下，亦會造成青江菜生質量與葉綠素濃度的降低，並可發現DBP於植體內的累積。
在青江菜的蛋白質體分析(proteomic analysis)與質譜儀分析(mass spectrometry analysis)的結果中發現，DBP會造成青江菜體內三種酵素濃度的升高，此三種酵素分別是：acyl-[acyl-carrier-protein] desaturase, root phototropism protein 3與ferredoxin-nitrite reductase，而此三種酵素在細胞內所負責的功能，分別是脂肪酸合成、光合作用的訊息傳導、以及亞硝酸鹽的同化作用。由此結果，可以推論DBP的處理，會誘發在化學品逆境下的青江菜，體內特定生理反應的異常改變。此外，DBP亦會造成青江菜體內三種生長酵素之異常降低，分別是：dihydroflavonol-4-reductase、aminoacyl-tRNA synthetase與ATP synthase subunit beta，此三種酵素在細胞體內是分別負責類黃酮素(flavonoid)的生合成、tRNA的胺醯化作用(aminoacylation)、與ATP的產生。因此，可以由此推論，青江菜經DBA處理後，所降低的這三種酵素，可能就是影響青江菜葉片黃化與生長受阻的主要原因。
本研究的結論指出，在經過DBP處理下的水耕系統中，小白菜與青江菜的生長情形、外觀型態以及葉綠素濃度，皆會因不同濃度的DBP處理，而產生顯著的劑量濃度反應關係。此外，由植物蛋白質體學方面的分析結果發現，DBP會經由影響小白菜與青江菜正常蛋白質的功能，進而造成其外觀型態、生長速率與生理代謝上的各種改變。

The effects of the endocrine disrupter, di-n-butyl phthalate (DBP), on the growth of leaf vegetable Bok choy (Brassica rapa subsp. chinensis) and Chinese cabbage (Brassica rapa var. chinensis) were investigated. The results showed that leaves of Bok choy became white in color with the occurrence of chlorosis and necrosis upon treating with 30 mg L-1 DBP in hydroponic culture for 42 days. Transmission electron microscopic images revealed that changes in the chloroplast structures accompanied the chlorosis. In addition, a decrease in biomass and chlorophyll, and accumulation of DBP, were found in DBP-treated Bok choy.
The proteome of the leaf tissue was analyzed using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Six protein spots were identified from 2-DE that showed reproducible differences in expression between the normal control and the DBP-treated Bok choy. Based on proteome level studies two protein spots increased were identified as superoxide dismutase (SOD) and peroxidase 21 precursor. These proteins are believed to increase in expression in response to free radical exposure as a detoxification mechanism. The other four protein spots disappeared on the leaf of treatment with DBP were identified as heat shock cognate protein 80, protein disulfide isomerase precursor, apocytochrome f precursor, and RNA polymerase beta subunit. These results are indicated that DBP might affect the polypeptide folding, electron transport chain and DNA transcription in the Bok choy cell and cause the restriction of growth and development in Bok choy.
Besides, the results of Chinese cabbage showed that leaves turned yellow in color with the occurrence of etiolation upon treating with 50 mg L-1 DBP for 42 days. Examination of grana structure in chloroplast under the TEM revealed the organelle unchanged in the tissue of yellow leaf. In addition, a decrease in biomass and chlorophyll, and accumulation of DBP, were found in DBP-treated Chinese cabbage.
There are also six protein spots were identified in 2-DE that showed reproducible differences in expression between the normal control and the DBP-treated Chinese cabbage. Three proteins were found or increased in amount and another three proteins were decreased or disappeared. Three protein spots increased were identified as acyl-[acyl-carrier-protein] desaturase (acyl-ACP desaturase), root phototropism protein 3 (RPT3) and ferredoxin-nitrite reductase (Fd-NiR). The three are responsible for the fatty acid biosynthesis, signal transduction of the phototropic response and nitrate assimilation in plant cells. These results are indicated that DBP seems to induce some physiological reactions increasing in the Chinese cabbage cell.
The other three protein spots that disappeared in Chinese cabbage on treatment with DBP were identified as dihydroflavonol-4-reductase (DFR), aminoacyl-tRNA synthetase (aaRS) and ATP synthase subunit beta. The results in this part revealed some damages or disorder of metabolism inside the Chinese cabbage cell that may partially be attributed to the change in the amounts of some proteins in the cell. Decrease of DFR indicated that DBP might affect the flavonoid biosynthesis and floral color development in the Chinese cabbage cell. This effect might the cause of Chinese cabbage leaf change to yellow. Decrease of aaRS indicated that DBP might affect the aminoacylation of tRNA and other transcriptional or translational regulation in the Chinese cabbage cell. This effect might cause some disorders of regular metabolism or development of Chinese cabbage. Decreased of ATP synthase subunit beta indicated that ATP synthesis in the Chinese cabbage cell might be affected by DBP and cause the restriction of growth and development in Chinese cabbage. From our results, we might say that DBP seems to induce some physiological reactions increasing and also cause the restriction of growth and development in Chinese cabbage. These results seem partially the same as that of Bok choy.
This study indicated that the growth and morphology of Bok choy and Chinese cabbage showed a significant dose-response relationship upon treatment with DBP in a hydroponic culture medium. Furthermore, DBP also affects the proteome formation as well as the physiology and the morphology of Bok choy and Chinese cabbage during growth.

TABLE OF CONTENTS I
LIST OF FIGURES IV
LIST OF TABLES VII
ABSTRACT IX
ABSTRACT IN CHINESE (中文摘要) XI
ABBREVIATIONS XIII


Chapter 1 Introduction 1
1-1 What are endocrine-disrupting compounds (EDCs) 1
1-2 Characteristics of phthalate esters 2
1-2.1 General description 2
1-2.2 Uses of PAEs 5
1-2.3 Disposal and releases into the environment 6
1-3 Characteristics of Di-n-butyl phthalate 6
1-3.1 Chemistry 6
1-3.2 Usage and exposure 7
1-4 Some toxicological and biological parameters of Di-n-butyl phthalate 11
1-4.1 Toxicity 11
1-4.2 Toxicokinetics 11
1-4.3 Genetic toxicity 12
1-4.4 Developmental toxicity 12
1-4.5 Reproductive toxicity 13
1-4.6 Toxicity of green plants 15
1-5 Proteomics and bioinformatics of DBP-treated plants 17
1-6 The tetrapyrrole pathway in DBP-treated plants 20
1-6.1 The tetrapyrrole synthetic pathway 20
1-6.2 Regulation of tetrapyrrole synthesis 22
1-7 Objectives of this research 23
1-8 Flowchart of this research 24

Chapter 2 Materials and methods 25
2-1 Chemicals 25
2-2 Plant culture 25
2-3 Chlorophyll (a+b) concentration determination 27
2-4 Residual DBP analysis 28
2-5 Leaf tissue observation 30
2-6 Protein extraction 30
2-6.1 Protein extraction 30
2-6.2 Protein desalting 31
2-6.3 Two-dimensional gel electrophoresis 32
2-6.4 Digestion of in-gel protein 35
2-6.5 Mass spectrometry analyses of proteome 35


Chapter 3 Results and Discussion 39
3-1 Bok choy (Brassica rapa subsp. chinensis) 39
3-1.1 Outer and inner injury of Bok choy leaf 39
3-1.2 Concentration of chlorophyll and accumulation of DBP in Bok choy leaf 41
3-1.3 Proteome differences of Bok choy 45
3-2 Chinese cabbage (Brassica rapa var. chinensis) 54
3-2.1 Outer and inner injury of Chinese cabbage leaf 54
3-2.2 Concentration of chlorophyll and accumulation of DBP in
Chinese cabbage leaf 56
3-2.3 Proteome differences of Chinese cabbage 60

Chapter 4 Conclusions 72

References 74

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