金屬硫蛋白 (Metallothionein, MT) 是一含有大量cysteine並且耐熱的小分子量蛋白質。這類蛋白質已經證實具有螯合¬二價重金屬的功用，除了可以調控必需重金屬鋅或銅的含量外，也可以在生物體內螯合非必需的重金屬如鎘或汞等，以減輕毒性，作為解毒機制的一環。從文獻知道，現存大部分物種從細菌到哺乳類動物皆發現有此類蛋白質，包括軟體、節肢及環節動物身上皆已証實有金屬硫蛋白的存在，然而於刺絲胞動物門的海葵至今是否存有金屬硫蛋白仍尚未被證實。本實驗中的動物，海葵Aiptasia pulchella 容易飼養並具有無性生殖的特性，可在實驗的過程中減少個體差異。經由實驗結果得知重金屬鎘除了會對海葵造成外觀上的傷害，也會進一步造成海葵部分累積較高量重金屬的組織脫落。鎘處理過的A. pulchella以immunoblotting可以偵測到三個耐熱的低分子量蛋白質，再以免疫組織染色可以更進一步看到金屬硫蛋白主要出現在海葵組織外胚層的部分，在重金屬鎘離子處理後明顯看到其被誘發表現的現象。雖然利用相近物種設計而來的核酸引子無法以RT-PCR在海葵身上找到確切的金屬硫蛋白序列，但是依據immunoblotting及免疫組織染色的結果，偵測到的蛋白質其分子量大小，耐熱特性以及表現模式，因此應可確認在海葵體內存在著金屬硫蛋白。

Metallothioneins (MTs) are cysteine rich, heat stable proteins with low molecular weight. They have been demonstrated that can chelate various divalent heavy metals. In addition to mediate homeostasis of essential metals Zn and Cu, MTs can bind with the nonessential metals, like Cd and Hg, to reduce their toxicities, which is recognized as a mechanism of detoxification. According to previous studies, MTs have been identified in a wide range of organisms, from bacteria to mammals. In invertebrate, MTs were proven existing in molluscs, crustaceans and annelids, but not in sea anemones. Sea anemone, Aiptasia pulchella, was chosen as the experimental organism in this study, because they are easy to be cultured in a laboratory system and have a life cycle with asexual reproduction, which reduce inter-individual variation. After exposed to cadmium, obvious morphological changes can be observed such as the shed tissues accumulated higher level of cadmium than normal tissues. From the results of immunoblotting, three heat stable, low molecular MT-like proteins were detected. Furthermore, the results of immunohistochemistry study revealed that the MTs expressed mainly in ectoderm and were induced after cadmium treatment. Although the exact MT sequence on A. pulchella was not obtained by using RT-PCR with primer combinations based on the immunobloting and immunohistochemistry results, the MT molecules were expressed in the rational location and their expression levels were induced after cadmium exposure. Therefore, it is strongly believed that MT molecules exist in A. pulchella.

口試委員會審定書 I
誌謝 II
Abstract IV
中文摘要 VI
Contents VII

1. Introduction
1.1. Heavy metals and cadmium 1
1.2. Cadmium pollution and toxicity 2
1.3. Protection mechanisms in animals 4
1.4. Characteristics of metallothioneins 4
1.5. Previous studies of vertebrate and invertebrate metallothioneins 6
1.6. Cnidarians and Aiptasia pulchella 8
1.7. Objective 9
2. Materials and Methods
2.1. Animals 11
2.2. Cadmium exposure 11
2.3. Measurement of cadmium bioaccumulation 11
2.4. RNA extraction and reverse transcription 12
2.5. Polymerase chain reaction (PCR) 13
2.6. Partial purification of metallothionein 13
2.7. SDS-PAGE and immunoblotting 14
2.8. Immunohistochemistry (IHC) 15
2.9. Statistic 17
3. Results
3.1. Morphological changes in A. pulchella during cadmium treatment 18
3.2. Bioaccumulation of cadmium in A. pulchella 18
3.3. Molecular analyses of metallothionein genes on A. pulchella 19
3.4. Detection of metallothioneins on A. pulchella by immunoblotting 20
3.5. Location of metallothioneins in A. pulchella 21
4. Discussion 23
5. References 29
Figures 41
Tables 52

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