本研究利用PCR將海葵 (Hexacorallia: Actiniaria) 及海鞘 (Ascidiacea: Aplousobranchia) 的粒線體DNA增幅，定序後獲得完整華美中海葵Mesactinia genesis、唸珠海葵Heteractis aurora和部份Calliactis sp. 以及海鞘Eudistoma gilboviride的粒線體DNA 序列，總長分別爲20,544 bp、19,800 bp、3,713 bp及14,203 bp。海葵環狀粒線體，包含了17 個基因，其中有13 個蛋白質基因，2 個rRNA，及2 個tRNA。所有基因均爲同方向轉錄。除了在其他海葵目 (Metridium senile、Nematostella sp.) 已知的蛋白質基因外，本研究所分析之三種海葵其基因組內有一段功能未明的ORF，介於cox2及nad4基因之間，H. aurora可轉譯一段長度646個胺基酸的蛋白質，M. geneisis 可轉譯兩段長度分別爲259及243個胺基酸的蛋白質，Calliactis sp.可轉譯兩段長度分別爲269及345個胺基酸的蛋白質，這是在其他海葵目，甚至刺細胞動物門所沒有發現的ORF。以RT-PCR檢視發現此段序列會被轉錄。此未知功能ORF 蛋白在N端可形成4個穿膜區域。在基因間區域igr13 可形成一髮夾構造可能是線粒體基因組控制區。海鞘Eudistoma gilboviride 包含36個基因，其中有11個功能蛋白基因，22個tRNA，2個rRNA，其中trnM、trnK 及trnV 基因在L-strand 轉錄，其餘基因皆在H-strand 轉錄。海鞘粒線體基因間排序差異非常的大，E. gilboviride 粒線體基因組和目前已發表的海鞘皆不相同，並發現不完整的atp8 及缺少nad6。利用海鞘綱與後口動物 (echinoderms, chordate and Xenoturbella) 和刺細胞進行親緣關係，發現海鞘在脊索動物門的分類與形態學相符合。

Complete DNA sequences were determined for the mitochondrial (mt) genomes of the sea anemones, Mesactinia genesis （20,544 bp）, Heteractis aurora (19,800 bp) and partial mtDNA between cox2 and nad4 of the Calliactis sp. (3,713 bp). In addition, complete mtDNA sequences were determined for the sea squirt, Eudistoma gilboviride (14,203 bp). The circular, sea anemones genomes contain the genes for 13 energy pathway proteins and two ribosomal RNAs and two transfer RNAs. H. aurora contains a previously undescribed ORF between the cox2 and nad4 genes encoding a putative protein of 646 amino acids. In M. geneisis and Calliactis sp. encodes two separate smaller ORFs of 259 and 243 as well as 269 and 345 in the corresponding regions, respectively. Possible control region of the mitochondrial genomes of M. geneisis and H. aurora were identified in the intergenic region 13. The sea squirt genomes contain the genes for 11 energy pathway proteins and two rRNAs and 22 tRNAs. All genes are encoded by the heavy strand, except for trnM, trnK, and trnV, which are encoded by the light strand. The ascidians showed frequent and extensive gene rearrangement. The gene order in E. gilboviride are very much different from the other ascidians mt-genome. The E. gilboviride mtDNA does not encode the nad6 and a tpyical atp8. Molecular phylogenetic analyses based on nucleic acid and amino acid sequences of the deuterostome (echinoderms, chordate and Xenoturbella), and cnidaria coincide with the morphological characters.

目錄
誌謝......................................................................................................... i
摘要........................................................................................................ ii
Abstract................................................................................................ iii
目錄....................................................................................................... iv
表目錄................................................................................................... vi
圖目錄.................................................................................................. vii
附錄....................................................................................................... ix
第一章 前言 ..........................................................................................1
1. 海洋多樣性與珊瑚礁生態..............................................................................1
2. 刺細胞動物的演化與粒線體基因組之特性..................................................3
3. 脊索動物的演化與粒線體基因組類緣關係研究..........................................5
4. 粒線體DNA 之特性與研究...........................................................................8
5. 研究目的........................................................................................................ 11
第二章 材料與方法 ............................................................................13
1. 樣品取得與保存............................................................................................13
2. DNA 的萃取..................................................................................................13
3. RNA 的萃取..................................................................................................16
4. PCR 擴增程式................................................................................................17
5. RT-PCR 反應.................................................................................................22
6. PCR 產物純化................................................................................................22
7. cloning ............................................................................................................23
8. 純化質體........................................................................................................25
9. 序列分析及系統重建....................................................................................25
第三章結果與討論 ............................................................................27
一、海葵 Mesactinia genesis 及Heteractis aurora 完整粒線體基因組分析
.............................................................................................................................27
1. 粒線體基因大小及排列................................................................................27
2. 基因的重新排序及introns...........................................................................28
3. tRNA 基因.....................................................................................................30
4. 編碼蛋白基因、密碼子特點........................................................................30
5. 系統演化樹分析............................................................................................31
6. 海葵粒線體基因組未知功能之ORF ..........................................................32
7. 非密碼序列及控制區分析............................................................................35
二、海鞘 Eudistoma gilboviride 完整粒線體基因組分析 .............................37
1. 粒線體基因大小及排列................................................................................37
2. 鹼基組成........................................................................................................38
3. 基因排序比較................................................................................................39
4. 編碼蛋白基因、密碼子特點........................................................................41
5. 非密碼區........................................................................................................42
6. tRNA 基因.....................................................................................................43
7. rRNA 基因.....................................................................................................44
8. Cytochome b ...................................................................................................46
9. F0-ATPase subunit 8......................................................................................46
10. NADH dehydrogenase subunit 6................................................................48
11. 系統演化樹分析..........................................................................................50
第四章 結論 ........................................................................................52
參考文獻...............................................................................................57
表目錄
表一、Mesactinia genesis, Heteractis aurora PCR 定序分析增幅短片段使用的
引子.............................................................................................................74
表二、Mesactinia genesis, Heteractis aurora PCR 定序分析增幅長片段使用的
引子.............................................................................................................75
表三、Eudistoma gilboviride PCR 定序分析增幅短片段使用的引子.............76
表四、Eudistoma gilboviride PCR 定序分析增幅長片段使用的引子.............77
表五、RT-PCR 定序分析增幅片段使用的引子及溫度....................................78
表六、海葵 M.genesis, H. aurora, M. senile 及Nematostella sp. 完整粒線體基
因組鹼基組成.............................................................................................79
表七、M. senile, M.genesis, H. aurora 粒線體基因之核甘酸組成成分表.......80
表八、海葵粒線體蛋白基因起始碼及終止碼的使用.........................................81
表九、未知功能 ORF 胺基酸序列相似度及核苷酸差異性分配......................82
表十、未知功能的 ORF1938 胺基酸3D 預測結果.........................................83
表十一、M. senile, M.genesis 及H. aurora 粒線體基因非密碼區域..............84
表十二、Eudistoma gilboiride 粒線體基因組蛋白質基因、rRNA 及非密碼區
長度及基因重疊區域.................................................................................85
表十三、被囊動物完整粒線體基因組鹼基組成.................................................86
表十四、被囊動物粒線體蛋白基因起始碼及終止碼的使用.............................87
表十五、被囊動物粒線體基因組非密碼區及最長非密碼區位置.....................88
表十六、核糖體 RNA 大小次單元之間的比較..................................................89
表十七、本研究使用物種及其 NCBI 代碼.........................................................90
圖目錄
圖一、珊瑚蟲綱依照粒線體基因組的結構區分演化地位.............................91
圖二、尾索動物不同時期的系統分類假說.....................................................92
圖三、根據 33 個哺乳類動物的粒線體基因分別將其保守度依顏色做分類
.............................................................................................................................93
圖四、本研究使用之海葵及海鞘生態照.........................................................94
圖五、RT-PCR 反應 primers 位置圖...........................................................95
圖六、華美中海葵粒線體基因的基因圖譜.....................................................96
圖七、唸珠海葵粒線體基因的基因圖譜.........................................................97
圖八、珊瑚蟲綱四個物種粒線體基因排序概要圖.........................................98
圖九、本研究中海葵轉譯基因之二級結構預測圖.........................................99
圖十、利用 UPGMA 法，粒線體13 個蛋白基因及2 個rRNA 基因在各物
種間完整基因組演化圖譜.......................................................................100
圖十一、利用鄰接法，粒線體13 個蛋白基因及2 個rRNA 基因在各物種間
完整基因組演化圖譜...............................................................................101
圖十二、利用鄰接法，粒線體13 個蛋白胺基酸序列在各物種間完整基因組
演化圖譜...................................................................................................102
圖十三、海葵 M. genesis、H.aurora 及Calliactis sp.之ORF 鹼積極胺基酸
序列排序。...............................................................................................103
圖十四、H. aurora RNA 與DNA 序列比較................................................105
圖十五、利用 TMHMM 預測海葵Mesactinia genesis 未知功能蛋白
ORF777 穿膜區域..................................................................................106
圖十六、利用BioEdit 預測海葵Mesactinia genesis 未知功能蛋白 ORF777
疏水性區域...............................................................................................107
圖十七、利用 TMHMM 預測海葵Heteractis aurora 未知功能蛋白
ORF1938 穿膜區域................................................................................108
圖十八、利用 BioEdit 預測海葵Heteractis aurora 未知功能蛋白 ORF1938
疏水性區域...............................................................................................109
圖十九、利用 TMHMM 預測海葵Calliactis sp. 未知功能蛋白 ORF807 穿
膜區域....................................................................................................... 110
圖二十、利用 BioEdit 預測海葵Calliactis sp. 未知功能蛋白 ORF807 疏水
性區域....................................................................................................... 111
圖二一、未知功能 ORF1938 胺基酸序列2D 預測結果........................... 112
圖二二、M. senile 和 M. genesis 可能控制區排序及 stem-loop 結構..... 114
圖二三、Eudistoma gilboiride 完整粒線體基因圖譜 .................................. 115
圖二四、Eudistoma gilboiride 轉譯基因之二級結構預測圖 ...................... 116
圖二五、海鞘粒線體基因排序比較分析.......................................................121
圖二六、E. gilboviride 粒線體基因組內疑似控制區之候選區域stem-loop 次
級結構.......................................................................................................122
圖二七、核糖體 RNA 大小兩次單元保守區排序.......................................123
圖二八、cytochrome b 蛋白質C-terminal 排序........................................124
圖二九、酵母菌、人類、果蠅、刺細胞、半索及囊被動物的 ATP8 完整蛋
白和海鞘的不完整ATP8 蛋白排序......................................................125
圖三十、利用最小進化法，粒線體 13 個蛋白基因及2 個rRNA 基因在各
物種間完整基因組演化圖譜...................................................................126
圖三一、利用鄰接法，粒線體 13 個蛋白基因及2 個rRNA 基因在各物種
間完整基因組演化圖譜...........................................................................127
圖三二、利用最小進化法，粒線體COX1 蛋白繪出各後生動物在演化圖譜
中的位置...................................................................................................128
圖三三、利用最小進化法，粒線體13 個編碼蛋白繪出各後生動物在演化圖
譜中的位置...............................................................................................129

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