水母是海洋中常見的大型浮游生物，為無脊椎動物之中的刺胞動物門的一員。目前已發現的水母共有兩百多種，分布與生存的棲息地相當廣泛，不管在鹹水或淡水，表層或深海，都可以發現水母的蹤跡。最近研究發現，水母的粒線體基因是呈線型，粒線體基因大部分是環型，只有真菌、植物、單細胞真核生物以及水母才具有線狀的粒線體基因。但是我們對於水母的生物多樣性的了解仍然相當少。因此本研究使用 PCR 與 Primer walking 的方式來把端鞭水母Acromitus flagellatus 的粒線體基因定序出來，此資料除了可以幫助充實水母的DNA 資料庫以外，也能夠以此序列進行分析，進一步了解水母各物種間的親緣關係。本研究之中所定出的端鞭水母粒線體基因長度為14631 鹼基，此長度並非粒線體基因全長。其中含有2個rRNA、2個tRNA和13個蛋白質基因。

Jellyfish are no-polypform of the Cnidaria. And also are typical free-swimming marine animals that has umbrella-liked bell and many tentacles.They can found in freshwater and ocean.
Recent research find that Jellyfish has linear mitochondrial DNA (mtDNA). Most mtDNA are circular molecule, but fungi, plants, unicellular eukaryotes, and jellyfish has the linear mtDNA. And many question are remain on the mtDNA in Jellyfish about transcription and evolution. To resolved these questions, this study use PCR and Primer walking to sequencing the mtDNA of Acromitus flagellatus. and 14631 bp sequence has been determined. The sequenced mtDNA containing the two rRNA, two tRNA, and 13 protein-coding genes. The genes of A. flagellates can separate in two clusters for transcription, and the transcription were proceeds to the ends of mtDNA.

謝辭 ..……………………………………………………………………………………i
摘要 ..………………………………………………………………………………...…ii
Abstract …………………………………………….…………………………..…..iii
目次 ..…………………………………………..………………………………………iv
圖次 ………………………………………………………………………………….vi
表次 …………………………………………………………………………………vii

一、前言 ………………………………………………………………………….…….1
1. 水母之定義及分類 ....................................................................................................1
1-1.解剖學 ...................................................................................................................1
1-2.分類學 ………………………………………………………………...................1
1-3.生態 ……………………………………………...................................................2
2. 粒線體DNA特性 ......................................................................................................3
3. 水母粒線體基因 ........................................................................................................4
4. 實驗目的 ....................................................................................................................5
二、材料與方法 ………………………………………………………………….…….6
1.樣品取得及保存 ……………………………………………………………..............6
2.水母DNA萃取 ……………………………………………………………………….6
2-1.Blood & Tissue DNA Extraction Kit (Viogene) 萃取法 …………………..........6
2-2.Phenol/chloroform DNA 萃取法 ……………………………………….............7
3.水母粒線體DNA短片段増幅 ………………………………………………...……..7
4.長片段DNA增幅定序 ………………….……………………………………….…...8
5.粒線體基因末端増幅定序 ………………………………………………………......8
6.粒線體DNA分析 …………………………………………………………………….9
三、結果 ............………………………………………………………………….……11
1.水母粒線體基因體組成以及大小、特性 …………………………………………..11
2.基因排序及間隔 …………………………………………………………………....11
3.蛋白質基因 ………………………………………………………………................12
4.基因鹼基組成及密碼子的使用 ……………………………………………………12
5.tRNA …………………………………………………………………………………13
6.親緣關係分析 …………………………………………………………………..….13
四、討論 ………………………………………………………………………………15
1.本次研究主要成果 ……………………………………………………………........15
2.水母間親緣關係 …………………………………………………………………....16
五、參考文獻 .............................................................................................................17
六、附錄 ...............................................................................................................41
附錄一、水母各綱之代表物種粒線體基因序列經Neighbor-Joining法分析後之結果 ...................................................................................................................41
附錄二、端鞭水母Acromitus flagellates 之外觀圖 ..................................................42
附錄三、RACE 法進行PCR步驟簡圖 .....................................................................43
附錄四、Acromitus flagellatus、Carybdea rastonii、Mastigias papua、Craspedacusta sowerbyi以及Phyllorhiza punctata 之16s基因序列比對 ..........................44
圖一、端鞭水母Acromitus flagellates粒線體基因排序..............................................21
圖二、本研究中所定出的端鞭水母粒線體基因序列 ...............................................22
圖三、端鞭水母與海月水母之tRNA-Met及tRNA- Trp的二級結構之比較 ........30
圖四、使用Neighbor-Joining法分析粒線體16S基因之DNA序列後所得的水母親緣關係圖 ...........................................................................................................31
圖五、使用Neighbor-Joining法分析粒線體cox1基因之DNA序列後所得的水母親緣關係圖 ...........................................................................................................32
圖六、使用Neighbor-Joining法分析粒線體cox1基因之胺基酸序列後所得的水母親緣關係圖 ...........................................................................................................33
圖七、使用Neighbor-Joining法分析粒線體cox2基因之DNA序列後所得的水母親緣關係圖 ...........................................................................................................34
圖八、使用Neighbor-Joining法分析粒線體cox2基因之胺基酸序列後所得的水母親緣關係圖 ...........................................................................................................35
圖九、使用Neighbor-Joining法分析粒線體12S基因之DNA序列後所得的水母親緣關係圖 ...........................................................................................................36
圖十、使用Neighbor-Joining法分析粒線體nad1基因之DNA序列後所得的水母親緣關係圖 ...........................................................................................................37
圖十一、使用Neighbor-Joining法分析粒線體nad1基因之胺基酸序列後所得的水母親緣關係圖 ...................................................................................................38
圖十二、使用Neighbor-Joining法分析粒線體nad4基因之DNA序列後所得的水母親緣關係圖 .......................................................................................................39
圖十三、使用Neighbor-Joining法分析粒線體nad4基因之胺基酸序列後所得的水母親緣關係圖 ...................................................................................................40
表一、端鞭水母Acromitus flagellates 粒線體基因位置及特性 …………………21
表二、六種水母之間的基因長度比較 ................................................................29

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