鱷目中屬於短吻鱷科的凱門鱷 (Caiman crocodilus apaporiensis)其LDH-A4 (muscle) and LDH-B4 (heart) 同功異構脢 cDNA序列已定序完成。 利用凱門鱷以及其他已知的脊椎動物LDH-A4 和 LDH-B4 cDNA 和胺基酸序列，配合ML、MP、ME以及NJ等演算方式建立分子親緣樹。結果再度證實龜系與鱷系屬於同宗，並且分支於有鱗類與鳥類趨異演化之後，並且再次將有鱗類置於爬蟲類親緣樹的基部。凱門鱷與短吻鱷的趨異演化 （divergence）時間為三千六百萬年前，而凱門鱷�短吻鱷系與龜鱉系的趨異演化時間為一億七千七百萬年前。

凱門鱷、北京鴨 (Anas platyrhynchos)，紅面番鴨 (Cairina moschata)以及家鵝 (Anser anser)之ε-crystallin/ Lactate dehydrogenase B cDNA序列已完成定序。結果再度證實這兩種蛋白質是經由同一個基因所轉譯出來的產品。並且更進一步證實在胺基酸序列上，除了北京鴨在115以及119的位置皆為Gly 之外，在其他物種中115以及119的位置皆分別為 Asn以及 Phe。這四個物種之ε-crystallin/ LDH-B 在經過蛋白活性測試與結構分析比較之後，並未發現北京鴨的ε-crystallin有任何異於其他的特性。因此推斷在LDH-B4 被徵募為水晶體蛋白的過程中，選擇性壓力是造成北京鴨ε-crystallin 在115以及119的位置皆突變為Gly的主因。

L-lactate dehydrogenase (LDH) cDNAs encoding for LDH-A4 (muscle) and LDH-B4 (heart) isozymes from caiman (Caiman crocodilus apaporiensis) belonging to the order Crocodilia were sequenced. The phylogenetic relationships of the newly determined cDNA and their deduced protein sequences, as well as the previously published sequences of vertebrate LDH isozymes were analyzed by various phylogenetic tree construction methods. These results indicated that Chelonia is indeed more closely related to Crocodilia. The divergent times between caiman and alligator, Chelonia and Crocodilia, were estimated to be approximately 36, 177 million years, respectively.

ε-crystallin/Lactate dehydrogenase B cDNA from caiman (Caiman crocodilus apaporiensis), Pekin duck (Anas platyrhynchos), Muscovy duck (Cairina moschata) and Greylag goose (Anser anser) eye lens were sequenced. Accorcding to cDNA sequences, duck lens ε–crystallin and heart LDH-B are the products of the same gene. In amino acid sequences, two residues Asn-114 and Phe-118 are well conserved inε-crystallin/ LDH-B among caiman, Muscovy duck and Greylag goose except in Pekin duck which are replaced by glycine residues. The lens protein composition, LDH activity andε-crystallin/ LDH B4 protein structure of caiman and three avians were analyzed and compared. The results show no significant differences in conformational or enzymatic properties between Pekin duck ε-crystallin and caiman, Muscovy duck and Greylag goose ε-crystallin. The unique replacement of both Asn-114 and Phe-118 by Gly residues in Pekin duck ε-crystallin amino acid sequence might therefore be due to the selective pressure during the recruitment processes of active enzyme into avian lensε-crystallins.

誌謝…………………………………………………………………..... 4
中文摘要……………………………………………………………..... 6
英文摘要……………………………………………………………..... 7
緒論………….……………………...…………………………………. 9
一， 爬蟲類簡介………………………………………………….. 9
二， 鱷目簡介……………………………………………………. 10
三， 乳酸脫氫脢簡介……………………………………………. 11
四， 鱷目分子親緣樹及演化……………………………………. 12
五， 水晶體蛋白簡介……………………………………………. 14
六， 乳酸脫氫脢水晶體蛋白之演化…………..………………... 15
材料與方法…………………………………………………………… 17
一， 動物樣本………………….…………………………….…... 17
二， 同功異構脢電泳分析……………..………………….….…. 17
三， RNA 萃取及單股 cDNA製備……………………….…… 18
四， 引子合成與聚合酵素連鎖反應 …………………………... 21
五， 非蛋白質轉譯區的序列分析………………………………. 23
六， PCR 產物的純化…………………………………………… 25
七， 勝任細胞製備.....................................................................… 27
八， cDNA選殖………………………………………………….. 27
九， 自動核酸序列定序…………………………………………. 29
十， SDS 聚丙烯硫脂凝膠電泳………………………………… 32
十一， 基因相關性之比對及演化樹之建立…………………… 33
十二， 蛋白質立體結構之電腦模擬…………………………… 33
結果……………………………………………………………………. 35
一， 凱門鱷 LDH-A 與LDH-B 同功異構脢電泳分析……… 35
二， 凱門鱷 LDH-A 與LDH-B cDNA序列定序…..………… 35
三， 鱷目之分子親緣樹建立…………………………………… 36
四， 鱷目演化時間的推測……………………………………… 37
五， 北京鴨以及家鵝 LDH-A 與LDH-B 同功異構脢電泳分析..37
六， 凱門鱷、北京鴨、紅面番鴨以及台灣家鵝ε-crystallin/Lactate dehydrogenase B 之獲得及定序………………………….. 38
七， 鳥類之分子親緣樹建立…………………………………… 39
八， ε-crystallin/Lactate dehydrogenase B 之蛋白純化、活性與結構分析…………………………………………………………… 40
討論與總結……………………………………………………………. 42
一， 鱷目之分子親緣樹及演化………………………………….. 42
二， 乳酸脫氫脢基因之演化…………………………………….. 43
參考文獻………………………………………………………………. 47
圖表……..……………………………………………………………...
表一，鳥類Synonymous/Nonsynonymous substitution rate 比較.. 54
表二，北京鴨，家鵝，凱門鱷LDH-B4/ε-crystallin之間氨基
.酸相似度 (identities)...交叉比較………………………. 55
圖一，傳統爬蟲類分類………………………………………… 56
圖二，顱孔數目以及位置分類………………………………… 57
圖三，鱷目的分類……………………………………………… 58
圖四，爬蟲類之傳統親緣樹…………………………………… 59
圖五，爬蟲類分子演化親緣樹………………………………… 60
圖六，凱門鱷 LDH-A與LDH-B同功異構脢電泳分析…….. 61
圖七，凱門鱷 LDH-A 完整轉錄區域序列…………………… 62
圖八，凱門鱷 LDH-B 完整轉錄區域序列…………………… 63
圖九a，LDH-A 胺基酸序列比對…………………………………… 64
圖九b，LDH-B 胺基酸序列比對……………………………………..65
圖十，NJ，ME 種緣重建演算法建立之分子親緣樹………………...66
圖十一，MP，ML 種緣重建演算法建立之分子親緣樹……………..67
圖十二，NJ，ME 種緣重建演算法(胺基酸序列)建立之分子親緣樹.68
圖十三，爬蟲類演化時間推測…………………………………………69
圖十四，北京鴨、鵝LDH-A與LDH-B同功異構脢電泳分析………70
圖十五，鵝ε-crystallin/LDH-B完整轉錄區域序列…………………..71
圖十六，紅面番鴨ε-crystallin/LDH-B部分轉錄區域序列…………..72
圖十七，ε-crystallin/LDH-B部分胺基酸序列比對…………………..73
圖十八，鳥類NJ種緣重建演算法建立之分子親緣樹………………..74
圖十九，凱門鱷、北京鴨、紅面番鴨、家鵝水晶體蛋白之SDS聚丙
烯硫脂凝膠電泳分析………………………………………….75
圖二十，電腦模凝凱門鱷ε-crystallin/LDH-B蛋白結構……………..76
圖二十一，電腦模凝家鵝ε-crystallin/LDH-B蛋白結構……………..77
圖二十二，凱門鱷、北京鴨、紅面番鴨、家鵝ε-crystallin/LDH-B 同
功異構脢電泳分析………………….…………………..... 78
圖二十三，爬蟲類 (鱷系與龜系) 之分子親緣樹………………….…79

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