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研究生:徐哲雄
研究生(外文):Che-Hsiung Hsu
論文名稱:綠鬣蜥之乳酸脫氫酶基因與蛋白質結構研究及爬蟲類之分子親緣關係
論文名稱(外文):Studies on L-Lactate dehydrogenase genes and protein structure of Iguana iguana and molecular phylogenetics among reptiles
指導教授:李水龍李水龍引用關係
指導教授(外文):Steven Shoei-Lung Li
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:101
中文關鍵詞:綠鬣蜥蛋白質序列演化乳酸脫氫酶爬蟲類
外文關鍵詞:protein sequencesevolutioniguanareptileLDH
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L-Lactate dehydrogenase (乳酸脫氫酶;LDH)為一管家基因 (house keeping gene)所轉譯出來的蛋白質,其同功異構酶 (isozyme)已知是一種穩定、演化緩慢而適合判定物種間親緣關係的分子標記。現已將綠鬣蜥(Iguana iguana) LDH-A4 (muscle) 與 LDH-B4 (heart) cDNA序列定序出;且澱粉凝膠電泳分析證實,在綠鬣蜥LDH-B具有兩種形式的同功異構酶,命名為LDH-B與LDH-b。建構LDH-B與LDH-b蛋白質單體結構模型後發現,除了五個胺基酸殘基 (residue)之外,其餘結構均相同。將綠鬣蜥與其餘已知LDH基因與蛋白質序列的脊椎動物,藉由Mega2與Phylip軟體建構其分子親緣關係樹。結果發現在蜥蜴的親緣關係中,根據傳統分類屬於不同屬的綠鬣蜥與Sceloporus woodi較屬於同屬的Sceloporus undulatus與Sceloporus woodi有較近的親緣關係。在演化分歧時間方面,Sceloporus undulatus與綠鬣蜥在188百萬年以前分歧;而綠鬣蜥與Sceloporus woodi則在98百萬年之前分歧。此外,分析這三種蜥蜴的粒線體DNA(12S、16S與ND1)發現,其親緣關係支持傳統動物分類的結果,同屬的物種較不同屬的物種有較近的親緣關係。因此,這兩種不同的結果未來仍須更進一步的確定。


L-Lactate dehydrogenases (LDH) are “house keeping” enzymes. The LDH isozymes are known to be a very stable, slow-evolving and suitable to be a model to elucidate the phylogenetic relationships among various species. The cDNA sequences of LDH-A4 (muscle) and LDH-B4 (heart) from green iguana (Iguana iguana) were determined. The results of isozyme electrophoresis demonstrated that there are two isoforms of Iguana iguana LDH-B isozyme and called LDH-B and LDH-b. The protein structures of LDH-B and LDH-b monomer were constructed, and these two proteins have the same structure except the 5 different amino acids. The phylogenetic relationships between green iguana and other vertebrates, whose LDH cDNA sequences published previously, were analyzed by phylogenetic tree construction methods Mega2 program as well as Phylip program. These results on the relationships among lizards indicate that Iguana iguana is closer to Sceloporus woodi than to Sceloporus undulates. The divergent times between Iguana iguana and Sceloporus woodi, Iguana iguana and Sceloporus undulatus were estimated about 98 and 188 million years, respectively. The sequences of mitochondrial DNA(12S, 16S and ND1) among these three lizards were also analyzed, and the results were consistent with the traditional phylogenetics that species in the same genus were closer. The unexpected relationship that different genus of Iguana iguana and Sceloporus woodi is closer than that within same genus of Sceloporus woodi and Sceloporus undulates by analyzing among vertebrates LDH isozymes remained to be further confirmed.


目錄(Contents)
中文摘要………………………………………………………… I
英文摘要………………………………………………………… II
壹、緒論………………………………………………………… 1
一、爬蟲綱簡介…………………………………………… 1
二、蜥蜴亞目簡介………………………………………… 2
三、喙頭目簡介…………………………………………… 3
四、松果體簡介…………………………………………… 3
五、乳酸脫氫酶簡介……………………………………… 4
六、脊椎動物乳酸脫氫酶基因及蛋白質結構簡介……… 4
七、喙頭目與蜥蜴亞目之分子演化及親緣關係………… 5
貳、材料與方法………………………………………………… 8
一、動物組織樣本………………………………………… 8
二、同功異構酶電泳分析………………………………… 8
三、RNA萃取……………………………………………… 9
四、單股cDNA製備………………………………………. 10
五、引子設計與合成……………………………………… 11
六、聚合酶連鎖反應……………………………………… 12
七、非蛋白質轉譯區序列分析…………………………… 13
八、PCR產物純化…………………………………………. 16
九、勝任細胞製備………………………………………… 17
十、cDNA選殖……………………………………………. 17
十一、自動核酸序列定序………………………………… 18
十二、基因相關性之比對及分子親緣關係樹之建立…... 21
參、結果………………………………………………………… 23
一、綠鬣蜥LDH-A與LDH-B同功異構酶電泳分析……… 23
二、綠鬣蜥LDH-A與LDH-B序列定序分析……………… 23
三、綠鬣蜥LDH-B的基因多樣性…………………………. 25
四、爬蟲類之乳酸脫氫酶分子親緣樹建立 (Mega2)……... 26
五、爬蟲類之乳酸脫氫酶分子親緣樹建立 (Phylip)……… 28
六、蜥蜴之粒腺體基因分子親緣樹建立………………… 29
七、爬蟲類分歧時間點的推測…………………………… 29
八、LDH-B與LDH-b之蛋白質結構電腦模擬……………. 30
肆、討論………………………………………………………… 32
一、綠鬣蜥乳酸脫氫酶異構型及胺基酸序列探討……… 32
二、綠鬣蜥乳酸脫氫酶(LDH-B、LDH-b)蛋白質結構探討…. 33
三、綠鬣蜥乳酸脫氫酶(LDH-B、LDH-b)基因多樣性對
蛋白質結構影響之探討………………………………. 34
四、蜥蜴亞目之分子親緣關係與演化……………………. 36
五、參考文獻…………………………………………………… 39
表…………………………………………………………………. 45
表一、傳統爬蟲類分類表………………………………….. 45
表二、綠鬣蜥LDH-A cDNA序列與其他物種相似度比較表 46
表三、綠鬣蜥LDH-A胺基酸序列與其他物種相似度比較表 47
表四、綠鬣蜥LDH-B cDNA序列與其他物種相似度比較表 48
表五、綠鬣蜥LDH-B胺基酸序列與其他物種相似度比較表 49
表六、綠鬣蜥LDH-B基因多樣性及胺基酸比較………… 50
表七、綠鬣蜥LDH-b cDNA序列與其他物種相似度比較 51
表八、綠鬣蜥LDH-b胺基酸序列與其他物種相似度比較表 52
表九、綠鬣LDH-B與LDH-b分子量、等電點與帶電荷基團
比較……………………………………………………..53
表十、綠鬣蜥LDH-B、LDH-b與caiman LDH-B胺基酸變異表 54
表十一、綠鬣蜥(iguana)LDH-B/LDH-b胺基酸屬性比較表… 55
表十二、爬蟲類乳酸脫氫酶功能性胺基酸比較表………… 56
圖…………………………………………………………………. 57
圖一、顱孔的數目、位置及其代表的分類……………….. 57
圖二、綠鬣蜥外貌圖……………………………………….. 58
圖三、喙頭目僅存物種-紐西蘭鱷蜥(tuatara)外觀圖………. 59
圖四、鼠LDH-A的exon、intron、氨基酸序列
及功能區對照圖……………………………………. 60
圖五、爬蟲類之傳統親緣關係樹………………………….. 61
圖六、爬蟲類之分子親緣關係樹………………………….. 62
圖七、綠鬣蜥LDH-A/LDH-B同功異構酶電泳分析圖…….. 63
圖八、綠鬣蜥LDH-A ---- cDNA序列與胺基酸序列……….. 64
圖九、綠鬣蜥LDH-B ---- cDNA序列與胺基酸序列……….. 65
圖十、綠鬣蜥LDH-b ---- cDNA序列與胺基酸序列………. 66
圖十一、利用LDH-A與LDH-B序列,去除前60個
高度變異區核酸配合Mega2軟體所建構之
NJ、ME親緣關係樹……………………………….. 67
圖十二、利用LDH-A與LDH-B序列,去除前60個
高度變異區核酸配合Mega2軟體所建構之
MP、ML親緣關係樹………………………………… 68
圖十三、利用LDH-A與LDH-B胺基酸序列,去除前
20個高度變異區胺基酸配合Mega2軟體所
建構之MP、ML親緣關係樹………………………… 69
圖十四、利用LDH-A與LDH-B胺基酸序列,配合Phylip軟體個別運算所建構之NJ親緣關係樹……………………. 70
圖十五、利用LDH-A與LDH-B胺基酸序列,配合Phylip軟體個別運算所建構之MP親緣關係樹…………………… 71
圖十六、利用LDH-A與LDH-B胺基酸序列,去除前
20個高度變異區胺基酸配合Phylip軟體所建
構之NJ親緣關係樹…………………………..……….. 72
圖十七、利用LDH-A與LDH-B胺基酸序列,去除前
20個高度變異區胺基酸配合Phylip軟體所建構
之MP親緣關係樹……………………………..………... 73
圖十八、利用粒線體12S、16S以及DN1核酸序列,
配合Mega2軟體所建構之NJ、ME、MP以
及ML親緣關係樹…………….……………………...... 74
圖十九、爬蟲類演化時間推測………………………………… 75
圖二十、蜥蜴之演化時間推測………………………………… 76
圖二十一、綠鬣蜥LDH-B、LDH-b單體結構…………………… 77
圖二十二、綠鬣蜥LDH-B、LDH-b蛋白質
四聚體正面圖與側面圖……………………………… 78
圖二十三、以LDH-B、LDH-b移動距離分析綠鬣蜥LDH-A、
LDH-B同功異構酶電泳圖………………………. 89
圖二十四、爬蟲類與其他脊椎動物LDH胺基酸序列排比…… 80
圖二十五、LDH-B單體與另一單體結合圖……….…………… 83
圖二十六、LDH-B單體第216、262胺基酸Asp、Lys,與其他
物種該兩處胺基酸Asn、Glu位置及側鍊比較……….… 84
圖二十七、LDH-B與LDH-b蛋白質單體模型重疊………..…… 85
圖二十八、LDH-B與LDH-b第12、17、52、53、308胺基酸
側鍊比較……………………………………………. 86
圖二十九、LDH-B與LDH-b蛋白質單體模第52、53胺基酸側鍊與NAD相對位置………………………………………. 88
圖三十、LDH-B單體與另一單體結合時第12、17胺基酸
及其側鍊與另一單體第302個胺基酸及側鍊的
相互位置…………………………………………… 89
圖三十一、LDH-b單體與另一單體結合時第12、17胺基酸
及其側鍊與另一單體第302個胺基酸及側鍊的
相互位置………………………………………… 91
圖三十二、鬣蜥科(Iguanidae)所包含的8種亞科……………… 93


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