臺灣博碩士論文加值系統

小鼠儲精囊液蛋白SVS I 與雙胺類氧化酶(DAO)在一級結構上具有56%的相似度，且兩者的基因Svs I與Abp1皆位於小鼠染色體相鄰的位置上。進一步基因組序列分析顯示小鼠染色體6B2.3區段內，Abp1-like genes具有分化叢集的現象。大鼠染色體4q23-4q24區段內的的Abp1-like genes也有相同的現象，但人類相對應的染色體7q36區段內僅有Abp1 的存在。演化樹的分析中顯示，Abp1-like genes是由共同的祖先所分化出來的。
小鼠的Abp1-like genes共含有四個完整的基因：Abp1、Svs I、Loc243375和Loc243376。其中Abp1主要表現在小鼠的子宮，而Svs I、Loc243375和Loc243376主要表現在雄性生殖道中。Abp1在子宮中的表現受到了動情素的調控。在儲精囊中，Svs I的表現量則超過Loc243375和Loc243376的一千倍。
進一步的研究而知DAO表現於小鼠子宮的腔內上皮細胞及腺體上皮細胞內。子宮液中的DAO以雙硫鍵的方式結合形成homodimer。在子宮正常週期中，動情期有最多的DAO表現，同時也具有最高量的DAO酵素活性。
SVS I蛋白大量表現於儲精囊，但儲精囊液卻不具有DAO酵素活性。顯示演化後的SVS I已失去DAO酵素活性。SVS I以雙硫鍵與儲精囊液中其他結構性蛋白結合，形成高分子量聚合物。此雙硫鍵的鍵結可增加SVS I 做為轉麩醯胺酶受質的能力。本論文證明了雙胺類氧化酶基因家族在小鼠中的分化產生了不同功能的基因以符合生理上的需求。

Four Abp1-like genes, including Abp1、Svs I and two hypothetic genes Loc243375、Loc243376 were identified in mouse chromosome 6B2.3. A phylogenic analysis suggested that these genes may evolve from an ancestral gene. The results of RT-PCR with cDNA from sexual glands revealed the exclusive expression of Abp1 in female and SvsI、Loc243375、Loc243376 in male, in particular seminal vesicle. Further, Loc243375 and Loc243376 were slightly expressed while Svs I was greatly expressed in seminal vesicle. The uterine expression of Abp1 was correlated with the level of ovarian steroid hormones during estrous cycle.
Mouse SVS I in seminal vesicle and amiloride binding protein (Abp) in uterus share 56 % identity in their primary structure. We prepared the antibody specific to each of these two proteins. Abp was immunolocalized in the luminal epithelium and glandular epithelium of uterus. This in line with the immunodetection of Abp in the uterine fluid demonstrated the secretion of Abp from the uterine epithelium. The result of diagonal electrophoresis manifested the homodimmer formation of Abp by disulfide linkage. Abp showed diamine oxidase (DAO) enzyme activity. Both the protein level and the enzyme activity reached climax in estrus stage during estrous cycle. On the other hand, SVS I showed no DAO activity. Instead, SVS I forms complexes with other components in seminal vesicle fluid by disulfide bond. SVS I may become a better substrate of transglutaminase as a result of the intermolecular disulfide bonds among SVS I、SVS II、and SVS III in the mouse seminal vesicle secretion. Apparently, duplication of the Abp1-like genes results in various functions in order to meet the physiological requirement.

縮寫表 V
摘要 VI
Abstract VII
第一章 緒論
1.1 生殖系統概述 1
1.1.2 雄性生殖系統 1
1.1.3 雌性生殖系統 3
1.2 多胺polyamines
1.2.1. Polyamines的基本性質 6
1.2.2. Polyamines的合成與代謝 6
1.2.3. Polyamine的生理功能 8
1.3 雙胺氧化酶
1.3.1. Copper-containing amine oxidase 9
1.3.2. Diamine Oxidase 10
1.4 論文研究緣起及方向 13
第二章 材料與方法
2.1 RNA的萃取 14
2.2 RNA品質的檢定 15
2.3 反轉錄聚合酶連鎖反應 17
2.4 即時聚合酶連鎖反應 18
2.5 洋菜膠體電泳 19
2.6 DNA片段溶離 20
2.7 DNA接合反應 (Ligation) 21
2.8 質體轉型 (Transformation) 22
2.9 小量質體抽取 23
2.10 限制酶反應 24
2.11 SDS-聚丙烯醯胺膠體電泳
25
2.12 Coomassie Blue G-250 染色 26
2.13 西方墨點法 27
2.14 免疫組織化學染色法 28
2.15 雙胺類氧化酶酵素活性 31
第三章 實驗結果
3.1 Abp1-like genes的基因結構 32
3.2 Abp1-like genes的演化樹分析 33
3.3 Abp1-like genes在各組織中的表現 33
3.4 Abp1在子宮動情週期中的表現 34
3.5 Svs I、Loc243376和Loc243375在儲精囊中的表現 34
3.6 Diamine oxidase於子宮中的表現 34
3.7 子宮液對角線電泳分析 35
3.8 DAO在正常動情週期中的表現 35
3.9 Diamine oxidase assay 35
3.10 儲精囊液對角線電泳分析 36
3.11 麩醯胺酶交聯反應 36
第四章 討論與未來展望
4.1 Abp1-like genes的分化與表現 64
4.2 DAO在子宮中的表現與功能探討 64
4.3 Abp1-like genes在儲精囊液中的表現與功能探討 65
4.4 未來的研究方向 67
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