臺灣博碩士論文加值系統

本論文利用掃描式電子顯微鏡觀察點帶石斑(Epinephelus coioides)和馬拉巴石斑 (E. malabaricus)卵表面微細構造包括卵門區域（micropyle region）及卵表壁孔（pore）構造；進一步利用基因選殖方法選殖馬拉巴石斑及點帶石斑的ZP3基因部分片段，並嘗試以5’和3’ Race基因選殖技術，完成此兩種石斑ZP3基因cDNA完整序列，並進一步利用生物資訊方法探討此ZP3蛋白質結構。
掃描式電子顯微鏡觀察石斑魚卵外觀全貌、卵門及卵表微細構造。點帶石斑魚卵呈圓形，直徑約642 μm，卵門孔徑約6.7 μm略呈圓形漏斗狀，壁孔呈菱形分布，分布密度約為54個/100 μm2。馬拉巴石斑魚卵呈圓形，直徑約680 μm，卵門孔徑約7.4 μm略呈圓形漏斗狀，壁孔呈不規則分布,分布密度約為67個/100 μm2。
點帶石斑ZP3基因片段（422 bp）可轉譯成140個胺基酸，馬拉巴石斑ZP3基因片段（319 bp）可轉譯成106個胺基酸。醣化位置在點帶石斑及青魚將 此段序列是缺乏的，在馬拉巴石斑有一個位置其胺基酸組合為NCSS（天門冬醯胺、半胱胺酸、絲胺酸），其他已發表魚類也有都具有一個位置，這些醣化位置的胺基酸組合都是由NVSS（天門冬醯胺、纈胺酸、絲胺酸）構成。
石斑魚ZP3胺基酸序列中也具有與其他魚種相同的一些蛋白質活性位置，例如：蛋白質激酶C磷酸化位置、酪胺酸激酶磷酸化位置、酪蛋白激酶II磷酸化位置、半胱胺酸數目…等。
另外，丙胺酸、纈胺酸、白胺酸、異白胺酸..等這些疏水性胺基酸皆存在於石斑魚ZP3胺基酸序列中，點帶石斑具有41個，佔其序列29.71％、馬拉巴石斑有23個，佔21.7％。再從胺基酸序列的相似度百分比來看，點帶石斑與馬拉巴石斑有較高的相同性，此可印證以外部型態所做的親緣關係。然而，調出ZP3全部基因核苷酸序列乃是進一步探討親緣關係所需的重要工作。

Microstructures of micropyle regions and pores on the egg surface of the groupers including Epinephelus coioides and E. malabaricus were observed by using electron microscopy. Sperm receptor ZP3 gene were cloned with polymerase chain reaction and 5’ and 3’ Race method. Furthermore, with the bioinformation method the protein structure of ZP3 was explored.
By using SEM to observe the whole egg E. coioides eggs were spherical with a diameter of 642 μm. The micropyle was funnel and it’s diameter was ca. 6.7 μm. The pore possessed a rhombus arrangement over egg surface and the density of the pore was about 54 per 100 μm2. E. malabaricus eggs were spherical with a diameter of ca. 680 μm. The micropyle was funnel and it’s diameter was ca. 7.4 μm. The pore distributed irregularly over egg surface and the density of the pore was ca. 67 per 100 μm2.
A segment of ZP3 gene of E. coioides was cloned to contain
422 bp nucleotides and translated to the 140 amino acids. A segment of ZP3 gene of E. malabaricus was cloned to contain 319 bp nucleotides and translated to the 106 amino acids. The glycosylation was not appeared in the ZP3 gene segment of E. coioides as well as Oryzias latipes. The glycosylation site of E. malabaricus ZP3 was composed of aspargine, cysteine and serine. In other fishes as carpio.…the glycosylation site was composed of aspargine, valine and serine.
The amino acid sequence of ZP3 gene of the groupers as other fishes had the active sites such as protein kinase C phosphorylation, tyrosine kinase phosphorylation, and casein kinase II phosphorylation and were with 8-13 cysteines.
The hydrophobic amino acids as alanine, valine, leucine and isoleucine were found in the ZP3 segments of the two groupers. The ZP3 of E. coioides and E. malabaricus had 41(29.7%) and 23(21.7%) hydrophobic amino acids, respectively. E. coioides and E. malabaricus were 13-19 % and 11-15 %, respectively same in the ZP3 amino acids as other fishes. However, the two groupers were ca. 50 % same in the ZP3 amino acids each other. The congeneric species of E. coioides and E. malabaricus had a closer phylogenetic relationship each other than with other generic fishes. The results agreed with the phylogentic relationship obtained from morphological data. However, the whole sequence of ZP3 needed to be cloned for phylogenetic inference.

中文摘要 …………………………………………………… i
英文摘要 ……………………………………………………… iii
第一章 前 言 …………………………………………… 1
一、文獻回顧 …………………………………………… 2
二、研究目的 ………………………………………… 12
第二章 材料與方法 ……………………………………… 13
一、實驗架構 …………………………………………… 13
二、材 料 …………………………………………… 14
三、方 法 …………………………………………… 15
第三章 結 果 …………………………………………… 23
第四章 討 論 …………………………………………… 29
第五章 結 論 …………………………………………… 34
第六章參考文獻 …………………………………………… 35
表 …………………………………………………………… 41
圖 …………………………………………………………… 47
附錄 ………………………………………………………… 86

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