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Title: 腎形真葉珊瑚 Piwi 基因之表現
Molecular cloning and characterization of Piwi gene as a marker for stem cells in the scleractinian coral, Euphyllia ancora
Authors: Hsiang-Ming Wang
Contributors: NTOU:Department of Aquaculture
Keywords: 腎形真葉珊瑚;幹細胞;生殖細胞;Piwi
corals (Euphyllia ancora);stem cells;germ cells;piwi
Date: 2013
Issue Date: 2013-10-07T02:43:03Z
Abstract: 珊瑚同時具有有性及無性生殖系統;在其他物種之有性生殖系統之中,生殖細胞的形成需仰賴生殖幹細胞不斷的進行自我新生,同時利用不對稱分裂分化成原始生殖細胞才能使珊瑚每逢生殖季時產生許多配子進而繁衍後代,而無性生殖中多能型幹細胞分化成各種細胞類型組成另一複製個體,顯示出幹細胞對於兩種生殖模式為不可或缺之角色,而幹細胞系統也被認為存在於珊瑚體內,但目前仍未有文獻證明珊瑚之幹細胞。為此,利用已知在許多物種的幹細胞研究中之 Piwi 基因做為珊瑚生殖幹細胞之標誌基因。而目前已成功選殖出腎形真葉珊瑚之兩型 Piwi 基因。在其他物種之 Piwi 基因之包含兩個高度保守之 PAZ 及 PIWI domain,而其兩型基因經比對後發現位於 PAZ 及 PIWI domain 位置有一定程度的相似性,進一步證明此兩型基因確實為 Piwi 基因。之後萃取其 RNA 反轉錄出 cDNA 並利用半定量 RT-PCR 觀察發現 EaPiwi1 及EaPiwi2 mRNA 在全年皆有表現,且在觸手、腸系膜及生殖腺三種主要組織的半定量 RT-PCR 皆可觀察到 EaPiwi1 及 EaPiwi2 的表現,而在原位雜合染色中發現於雌株及雄株個體主要為生殖細胞表現EaPiwi1 mRNA,隨後為觀察 Piwi 蛋白表現之位置及細胞類型遂製備抗 EaPiwi1及抗 EaPiwi2 抗體。進行免疫組織染色時,於雌株發現 Piwi 蛋白主要表現於早期卵細胞,而直徑大於125微毫米的卵細胞僅出現微弱甚至沒有表現。而雄株主要初級精母細胞有明顯訊號,但次級精母細胞、精細胞及精子僅有微弱表現甚至沒有訊號出現。此外,於觸手及腸系膜也有其他表現 Piwi 蛋白的細胞類型被偵測出,而此結果與Piwi表現於後生動物的生殖細胞及多能型幹細胞的特徵相似,推測多能型幹細胞在此物種的可能性。
Coral species reproduce themselves in both sexually and asexually. Under these reproductive systems, multipotent stem cells, which have abilities to self-renew and differentiate into diverse specialized cell types including germ cells, are assumed to exist in their bodies; however, so far, little research has been conducted. To address this issue, we here identified and characterized the piwi gene homolog of a scleractinian coral, Euphyllia ancora, as a candidate stem cell marker. The full-length of E. ancora piwi (Eapiwi) cDNA was elucidated, and a specific antibody against the Eapiwi was successfully generated. Immunohistochemical analysis revealed that anti-Eapiwi immunoreactivity (irEapiwi) in the early oocyte, and developing oocyte, but only faint or undetectable reactivity in developing oocytes that were >125 μm in diameters. In males, irEapiwi1 and irEapiwi2 could be detected in the primary spermatocytes but was only faintly detectable in the secondary spermatocytes, spermatids, and sperms. Furthermore, small number of irEapiwi cells could also be detected in the tentacles and the mesenterial filaments in both sexes. These results demonstrated that Piwi is expressed not only in germ lineage but also in other type of cell lineage in the coral body. Because Piwi have been shown to be expressed specifically in germline and multipotent stem cell in various metazoan animals, raising a possibility that the irEapiwi cells observed in this study might contain the multipotent stem cells in the coral.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0010033027
Appears in Collections:[水產養殖學系] 博碩士論文

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