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標題: | 斑馬魚早期胚胎發育相關蛋白質之研究 Studies on Early Embryonic Development-Related Proteins in Zebrafish (Danio rerio) |
作者: | Hua-Shan Shih 施驊珊 |
指導教授: | 莊榮輝 |
關鍵字: | 母源-胚源轉換期,蛋白質體學,二維電泳,能量代謝,斑馬魚, Maternal-to-zygotic transition (MZT),Proteomics,Two-dimensional electrophoresis (2-DE),Energy metabolism,Zebrafish, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 動物早期胚胎發育過程中,母源-胚源轉換期 (maternal-to-zygotic transition, MZT),對於胚胎的正常發展扮演重要角色。本論文利用蛋白質二維電泳,分析斑馬魚 (Danio rerio) 胚胎MZT前後之關鍵調控蛋白質。取得班馬魚256-細胞期及球體期胚胎分別作為MZT前後之標準樣本,萃取此兩時期胚胎的整體蛋白質,進行二維電泳分析,觀察MZT前後之蛋白質體差異。於二維電泳圖譜中比對出17個表現差異之蛋白質,取其中差異最顯著之六個蛋白質色點,進行ESI-QUAD-TOF身份鑑定。以等電點及分子量比對後,鑑定出五種蛋白質,分別為:hypothetical protein LOC768289、mitochondrial processing peptidase alpha (MPP-alpha)、aldehyde dehydrogenase 2b (ALDH2b)、methylmalonate-semialdehyde dehydrogenase (MMSDH) 及ubiquitin C-terminal hydrolase L1 (UCHL1)。這些蛋白質可能和粒線體運輸、支鏈胺基酸代謝或細胞週期調控相關。此外,針對這幾個差異性蛋白質及lactate dehydrogenase (LDH) 上游mRNA,進行半定量RT-PCR,發現在MZT前後其mRNA消長與蛋白質一致,其中UCHL1、MPP-alpha及LDH在統計上具有顯著差異。為了瞭解這些關鍵蛋白質對MZT的影響,利用顯微注射技術,將LDH及UCHL1的專一性抑制劑:oxamate及LDN57 444,注射入斑馬魚胚胎,導致胚胎分別有細胞分裂延遲及體節縮短的現象。另外,測定LDH在MZT前後的酵素活性,在256-細胞期的LDH活性高於球體期者。這些差異性蛋白質可能和早期胚胎粒線體之能量代謝相關,未來可朝早期胚胎代謝體學的方向深入調查,可藉由專一性單株抗體、利用轉殖基因動物或RNA干擾等技術,進一步了解這些蛋白質調控胚胎發育之生理意義。 Maternal-to-zygotic transition (MZT) plays an important role in early embryogenesis. In this study, zebrafish embryos at stages of 256-cell and sphere were used as the standard samples for pre- and post-MZT. Totally, 17 proteins expressed differentially during MZT were picked out by two-dimensional electrophoresis (2-DE). Six of these proteins were identified as hypothetical protein LOC768289, mitochondrial processing peptidase alpha (MPP-alpha), aldehyde dehydrogenase 2b (ALDH2b), methylmalonate-semialdehyde dehydrogenase (MMSDH) and ubiquitin C-terminal hydrolase L1 (UCHL1) by using ESI-QUAD-TOF. These proteins might relate to mitochondrial protein import pathway, branched-chain amino acid metabolism, or cell cycle regulation. Furthermore, we detected the mRNA expression of MPP-alpha, ALDH2b, MMSDH, UCHL1 and LDH (lactate dehydrogenase) by semi quantitative RT-PCR. These results were comparable to 2-DE data. The expression differences of UCHL1, MPP-alpha and LDH during MZT were statistically significant. In an attempt to study the function of these proteins during MZT, we used the specific inhibitors, oxamate and LDN57 444, to inhibit LDH or UCHL1, respectively. The phenotypes of cleavage delay and dorsalis were observed in inhibitors treated embryos. Additionally, we found that LDH activity in 256-cell was higher than that in sphere by LDH activity assay. In conclusion, our results demonstrated that the change of the protein profile during MZT may relate to mitochondrial energy metabolism. These proteins can be further studied by using specific monoclonal antibody, transgenic animal model, or RNA interference. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41498 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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