斑馬魚雙氫雙烴基去氫酶在胚胎發生過程當中之表現

Expression of Dimeric Dihydrodiol Dehydrogenase of Danio rerio during embryo devolopement

劉兆坤(Chao-Kun Liu)

指導教授 ： 劉德中

中山醫學大學/健康管理學院/營養學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00051

摘要

利用生物資訊資料庫NCBI (National Center for Biotechnology Information)中的搜尋比對工具BLAST (Basic Local Alignment Search Tool)，搜尋比對到一組相對應的斑馬魚(編號BC081393/ AAH81393)與人類(編號NM_014475/NP_055290) 雙氫雙烴基去氫酶。斑馬魚雙氫雙烴基去氫酶cDNA之開放閱讀框架(Open Reading Frame)含有1005個鹼基對，可轉譯成334個胺基酸，估計分子量36.740 kDa ;而人類已完全被放大出來，人類雙氫雙烴基去氫酶cDNA之開放閱讀框架含有1005個鹼基對，可轉譯成334個胺基酸，估計分子量36.740 kDa。
藉由pGEM-T表達質體系統，將斑馬魚雙氫雙烴基去氫酶表現出來。以此正意股的cDNA作為模版設計一條以DIG-UTP標定反意股的cDNA，為我們設計DIG-UTP標定的探針(probe)。接著，收集斑馬魚受精後24、48、72、96小時(hpf: hours post fertilization)的胚胎，和DIG-UTP標定的探針進行原位雜合(whole mount in situ hybridization)實驗，結果顯示在斑馬魚肝臟(liver)和腎管(pronephric ducts) 位置處有雜合反應的訊息，在24hpf胚胎無反應訊息。推測此新穎的酵素存在於48hpf、72hpf、96hpf斑馬魚胚胎的肝臟和腎管中，並且發現在更早期的斑馬魚胚胎（如: 24hpf；咽部期）中是不存在的。

關鍵字

NCBI、雙氫雙烴基去氫酶、原位雜交

並列摘要

In this study, the search tool BLAST (Basic Local Alignment Search Tool), based on NCBI (National Center for Biotechnology Information) was used against nucleotide and protein databases. The analysis found a Dimeric Dihydrodiol Dihydrogenase (dimeric DD) of zebrafish (NCBI accession number BC081393/ AAH81393) and human (NCBI accession number NM_014475/NP_055290). The dimeric DD cDNA of zebrafish and human were successfully amplified with a continuous open reading frame of 1005 bps and 1005 bps encoding a protein of 334 and 334 amino acids with a calculated molecular mass of 36.740 kDa and 36.740 kDa respectively.
Zebrafish dimeric DD was cloned with a pGEM-T vector. To design a probe, which cdna of a antisense with DIG-UTP. Then, Zebrafish embryo were collected 24 hpf (hpf: hours post ertilization)、48 hpf、72 hpf and 96 hpf . Whole mount in situ hybridization analysis showed that dimeric DD is expressed in liver and pronephric ducts of 48 hpf、72 hpf and 96 hpf of Zebrafish embryo, not in 24 hpf of Zebrafish embryo. Therefore, this novel Dimeric Dihydrodiol Dihydrogenase is expressed in liver and pronephric ducts of larval Zebrafish not in more early stage.

並列關鍵字

National Center for Biotechnology Information, Dimeric Dihydrodiol Dihydrogenase, whole mount in situ hybridization.

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