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研究生:梁瓊之
研究生(外文):Cyong-Jhih
論文名稱:斑馬魚中cyclin-dependent kinase like 1(CDKL1)蛋白的選殖、表現模式及功能分析
論文名稱(外文):Identification, expression pattern and characterization of zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1)
指導教授:許立松
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:75
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Cyclin-dependant protein kinase (CDK) 家族在許多生物功能中扮演著重要的角色,例如:細胞週期的進行,RNA的轉錄,代謝,細胞凋亡及神經功能。此篇論文中,我們在斑馬魚中找到ㄧ個新的CDC2相關蛋白,稱為cyclin-dependent protein kinase like 1 (zCDKL1)。zCDKL1能轉錄350個胺基酸並具有X-Ser-Pro-X 區域, 此蛋白屬於proline-directed Serine/Threnine 激酶家族中的 KKIALRE。在胺基酸比對下,斑馬魚的CDKL1與其他物種有50%到78%的同源性。利用RT-PCR,發現在受精後0到48小時斑馬魚胚胎中皆可偵測到zCDKL1 mRNA 的表現,而其成魚表現的位置主要在腦、眼睛、睪丸和卵巢。另外用全胚體原位雜合技術觀察,則zCDKL1出現在受精後24到48小時胚胎中的 floor plate,背部神經節 和hypocord 。而僅在受精後24到36小時中,zCDKL1會表現在腎原管。在HEK293細胞中,zCDKL1 蛋白散佈在細胞核與細胞質。利用體外激酶活性測試(In vitro kinase assay),zCDKL1 蛋白具有磷酸化 髓鞘鹼性蛋白 (myelin basic protein) 和 組蛋白H1 (histone H1)的功能。藉由注射專一性的反義寡核酸將斑馬魚胚胎 CDKL1基因抑制,發現會造成胚胎中體節的突變,身體軸線的彎曲和尾巴變短等現象。而加入CDKL1 mRNA能具專一性修復注射CDKL1反義寡核酸所造成的傷害。這些結果證實,zCDKL1蛋白在斑馬魚的神經發育中,扮演著重要的角色。

Members of the cyclin-dependent kinase (Cdk) family play important regulatory roles in the control of cell-cycle progression, transcription metabolism, apoptosis, and neuronal function. In this study, we have isolated a novel protein kinase termed cyclin- dependent kinase like 1 in zebrafish (zCDKL1). zCDKL1 which is a Cdc2-related protein kinase, encoded polypeptide that is 350 amino acids with the X-Ser-Pro-X motif, and indicated that this protein belonged to the proline-directed Ser/Thr protein kinase family (KKIALRE). Based on amino acids sequence alignment, we have found that the zebrafish CDKL1 shared 50% to 77% homology to orthologs from other species. By reverse transcription-PCR analysis, zCDKL1 mRNA was expressed from fertilization to 48 hours post fertilization of zebrafish. Transcript of zCDKL1 was expressed in all adult tissues and enriched in brain, eyes, testes and ovary. In situ hybridization showed that CDKL1 was expressed in the floor plate, dorsal neuron tube, hypocord at 12 to 48 hours post fertilization whereas expression at pronephric tube was only found in 24 and 36 hour post feterlization. The zCDKL1 was diffusely expressed in nucleus and cytoplasm when overexpressed in HEK293 cells. In vitro kinase assay, the ability of zCDKL1 was toward phosphorylation of myelin basic protein and histone H1. Embryos injected with MOs directed against CDKL1 (cdkl1 MOs) showed developmental defects, including deformed somites, body axis curvature and a shortened trunk by 24hpf. These results suggested that CDKL1 may play a pivotal role in the development of ventral nervous system in zebrafish embryos.

目錄

壹、中英文摘要.............................. 1
貳、序論.....................................3
參、研究動機.................................9
肆、實驗方法與材料...........................10
伍、實驗結果.................................35
陸、討論.....................................41
柒、參考文獻.................................47
捌、圖表與圖表說明...........................53


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