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研究生:孫振傑
研究生(外文):Jhen-Jie Sun
論文名稱:斑馬魚camsap基因在胚胎及成體組織的基因表現
論文名稱(外文):Expression of zebrafish camsap genes in the embryos and adult tissues
指導教授:林文文林文文引用關係
指導教授(外文):Wen-Wen Lin
口試委員:鄭至玉 , 高孝偉
口試委員(外文):Chih-yu Cheng , Hsiao-Wei Kao
口試日期:2014-07-28
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:海洋生物技術研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:53
中文關鍵詞:斑馬魚基因表現
外文關鍵詞:zebrafishcamsap
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攜鈣素調控血影蛋白聯合蛋白(calmodulin regulated spectrin-associated protein, CAMSAP)是一種微小管負端的結合蛋白。在斑馬魚中有四個基因,分別是camsap1a、camsap1b、camsap2,以及camsap3。它們在蛋白質的C端都具有可以和微小管結合的CKK結構區域。動物細胞中的CAMSAP可以穩定並調節非中心體微小管(non-centrosomal microtubule)負端的解聚合及聚合作用。由於camsap在胚胎發育時期的表現與基因功能尚未明瞭,因此我們選擇斑馬魚作為研究動物,以反轉錄聚合酶連鎖反應與全覆式定位雜交,分析斑馬魚camsap在成魚組織與胚胎中的基因表現型式。我們最先比對了四個斑馬魚camsap cDNA及胺基酸序列,發現它們與哺乳類相似,在C端也具有保守的CKK結構區域,在蛋白質的中間區域則是多變異的。由成魚組織RT-PCR分析,我們發現四個基因的表現位置不盡相同:camsap1a表現在腦中;camsap1b在肌肉與精巢;camsap2在腦、肌肉、精巢與眼;camsap3在肌肉、卵巢及精巢。在不同時期的胚胎RT-PCR分析,camsap四個基因的表現則較為相似,幾乎都從一開始的卵裂期就有表現。由全覆式定位雜交的結果得知,所有的camsap基因都表現在最初的卵裂期、囊胚期及原腸期胚胎細胞中。然後到了體節期、咽鰓期及孵化期,逐漸不在整個胚體表現。camsap1a與camsap1b的表現位置相似,都在中腦、後腦、耳囊、視網膜、胸鰭、與卵黃融合細胞層(YSL)有表現,不同的是camsap1b在卵黃融合細胞核(YSN)與腸道的表現普遍比camsap1a明顯。camsap2與camsap3在胚胎上也是在中腦、後腦、耳囊、視網膜、胸鰭、與卵黃融合細胞層上表現得較明顯,但camsap3在視網膜和發育後期的卵黃融合細胞層表現較不明顯。
CAMSAP, calmodulin regulated spectrin-associated protein, is a microtubule minus-end binding protein. In zebrafish, there are four camsap genes, camsap1a, camsap1b, camsap2 and camsap3. Mammalian CAMSAPs stablilize and regulate the polymerization of minus-end of noncentrosomal microtubule via a conserved CKK domain in their C-termini. The expression profiles and functions of camsap genes during embryonic development are unclear. In this study, the spatiotemporal expression patterns of zebrafish camsap genes were studied. We analyzed the nucleotide and amino acid sequences of four zebrafish camsaps and found that they all contain the conserved CKK domain in their C-termini and a variable region in the middle of proteins. RT-PCR analyses of adult tissues showed that camsap1a is expressed in the brain, camsap1b expressed in the muscle and testis, camsap2 expressed in the brain, muscle, testis and eyes, and camsap3 expressed in the muscle, ovary and testis. RT-PCR analyses of embryos at various stages showed that all four camsap genes are maternally expressed and deposited in the embryos at cleavage and blastula stages. Whole-mount in situ hybridization further revealed that the transcripts of all four camsap genes are initially maternally deposited in the cleavage and blastula embryos. The zygotic expressions of camsap genes start to differentiate from gastrula to hatching stages. Though camsap1a and camsap1b are all expressed in the midbrain, hindbrain, otic vesicles, retina, pectoral fins and yolk syncytial layer, the expression of camsap1b in the yolk syncytial nuclei and gut primordium is weaker. camsap2 and camsap3 are also expressed in the midbrain, hindbrain, otic vesicles, pectoral fins, and yolk syncytial layer, but the expression of camsap3 is lower in the retina and yolk syncytial layer. 
中文摘要 ............................................................................................................................... 1
英文摘要 ............................................................................................................................... 2
前言 ...................................................................................................................................... 3
一、CAMSAP的蛋白質結構 ...................................................................................... 3
二、camsap基因家族成員的親緣關係圖 .................................................................. 5
三、斑馬魚作為實驗模式動物 ................................................................................... 8
四、斑馬魚胚胎發育 ................................................................................................... 9
五、微小管作為細胞骨架在細胞中的功能 ............................................................. 13
六、CAMSAP在細胞中與微小管的交互作用 ........................................................ 16
七、實驗目的 ............................................................................................................. 20
材料與方法 ......................................................................................................................... 21
一、實驗動物:斑馬魚成魚與胚胎 ......................................................................... 21
二、CAMSAP引子對設計 ........................................................................................ 21
三、反轉錄聚合酶鏈鎖反應 ( RT-PCR ) ................................................................. 22
萃取總核醣核酸 (total RNA extraction) ........................................................... 22
RT - PCR (reverse transcription)反應成cDNA .................................................. 22
四、核醣核酸探針的製備 ......................................................................................... 23
詴管內轉錄反應(in vitro transcription) 製作RNA探針 ................................. 23
五、全覆式定位雜交(whole-mount in situ hybridization ) ....................................... 24
六、轉型(transformation) ........................................................................................... 26
結果 .................................................................................................................................... 27
一、斑馬魚camsap的cDNA序列比對 .................................................................. 27
二、斑馬魚camsap的多重胺基酸序列比對(multiple amino acid sequence alignment) .................................................................................................................................... 27
三、RT-PCR分析camsap在斑馬魚成體的表現..................................................... 28
四、RT-PCR分析camsap在斑馬魚胚胎的表現..................................................... 28
五、全覆式原位雜交分析camsap在斑馬魚胚胎發育過程中的基因表現型式 ... 29
<一、camsap1a的表現> ................................................................................... 30
<二、camsap1b的表現> ................................................................................... 30
<三、camsap2的表現> ..................................................................................... 31
<四、camsap3的表現> ..................................................................................... 31
討論 .................................................................................................................................... 32
參考文獻 ............................................................................................................................. 37
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