- 學位論文
斑馬魚中Camodulin-dependent protein kinase II inhibitor蛋白的選殖、表現模式及功能分析
Cloning, expression pattern and biological function of zebrafish calcium/calmodulin dependent protein kinase II inhibitor
摘要
攜鈣素依賴蛋白激酶II (CaMKII)是調控細胞週期、神經發育與訊息傳遞的重要中樞蛋白，最近研究在大鼠與人類發現了內生性攜鈣素依賴蛋白激酶抑制胜肽(CaMKIIN)，CaMKIIN會與CaMKII專一性結合並抑制其活性，但目前為止CaMKIIN在發育及相關基因表現的角色在脊椎動物仍然尚未清楚。本實驗發現在斑馬魚存在兩個攜鈣素依賴蛋白激酶抑制胜肽(zcamkiin 1, zcamkiin 2)，在與人類及其他物種蛋白質進行胺基酸比對之下具有高達70%以上的相似度，其中對於抑制激酶最重要的27個保守抑制胺基酸序列是完全一樣的。在半定量PCR結果發現，兩個基因在五個體節前表現量較不明顯，隨著胚胎發育表現量有逐依上升的情況。在成魚組織方面，兩個camkiin基因都會表現在腦部與眼睛。透過原位雜合實驗發現，camkiin 1在受精後24小時會表現在整個中樞神經系統，其中端腦(telencephalon)訊號較強，隨著受精後36與48小時後腦的神經細胞也有表現。camkiin 2也是在胚胎腦部有表現，較不同的是中後腦部位訊號表現較高，後期時會分布在複雜的中樞神經系統中。利用綠色螢光蛋白融合蛋白(GFP-zcamkiin)發現細胞質與細胞核均有表現。體內(in vivo)與體外(in vitro)實驗我們亦證實zCaMKIIN會與zCaMKII進行結合，體外活性試驗(In vitro kinase assay) 顯示與camkiin結合會抑制camkii的活性。透過反義核苷酸將胚胎的CaMKIIN基因抑制結果發現，24小時授精後兩個基因均會造成中-後腦疆界區域(Midbrain -Hindbrain boundary, MHB)有發育不正常現象，而camkiin1基因抑制時，會造成中腦與後腦會隆起。將camkiin 2基因抑制時會導致胚胎腦部呈現扁形型態與眼睛變小。將兩個基因同時進行抑制時，則會呈現兩者各自的抑制形態。因此兩個基因在胚胎中樞神經系統可能參與各自不同的發育機制。利用原位雜合實驗，我們發現抑制camkiin 1基因表現會導致MHB發育不正常且減少neurogenin-1在腦部的表現，這些不正常可以藉由共同注射zCaMKIIN 1 mRNA而回復。相對而言，抑制zcamkiin 2基因不會改變有關於前腦、中腦及後腦的標誌的表現。綜合以上結果，在本研究我們首先分離出斑馬魚兩個camkiin基因並分析它們的表現模式，結果顯示此兩個camkiin基因在腦部組織有各自獨特的表現，我們的研究亦指出此兩基因在斑馬魚的腦部發育中扮演重的角色。
並列摘要
Ca2+/Calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in a wide range of cellular function including cell cycle and neuron development, and signal transduction. Recently, two proteins, termed Ca2+/Calmodulin-depent protein kinase inhibitor (CaMKIIN) α and β, specific bound to and inhibited CaMKII activity has been reported in vertebrate. The role of CaMKIINs in development still remained to be explored. Herein, we demonstrated the isolation and expression patterns of zebrafish CaMKIINs, named as camkiin 1 and camkiin 2. camkiin 1 and camkiin 2 gene both encoded 79 amino acids and shared over 70% similarity to corresponded genes in mammalian, amphibian and others fish species. Multiple protein alignment indicated that the 27 residues conserved inhibitory region (CIR) shared highest homology. Semi-quantitative RT-PCR analysis revealed that both genes were weakly or undetectable before five somites stages whereas enriched in 24 hour post-fertilization (hpf) and persistently expressed thereafter. Both camkiin 1 and camkiin 2 were found to be highly expressed in adult eyes and brain. RNA transcripts of camkiin 1 were prominently expressed in forebrain and hindbrain region, especially in telencephalon. camkiin 2 was detected in anterior brain region and hindbrain nucleous. Through GST-pull down, co-immunoprecipitation and kinase assay, we have demonstrated that zcamkiins can bind to and reduce CaMKII activity. Morpholino antisense oligonucleotides were used to knockdown the zcamkiin expression. Both camkiin morphant have midbrain hidbrain boundary (MHB) abnormity. camkiin 1 morphant resulted in midbrain defect and enlarged hindbrain. Small eyes and flat head were found in camkiin 2 morphant. No synergistical effects were observed in morphont knockdown of both genes. Malformation of MHB and reduction of neurogenin-1 signals were observed in camkiin 1 but not camkiin 2 morphants. Co-injected with camkiin 1 mRNA can rescue the deformities. However, no significant alternation of markers for forebrain, midbrain, and hidbrain was found in camkiin 2 morphants. Taken together, our results provided the first evidences of identification and expression of zebrafish camkiin 1 and camkiin 2 genes. Distinct spatial expression pattern of camkiin 1 and camkiin 2 were observed by whole-mount in situ hybridization. Our results also suggested that these two genes may play an important role in zebrafish brain development.