臺灣大學: 生化科學研究所黃銓珍楊仲翔Yang, Chung-HsiangChung-HsiangYang2013-03-212018-07-062013-03-212018-07-062011http://ntur.lib.ntu.edu.tw//handle/246246/251029Zona pellucida (透明帶)為包覆在卵外面的一個聚合結構，其功能是避免精子重複進入卵中而特化出來的。多年來的研究顯示，哺乳類的卵上的透明帶是由特殊的蛋白所構成，分別是ZP1，ZP2以及ZP3。將這些蛋白進一步的比對分析其胺基酸序列可以發現到一個高度保留的ZP domain (透明帶結構域)，而且研究也顯示這個區域與蛋白的功能息息相關。透明帶結構域已知是許多細胞外蛋白質所共同擁有的特殊構造之一。 β-tectorin為336個胺基酸所組成，並帶有一個高度保留的ZP domain(透明帶結構域)， 它是內耳中tectorial membrane("覆膜")的主要成分之一: 覆膜在內耳中專司將聲音訊號轉換為神經訊號，為一個細胞外構造並與內耳中的聽毛細胞連結在一起。我的研究著重於分析β-tectorin 這一類帶有透明帶結構域的蛋白質，是否在斑馬魚的內耳發育上扮演著重要的角色。首先，藉由生物資訊學的方法分析斑馬魚的基因資料庫並成功取得了斑馬魚β-tectorin的胺基酸序列。進一步比對分析，發現斑馬魚的β-tectorin與其他物種有接近相似度以及保有一段在哺乳類以及鳥禽類β-tectorin所保留的透明帶結構域。利用斑馬魚原位雜交法以及反轉錄酵素聚合酶連鎖反應，分析β-tectorin在時間和空間上的表現，發現β-tectorin專一的表現在內耳的感覺器官macula中。由morpholino 來knockdown β-tectorin的表現，會影響發育的過程中內耳的耳石位置和數量，並且影響到內耳內部構造的型態。觀察這些耳石異常的斑馬魚可以發現牠們在游泳的行為也有所異常。 除了β-tectorin的研究外，我們也研究了另一個帶有透明帶結構域的zpDL1蛋白。同樣地藉由生物資訊學的方法分析斑馬魚的基因資料庫取得了zpDL1蛋白的序列。由於關於zpDL1蛋白的研究尚未有任何文獻報導，首先利用了原位雜交法探測zpDL1的表現位置。zpDL1專一地表現在內耳的感覺器官crista，呈現出三個點狀。觀察我們所建構以啟動子 (promoter)穩定表現綠色螢光蛋白的斑馬魚亦可以得到與原位雜交法一樣的結論。而這樣的Tg(zpDL1:GFP)有助於我們觀察感覺器官crista的發育，或者是進行以雷射進行破壞crista進而觀察hair cell是否能再生的實驗。 我的研究結果顯示了斑馬魚β-tectorin蛋白專一的表現在內耳的macula並且在斑馬魚內耳發育扮演重要的角色。一旦缺乏β-tectorin將會導致內耳發育上的缺失，耳石形成和功能上的異常。此外，從zpDL1研究的也顯示了zpDL1表現在另一種內耳內的感覺器官 crista的hair cell中，意謂著ZP domain蛋白與這些感覺器官的功能息息相關。整體而言，我的研究成果有助於了解含有ZP domain蛋白在內耳中所扮演的角色。The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is one component of a gel-like structure called tectorial membrane which is involved in transmitting sound wave into neuronal signals and is important for normal auditory function. β-tectorin is specifically expressed in mammalian and avian inner ear. We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin mRNA was expressed in the otic placode and in the specialized sensory patch of the inner ear during zebrafish embryonic stages. Analysis by western blot and immunofluorescence staining suggested that β-tectorin may undergo some posttranslational modifications. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ear of morphants. Finally, the swimming behaviors of β-tectorin morphants were abnormal since the development of inner ear was compromised. In addition to studies of β-tectorin, we had also studied another ZP domain containing protein, zpDL1. Through bioinformatics method, we predicted and identified zpDL1. Since zpDL1 has not been reported in any literature yet, we first characterized its expression by whole-mount in situ hybridization. The expression profile of zpDL1 displayed a specific pattern in the three crista of the inner ear, with an appearance correlated to the onset of crista emerging (about 48 hpf to 72 hpf ). A transgenic Tg(zpDL1:GFP) line was generated using 2 kilo bps promoter region upstream the start codon to drive GFP (green fluorescent protein) expression. Specific expression of GFP in the crista will be used to label the hair cells in the crista and further applied in hair cell ablations by intense laser beam. Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, specifically in the macula, and is important for regulating the development of zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects of inner ear, formation of otoliths and function. Furthermore, zpDL1, another ZP domain containg protein, also showed expression pattern in hair cells related sensory organ, crista. Taken together, our works contributed to better knowledge of the role of ZP domain containing proteins in the zebrafish inner ear.2838932 bytesapplication/pdfen-USZP domain 蛋白斑馬魚內耳發育發育生物學ZP domain proteinzebrafishinner ear developmentdevelopmental biologythesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/251029/1/ntu-100-D96b46002-1.pdf