斑馬魚gjb4基因之鑑定與功能分析

Identification and function analysis of zebrafish gjb4 gene

徐靜軒(Ching-Hsuan Hsu)

指導教授 ： 楊建洲

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00129

摘要

間隙連接（gap junction）的基本組成單位是連接蛋白（CXs）。連接蛋白是包含有4個穿膜結構區域的蛋白質。細胞間隙連接（GJIC）擁有許多的功能，不同CX所組成的間隙連接在特定的器官、組織或細胞中可以通透的物質會有所不同。這些間隙連接是相鄰兩個細胞間主要的運輸通道，可以允許小於1千道爾頓（dalton）的小分子通過。在本篇研究當中，我們利用斑馬魚觀察人類CX30.3 （GJB4）的同源基因cx34.4 (zfcx34.4; gjb4)，其相似度為62%。在斑馬魚胚胎發育時期從受精後1.5小時至受精後五天的斑馬魚胚胎皆有zfcx34.4基因表現，其中在受精後12至48小時表現量特別多。另外，從全胚胎原位雜交實驗以及石蠟切片的結果我們發現zfcx34.4基因在受精後24小時之前表現於全身，但是在受精後36小時則可明顯看到表現於斑馬魚骨骼肌當中的慢肌，但是在成魚肌肉組織當中zfcx34.4基因的表現量並不明顯，因此我們推測zfcx34.4可能只參與了早期胚胎的肌肉發育。另一方面我們也利用顯微注射的方式打入反股的Morpholino（MO）去抑制zfcx34.4的轉譯，觀察其外表形可發現有22％的胚胎體軸有嚴重的彎曲。而隨著MO劑量的增加，雖然外表形體軸嚴重彎曲的情況並沒有增加，但是其死亡率會隨著MO劑量的增加而增加。從以上結果推測zfcx34.4在胚胎早期肌肉發育當中可能扮演重要的角色。然而，zfcx34.4基因的功能是否有與人類GJB4相同或相似的地方則需要更進一步的研究。另外一部分，我們實驗室對於非症候群聽障的研究，在聽障病人的身上已發現幾個基因有突變的發生。另外在細胞模式研究中也發現其突變點會影響基因的正常功能。因此我們希望藉由模式動物來尋找內耳專一表現的啟動子，進而可以建立探討聽障基因功能的動物模式。首先我們先利用生物資訊學的方式，在ZFIN資料庫尋找到已經有文獻指出參與內耳發育前期的基因，從Ensemble Zebrafish資料庫搜尋mRAN序列，再以Neural Network Promoter Prediction進行啟動子區域的預測。之後我們將promoter建構到pGL3-basic vector質體上利用 Luciferase activity測定及β-gal assay分析promoter的活性。首先我們針對otolin 1a進行分析。 所預測出的啟動子區域為-110至-2627，其所測得的活性表現與negative control有明顯的差別。進而我們利用序列刪除的方式縮小promoter可能存在的區域，結果推測其啟動子區域可能在-275至-1區間。進一步的我們將活性較高之-1134至-110片段建構至pGL3 basic-EGFP載體利用顯微注射初步觀察在斑馬魚胚胎內表現位置。結果不論在受精後24或者96小時皆只可看到其表現於耳囊周圍。未來我們將利用Tol 2基因轉殖系統來確認我們所預測的啟動子區域，若能確定於內耳專一表現，將可以用來探討我們實驗室在非症候群聽障病人當中發現有突變發生的基因之功能。

關鍵字

間隙連接 ； 連接蛋白 ； 非症候群聽障 ； 斑馬魚

並列摘要

Connexins （Cxs） are homologous four transmembrane domain proteins that are the major components of gap junctions. Gap junctional intercellular communication （GJIC） has numerous functions, each of which meets the particular needs of organs, tissues or groups of cells. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both electrical and chemical signals. In this study, we presented expression pattern of human CX30.3 （GJB4） homologous gene zebrafish cx34.4 (zfcx34.4; gjb4). This gene shared 62% similarity with CX30.3 gene of Homo sapiens. The cDNA were isolated from zebrafish embryos by semi-quantitative RT-PCR. During embryogenesis, zebrafish cx34.4 gene expressed from 1.5 hours post-fertilization （hpf） to 5 days post-fertilization （dpf）, especially in 12hpf to 48hpf. In addition, whole-mount in situ hybridization and parafilm section revealed that zfcx34.4 was expressed in slow muscle of 36 to 48 hpf. Moreover, we injected zebrafish cx34.4 anti-sense Morpholino (MO) knockdown zebrafish cx34.4 gene at zebrafish one-cell stage. Observed at 72hpf, 22% of the knockdown embryos showed severe alterations in their phenotype. In addition, we increase the concentration of MO, the mortality rate showed dose dependent. Our results demonstrate the importance of gjb4 gene in zebrafish embryonic early stage. From our results, we suggested that zfcx34.4 probability affect the myogenesis in zebrafish development early stage. However, zebrafish cx34.4 gene plays role is similar human GJB4 or has another function needs to be investigated further. Another part of this study, our laboratory for non-syndromic hearing loss research has already found several genes with point mutations. Previously, we have established a cell model (HeLa cell) and tet-on protein expression system to study the pathogenesis of deafness-gene mutations. Therefore, we hope to find the inner ear specific expression promoter and creating an animal model to investigate gene function of non-syndromic hearing loss. First we used ZFIN database to find literature that have been specifically expressed genes in the inner ear, and used Ensemble database to search Zebrafish mRAN sequence, and then used Neural Network Promoter Prediction to predict promoter region. After that we constructed the prediction region to pGL3-basic vector and then detected the Luciferase activity. First, we analyzed for otolin 1a. The prediction promoter region was -110 to -2627, the results of luciferase activity showed that this region expressed the highest promoter activity than negative control. To reduce region of the promoter, we did the series deletion. Our results suggest that promoter region might exist in -275 to -110. To preliminary observe the prediction region expression in zebrafish, we chose the higher activity of -1134 to -110 fragment construct into pGL3 basic-EGFP vector. The results show that there are both 24 or 96 hpf embryos, its performance can be seen around the otic vesicle. In the future, we will use the Tol 2 transgenic system to validate our predicted promoter region, to determine if specific expression in the inner ear that will help to explore our lab in the non-syndromic hearing loss mutations genes function.

並列關鍵字

gap junction ； connexin ； nonsyndromic hearing loss ； zebrafish ； GJB4

參考文獻

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