酵母菌過氧化體生合成因子PEX13基因刪除突變株的構築及鑑定

The construction and identification of a peroxisome biogenesis factor PEX13 gene deletion mutant in Saccharomyces cerevisiae

許維邦(Wei-Pang Shu)

指導教授 ： 蔡榮宗

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2012年)

https://doi.org/10.6834/CSMU.2012.00037

摘要

PEX13為過氧化體生合成的重要基因，其轉譯之蛋白Pex13p主要功能為過氧化體膜上的停靠蛋白複合體（Docking complex）其中之一員，在過去的文獻中發現在Pex13p刪除的倉鼠卵巢細胞中會影響過氧化體基質蛋白進入過氧化體中，而在Zellweger syndrome的個案也有發現是由於PEX13基因的缺陷所造成，我們想要進一步建立過氧化體Pex13p的功能性分析。所以在本篇研究裡我們改造了含有PEX13啟動子以及PEX13基因的質體，透過酵母菌轉型作用，觀察質體是否有表現其蛋白量，接著改造將PEX13啟動子以及PEX13基因刪除的質體，利用酵母菌轉型作用建立生合成因子PEX13基因刪除突變株的酵母菌W303.1a(ΔPEX13)，利用酵母菌代謝油酸去觀察過氧化體功能之分析，同時PEX13基因刪除突變株的酵母菌再加入我們先前改造之質體，從代謝油酸的培養基觀察去功能有沒有互補，再利用螢光蛋白實驗確認將PEX13基因剔除掉後對於過氧化體生合成的影響，從實驗結果可以得知我們成功的建立了PEX13基因刪除突變株，可以使得未來實驗室能夠更深入的探討有關Pex13p的功能。

關鍵字

PEX13

並列摘要

PEX13 gene is an important gene for peroxisomal biogenesis. The Pex13p protein is a component of the docking complex located in peroxisome membrane for peroxisome matrix protein transport. In previous paper, the finding about Pex13p function has suggested that peroxisome matrix protein transport was abolished in pex13 gene knock-out hamster ovary cell. The human genetic disorder Zellweger sydrome is caused by the mutation of the PEX13 gene. In this study, we constructed pRS416-Ppex13-Pex13-HA, containing the PEX13 promoter and the PEX13 gene, and transformed this plasmid into wild-type W303.1a by yeast transformation technique to observe whether the Pex13p protein was expressed. In order to understand the function of Pex13p in this research, we are going to deliver pRS406ΔA-pex13-us-ds, including the upper-stream and downstream DNA fragments of PEX13, into the wild type W303.1a, and then PEX13 gene will be deleted by homologous recombination. This PEX13 gene deletion mutant was named ∆pex13. Then, we used the oleic acid selective medium to assy the peroxisome function. Through this experiment, we will observe if Δpex13 can be restored to use oleic acid by pRS416-PPex13-Pex13-HA complementation. Furthermore, we are going to use GFP-PTS1 (Peroxisome Targeting signal 1) to assay peroxisome formation by the fluorescence microscopy technique. The GFP-PTS1 encoding pRS424-Gal-GFP-PTS1 was transformed into the wild type, ∆pex13 mutant and the ∆pex13 (pRS416-PPex13-Pex13-HA) and these strains were assayed by the fluorescence microscopy. So, by these two methods we can conclude whehter the ∆pex3 mutant is correct.

並列關鍵字

PEX13

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