酵母菌過氧化體生合成因子PEX3基因刪除突變株的構築及鑑定

Pex3p在過氧化體生合成中扮演著極為重要的角色，酵母菌的Pex3p對於過氧化體的合成和維持過氧化體的完整性是不可缺少的膜蛋白，Pex3p會將Pex19p和過氧化體膜蛋白(peroxisome membrane protein，簡稱PMP)形成的複合體停靠在過氧化體上，接著將膜蛋白插入過氧化體的膜上。 當Saccharomyces cerevisiae生長時利用油酸作為唯一碳源，過氧化體成為生長的關鍵，因為過氧化體是在酵母菌中的脂肪酸代謝的唯一場所。為了進行Pex3p功能上的探討，在本研究中，利用包含PEX3基因上下游的DNA片段的質體pRS406ΔA-pex3-us-ds將其送入野生株W303.1a，經由同源重組的方式將PEX3基因刪除，構築突變株Δpex3，以便於將來透過此突變株對於Pex3p進行功能性探討。另外，我們將PEX3基因及啟動子(promoter)接入pRS416的載體，改造得到pRS416-PPex3-Pex3-HA，將pRS416-PPex3-Pex3-HA送入野生株進行蛋白表達，實驗結果確認此質體可以成功的表達Pex3p的蛋白。接著，利用油酸培養基培養觀察突變株Δpex3其代謝油酸能力是否受到影響，並觀察是否會因為我們送進去的pRS416-PPex3-Pex3-HA而恢復代謝油酸的功能，以判定突變株是否正確。另外將帶有PTS1(Peroxisome Targeting signal 1)綠色螢光蛋白基因的質體pRS424-Gal-GFP-PTS1送入野生株、PEX3突變株及互補株中直接觀察過氧化體的形成，以判定突變株是否正確。 藉由上述兩種分析S. cerevisiae過氧化體功能的方法，我們確認所構築的PEX3基因刪除的突變株Δpex3是正確的， 此突變株Δpex3將來可以作為分析Pex3p蛋白功能的工具之一。

Pex3p plays an extremely important role in peroxisome biogenesis, and it is an integral membrane protein involved in the early stages of peroxisome biogenesis formation and the maintenance of the integrity of peroxisome biogenesis. Pex3p is involed in the docking of the complex formed by Pex19p and peroxisome membrane protein (peroxisome membrane protein, PMP) on peroxisome membrane, and then Pex19p will promote the insertion of PMP into the peroxisome membrane.When Saccharomyces cerevisiae uses oleic acid as the sole carbon source for its growth, peroxisome becomes an essential organelle because it is the only place in which the fatty acid metabolizes in yeast. In order to understand the function of Pex3p in this research, we are going to deliver pRS406ΔA-pex3-us-ds, including the upper-stream and downstream DNA fragments of PEX3, into the wild type W303.1a, and then PEX3 gene will be deleted by homologous recombination. This PEX3 gene deletion mutant was named ∆pex3. In addition, we are going to clone PEX3 and its promoter into pRS416 to gain pRS416-PPex3-Pex3-HA, and then send pRS416-PPex3-Pex3-HA into the wild type, and observe its protein expression. The experimental results confirmed that pRS416-PPex3-Pex3-HA can express Pex3p successfully. Then, we used the oleic acid selective medium to assy the peroxisome function. Through this experiment, we will observe if Δpex3 can be restored to use oleic acid by pRS416-PPex3-Pex3-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, ∆pex3 mutant and the ∆pex3 (pRS416-PPex3-Pex3-HA) and these strains were assayed by the fluorescence microscopy. So, by these two methods we can conclude whehter the ∆pex3 mutant is correct.Through the two above analytical methods of peroxisomal functions, we have confirmed that the mutant Δpex3 is correct, and the mutant Δpex3 can be used as one of the tools to analyze the function of Pex3p in the future.