The contruction and identification of a peroxisome biogenesis factor PEX3 gene delection mutant in Saccharomyces cerevisiae.

• Main Authors: Yung-Yuan, 陳永原
• Other Authors: 蔡榮宗
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/67311356248711140079

碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 100 === 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.