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標題: | 二號吡咯啉-5-羧酸鹽還原酶之表達與功能性分析 Expression and functional analysis of Pyrroline-5-carboxylate reductase 1b |
作者: | 熊俐伃 Li-Yu Hsiung |
指導教授: | 李士傑 Shyh-Jye Lee |
關鍵字: | 斑馬魚,pycr1a,pycr1b,脯氨酸,衰老,早衰症,畸型小頭症, zebrafish,pycr1b,pycr1a,proline,aging,progeria,microcephaly, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 吡咯啉-5-羧酸鹽還原酶(Pyrroline-5-carboxylate reductase, PYCR)普遍被認為是管家酵素,可與NAD(P)H共同催化脯氨酸的生成。在斑馬魚有三種PYCR同源蛋白,分別是Pycr1a、Pycr1b與Pycr3。分別由Pycr1a/Pycr1b 和 Pycr3兩條路徑來催化脯氨酸的生成。Pycr1a與Pycr1b位於粒線體內而Pycr3位於細胞質中。研究顯示Pycr1a與Pycr1b可保護細胞抵禦氧化傷害,而Pycr1a與Pycr1b基因突變皆會影響粒線體的功能,進而造成早衰症及畸形小頭症等相關疾病,而本論文之目的乃探討Pycr1b在斑馬魚發育過程中之基因及蛋白質表現及分布。在此論文中,我以反轉錄聚合酶鏈式反應(RT-PCR)發現pycr1b基因從胚胎早期發育階段與成魚所有組織中皆有高度表現。而在原位雜合(in situ hybridization)實驗中我觀測到pycr1b基因在腦部及其特定區域有高度表達。我以Pycr1b C端約一半的序列製作了一個GST融合重組蛋白,並以之為抗原製作了一支抗體。利用西方墨點法,此Pycr1b抗體可以專一的辨識到一條約34 kDa的斑馬魚粒線體蛋白,而在一天大的斑馬魚胚胎粒線體蛋白中Pycr1b的條帶在Pycr1b (-/-)基因剔除魚比野生型的魚明顯減弱。此證明了此Pycr1b (-/-)突變魚是Pycr1b基因缺失突變魚,後續可更進一步用以檢測Pycr1b功能並進行其他實驗。 Pyrroline-5-carboxylate reductase (PYCR) is a housekeeping enzyme catalyzing the reduction of delta (1)-pyrroline-5-carboxylate (P5C) to proline with NAD(P)H as a cofactor. In zebrafish, there are three PYCR family genes, including pycr1a, pycr1b and pycr3, to catalyze the production of proline in two separate reactions by Pycr1a/Pycr1b or Pycr3. Pycr1a and Pycr1b are located in mitochondria and Pycr3 is a cytosolic protein. Pycr1a and Pycr1b can protect cells from overt oxidative stress. Mutations in the pycr1a or pycr1b gene can alter mitochondrial function and result in progeria and microcephaly-related diseases. The objective of this thesis is to understand the spatial and temporal expression of pycr1b gene during development in zebrafish. Using RT-PCR, I found that the Pycr1b is highly expressed from early developmental stages and different adult tissues. Embryos or larval zebrafish were fixed at designated stages and subjected to whole-mount in situ hybridization analysis against pycr1b. I found that pycr1b was highly expressed in the brain. To carry out the project, I also generated and characterized a Pycr1b antibody specifically recognizing a GST-fusion protein containing the C-terminal half of zebrafish Pycr1b. By Western blotting, the generated Pycr1b antibody can recognized a ~ 34 kDa protein from a mitochondria fraction of zebrafish embryos. I further observed that the Pycr1b band was weaker in pycr1b (-/-) than that of wildtype 1-day old zebrafish embryos. This suggests that the pycr1b (-/-) mutant is a null mutant which can be used for Pycr1b functional assay. The Pycr1b antibody may also be used to further analyze protein expression pattern and other functional test for Pycr1b. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77260 |
DOI: | 10.6342/NTU201902510 |
全文授權: | 未授權 |
顯示於系所單位: | 生命科學系 |
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