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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孫錦虹(Chin-Hung Sun) | |
| dc.contributor.author | Meng-Hsuan Lin | en |
| dc.contributor.author | 林孟萱 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:13:53Z | - |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-13 | |
| dc.identifier.citation | 1. Adam, R. D. (1991) The biology of Giardia spp. Microbiological reviews 55, 706-732
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(2005) The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC evolutionary biology 5, 1 60. Zhuge, C., Chang, Y., Li, Y., Chen, Y., and Lei, J. (2011) PDCD5-regulated cell fate decision after ultraviolet-irradiation-induced DNA damage. Biophysical journal 101, 2582-2591 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18596 | - |
| dc.description.abstract | 梨形鞭毛蟲是一種廣泛存在全球的致病性原蟲。梨形鞭毛蟲生活史中有一段細胞分化的過程稱之為囊體化，此時是滋養體轉變為囊體的過程。屬於AP-1蛋白家族的Maf可以調控許多物種的細胞分化。我們在梨形鞭毛蟲中找到一個近似Maf的蛋白質，經胺基酸序列分析發現蛋白質C端部分具有規律出現的leucine，符合Maf zip的一大特徵，且在這些Leucine前端具有一段胺基酸序列與人類Maf進行DNA binding的序列相似，於是我們將其命名為G. lamblia Maf-like protein 1(glMafl1)，並建立過量表現glMafl1之細胞株pPglMafl1。實驗結果發現此細胞株的囊體壁蛋白CWP1也大量增加。為了探究glMafl1的功能，我們將其DNA binding序列與zip序列設計突變，建立突變株pPglMafl1-m1與pPglMafl1-m2。我們發現大量表現glMafl1時，CWP1-3及調控囊體化之轉錄因子Myb2蛋白質及RNA表現量都會上升，促進生成囊體，然而突變株的CWP1-3、Myb2表現及囊體生成數目則減少。透過免疫沉澱法以及GST融合蛋白質沉澱法我們發現glMafl1會和其他調控囊體化之轉錄因子，如：Pax1、WRKY、PDCD5、Myb2直接結合。因此我們認為glMafl1可能參與一個許多轉錄因子結合形成的複合體，共同調控囊體化相關基因以促進囊體化進行。 | zh_TW |
| dc.description.abstract | Giardia lamblia is one kind of worldwide protozoan pathogens. There is a cell differntiation stage in Giardia life cycle, named encystation, which is the transformation between trophozoite and cyst. Maf, a member of AP-1 family, can regulate cell differntiation in many spieces.. We found a G. lamblia protein similar to well studied mammalian Maf. By amino acids analyzing, we observed that there were several leucines regularly existed at the C terminal of this G. lamblia protein, just like the higher evolution organisms’ Maf Zip domain. Moreover, there are some amino acids upstream of those regular leucines similar to the human Maf DNA binding site basic residues. Based on these amino acid analyzing, we named this protein G. lamblia Maf-like protein 1 (glMafl1), and constructed the Giardia cell strain, pPglMafl1, to overexpress it. The data showed that the cyst wall protein 1 (CWP1) expressing were also up-regulated in pPglMafl1. To understand the function of glMafl1, we mutated the putative basic DNA binding site and leucine zipper of the glMafl1 leading to mutant cell strains of pPglMafl1-m1 and pPglMafl1-m2. We observed that the cyst wall protein 1-3 (CWP1-3) and encystation regulating-Myb2 gene was up-regulated in pPglMafl1 at protein and RNA level. Overexpressing glMafl1 could induce cyst forming. Moreover, the CWP1-3, Myb2 expression and cyst forming decreased in two mutants. Using immunoprerecipitation assays and GST pull down assays, we found that glMafl1 directly interacts with other encystation relating transcription factors, Pax1, WRKY, PDCD5 and Myb2. We thus suggest that glMafl1 participates in a transcription factor complex, which plays a role in the induction of encystation relating genes expression, and thus promoting encystation of G. lamblia. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:13:53Z (GMT). No. of bitstreams: 1 ntu-103-R01445205-1.pdf: 5625942 bytes, checksum: 245be680916fdf895dbe7ce9eaaf8397 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii Abstract iii 摘要 iv 目錄 v 第一章 前言 1.1梨形鞭毛蟲簡介 1 1.2梨形鞭毛蟲囊體化 1 1.3 梨形鞭毛蟲囊體化之分子調控 2 1.4 轉錄因子Maf 2 1.5 轉錄因子Pax 4 1.6 轉錄因子WRKY 5 1.7 轉錄因子Myb 6 1.8梨形鞭毛蟲PDCD5蛋白質 7 1.9 研究動機 7 第二章 材料方法 2.1梨形鞭毛蟲滋養體及囊體化時期培養(Giardia lamblia culture) 9 2.2轉殖質體之建構(Plasmid construction) 9 2.2.1 5’∆5N-pac 9 2.2.2 pPglMafl1 9 2.2.3 pPglMafl1-m1 9 2.2.4 pPglMafl1-m2 10 2.3梨形鞭毛蟲細胞轉染與細胞株篩選(Transfection and Selection) 11 2.4重組蛋白質之表現與純化 (Expression and Purification of Recombinant glMafl1 protein) 11 2.4.1重組glMafl1與重組glMafl1突變蛋白質之質體建構 11 2.4.2重組蛋白質之表現與純化 13 2.5免疫螢光染色 (Immunofluorescence assay) 14 2.6 染色質免疫沉澱 (Chromatin Immunoprecipitation) 14 2.7 GST融合蛋白質共沉澱 (GST pull down assay) 16 2.8 西方點墨法與Coomassie blue染色(Western blot and Coomassie blue stain) 17 2.9 反轉錄聚合酶鏈式反應 (RT-PCR) 18 2.10 即時定量反轉錄聚合酶鏈式反應 (Real time PCR) 18 2.11 囊體計數 (Cyst count) 19 2.12 免疫沉澱法 (Immunoprecipitation) 20 第三章 實驗結果 3.1 梨形鞭毛蟲ORF3731序列分析並命名為glMafl1 21 3.2鑑定glMafl1在滋養體時期與囊體化時期的RNA表現量 25 3.3建立過量表現glMafl1蛋白質之梨形鞭毛蟲細胞株 25 3.4 glMafl1與glMafl1、cwp1、cwp2、Myb2基因啟動子結合 26 3.5 glMafl1蛋白質可形成同雙聚合子 26 3.6建立過量表現glMafl1突變蛋白質之細胞株m1 26 3.7建立過量表現glMafl1突變蛋白質之細胞株m2 27 3.8 Leucine zipper受到破壞之glMafl1-m2蛋白質 無法與glMafl1蛋白質聚合形成同雙合子 28 3.9 glMafl1正向影響囊體化進行 28 3. 10 glMafl1與囊體化轉錄因子交互作用 31 3.11 glMafl1與轉錄因子Pax1、WRKY、PDCD5直接交互作用 32 第四章 討論 4.1啟動囊體化相關物質轉錄的一把鑰匙─Maf 33 4.2 glMafl1可能參與並促進囊壁蛋白CWP1的轉錄 33 4.3 glMafl1可能於轉錄層面調控囊體化相關基因 33 4.4 glMafl1可能參與調控囊體化基因之轉錄因子複合體 33 附圖 36 參考文獻 59 | |
| dc.language.iso | zh-TW | |
| dc.title | 鑑定與探討梨形鞭毛蟲Maf-like轉錄因子之特性 | zh_TW |
| dc.title | Identification and characterization of a Maf-like transcription factor in Giardia lamblia | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李財坤(Tsai-Kun Li),詹迺立(Nei-Li Chan) | |
| dc.subject.keyword | 梨形鞭毛蟲,Maf,囊體化,囊體壁蛋白CWP1-3,Pax1,PDCD5,Myb2,WRKY, | zh_TW |
| dc.subject.keyword | Giardia lamblia,Maf,encystation,CWP1-3,Pax1,PDCD5,WRKY,Myb2, | en |
| dc.relation.page | 66 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2014-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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