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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 沈偉強 | |
| dc.contributor.author | Bei-Jia Su | en |
| dc.contributor.author | 蘇貝佳 | zh_TW |
| dc.date.accessioned | 2021-06-13T02:14:16Z | - |
| dc.date.available | 2012-05-24 | |
| dc.date.copyright | 2007-05-24 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-05-15 | |
| dc.identifier.citation | Aberg, J.A., and Powderly, W.G. (1997a) Cryptococcal disease: implications of recent clinical trials on treatment and management. AIDS Clin Rev: 229-248.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30745 | - |
| dc.description.abstract | 光線為一重要的環境因子，調控許多生物的生長、生理及發生過程。在光反應研究中得知，大多數的真菌多只能感受單一波段範圍的光線，而其中以藍光反應最為普遍。隱球菌 (Cryptococcus neoformans) 為一重要人體伺機性病原真菌，屬於異宗交配型 (heterothallic) 的擔子菌，在氮素源缺乏的環境下，不同交配型 (MATa 和 MATα) 的細胞會進行融合，產生雙核菌絲，最終形成擔子柄及擔孢子，完成其有性生殖。本實驗室過去的研究發現，隱球菌雙核菌絲的形成，會受到藍光的抑制，為了探討隱球菌藍光光反應的機制，已由隱球菌基因體中選殖出紅色麵包黴 (Neurospora crassa) wc-1與wc-2的同源基因 CWC1 (C. neoformans WC-1) 與 CWC2 (C. neoformans WC-2)。由胺基酸序列的分析得知，Cwc1具有與 chromophore 結合之保守性 LOV (Light, Oxygen, or Voltage) 功能區塊 (domain)，兩個 PAS (Per-Arnt-Sim) 功能區塊，以及 NLS (nuclear localization sequence) 序列，推測其為隱球菌的藍光接收器；Cwc2 胺基酸序列具有 PAS 和 GATA-type zinc finger DNA-binding motif 兩個重要的保守性功能區塊，Cwc1 及 Cwc2 二者可形成複合體，共同調控隱球菌的藍光反應。為進一步探討及確認 Cwc1 及 Cwc2 蛋白質功能區塊的重要性，本研究利用持續表現基因 GPD1 的啟動子，進行 Cwc1 及 Cwc2 系列突變蛋白質之表達試驗，並進行相關表現型之分析。結果顯示，Cwc1 蛋白質已知推定的保守性功能區塊 LOV、PAS，以及包含 NLS 的功能區塊發生突變，均使隱球菌雙核菌絲的形成，不再受到藍光的抑制，因此證明，此三個保守性區塊為 Cwc1 蛋白質必需之重要功能區塊；而針對 Cwc2 蛋白質推定之保守性功能區塊 PAS 及 GATA-type zinc finger DNA-binding motif 進行突變後，雙核菌絲的形成，亦不受藍光抑制，所以亦證明此兩個保守性區塊，對 Cwc2 蛋白質功能之重要性。除了這些已知的功能區塊外，另外本研究也發現部分蛋白質區域亦為二 Cwc 蛋白質，調控雙核菌絲生成所必須的重要功能區域。 | zh_TW |
| dc.description.abstract | Light regulates growth, physiolophy and differentiation in various organisms including fungi. Fungi usually perceive light within a particular region of radiant energy spertrum. One of the most widesperead and pronounced phenomena in fungi is the blue light photoresponses. Cryptococcus neoformans, a heterothallic basidiomycetous fungus, primarily infects the immunocompromised individuals. Under nitrogen limitation conditions, strains of opposite mating types, MATa and MATα, produce conjugation tubes and fuse to form dikaryotic filaments, and subsequently produce the basidia and meiotic progeny basidiospores to complete its sexual cycle. Our previous studies have demonstrated that the production of sexual dikaryotic filaments is inhibited by blue light via the Cwc1 (C. neoformans WC-1) and Cwc2 (C. neoformans WC-2) proteins, homologues of Neurospora crassa WC-1 and WC-2. The deduced Cwc1 amino acid sequence contains a specialized chromophore binding domain known as LOV (for Light-Oxygen-Voltage) domain, two putative PAS (Per-Arnt-Sim) domains, and a putative nuclear localization sequence (NLS). A putative PAS domain and GATA-type zinc finger DNA-binding motif were also identified in the Cwc2 protein. To confirm the roles of putative domains and also identify regions important for the Cwc1 and Cwc2 function, a series of partially deleted CWC1 and CWC2 genes under the constitutively expressed C. neoformans GPD1 promoter were generated in this study. These constructs were transformed into the MATα wild-type, cwc1 and cwc2 mutant strains and sexual filamentation and other phenotypes were examined and compared under light and dark conditions. Our results showed that LOV domain, PAS domains, and the region containing the NLS are crucial for the function of Cwc1 protein, and PAS domain and GATA-type zinc finger DNA-binding motif are also required for the proper execution Cwc2 protein. In addition, regions important for the CWC proteins were also identified. In this study, we used C. neoformans as a model to dissect the blue light photoregulation. To the end, we hope to reveal the functional domains important for the evolutionally conserved blue light regulators in the basidomycetous fungi. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T02:14:16Z (GMT). No. of bitstreams: 1 ntu-96-R93633018-1.pdf: 5349222 bytes, checksum: a21045ba5b0316799afd65f219e69701 (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 第一章 研究動機………………………………………………………………14
第二章 前人研究………………………………………………………………16 2.1. 紅色麵包黴 (Neurospora crassa) 的藍光光反應…………………………..16 2.1.1. 紅色麵包黴 wc-1 與 wc-2 基因的選殖及其胺基酸特性………….16 2.1.2. 紅色麵包黴WC-1及WC-2蛋白質之交互作用…………………….17 2.1.3. WC-1是紅色麵包黴的藍光接收器…………………………………...18 2.1.4. 紅色麵包黴WC-1蛋白質之功能區域分析…………………………..20 2.1.5. 紅色麵包黴WC-2蛋白質之功能區域分析…………………………..21 2.1.6. 紅色麵包黴之蛋白質激酶 (Neurospora protein kinase C, NPKC) 藉由WC-1調控光應……………………………………………………21 2.2. 松露 (Tuber borchii, whitish truffle) 光反應的研究………………………….22 2.3. 深綠木黴菌 (Trichoderma atroviride) 光反應的研究………………………..23 2.4. 水稻稻熱病菌 (Magnaporthe grisea, Magnaporthe oryzae) 光反應的研究....24 2.5. 水稻胡麻葉枯病菌 (Bipolaris oryzae) 光反應的研究………………………..25 2.6. 灰蓋鬼傘 (Coprinopsis cinerea, Coprinus cinereus) 光反應的研究………....26 2.7. 鬍鬚黴 (Phycomyces blakesleeanus) 光反應的研究………………………….27 2.8. 卷枝毛黴 (Mucor circinelloides) 光反應的研究……………………………...28 2.9. 隱球菌 (Cryptococcus neoformans) ……………………………………………29 2.9.1. 隱球菌的生命週期 (life cycle) ……………………………………….....29 2.9.2. 隱球菌之病原性……………………………………………………….....30 2.9.3. 致病因子 (virulence factors) …………………………………………….31 2.9.4. 隱球菌光反應之研究………………………………………………….....32 第三章 材料方法………………………………………………………………....35 3.1. 實驗材料及培養條件…………………………………………………………...35 3.2. CWC1基因各區塊突變質體之建構……………………………………………35 3.2.1. Cwc1蛋白質第1 ~ 102個胺基酸區塊之突變…………………………36 3.2.2. Cwc1蛋白質第103 ~ 250個胺基酸區塊之突變………………………36 3.2.3. Cwc1蛋白質第251 ~ 410個胺基酸區塊之突變………………………37 3.2.4. Cwc1蛋白質第411 ~ 567個胺基酸區塊之突變………………………37 3.2.5. Cwc1蛋白質第568 ~ 680個胺基酸區塊之突變………………………38 3.2.6. Cwc1蛋白質第681 ~ 766個胺基酸區塊之突變………………………39 3.2.7. Cwc1蛋白質第767 ~ 930個胺基酸區塊之突變………………………40 3.2.8. Cwc1蛋白質第931 ~ 1141個胺基酸區塊之突變……………………..40 3.3. CWC2基因各區塊突變質體之建構……………………………………………41 3.3.1. Cwc2蛋白質第1 ~ 71個胺基酸區塊之突變…………………………..41 3.3.2. Cwc2蛋白質第72 ~ 140個胺基酸區塊之突變………………………..42 3.3.3. Cwc2蛋白質第141 ~ 231個胺基酸區塊之突變………………………42 3.3.4. Cwc2蛋白質第231 ~ 348個胺基酸區塊之突變………………………43 3.3.5. Cwc2蛋白質第349 ~ 373個胺基酸區塊之突變………………………44 3.3.6. Cwc2蛋白質第374 ~ 392個胺基酸區塊之突變………………………44 3.4. 血清型A CWC1質體大量表現質體之建構…………………………………..45 3.5. 基因槍轉殖技術 (Biolistic transformation) …………………………………...46 3.5.1. 轉殖菌株之培養………………………………………………………....46 3.5.2. 轉殖金粉 (Gold particle；microcarrier)………………………………..46 3.5.3 DNA之製備………………………………………………………………47 3.5.4. 基因槍之操作流程……………………………………………………....47 3.6. 隱球菌少量基因體DNA之抽取………………………………………………48 3.7. 隱球菌總RNA之抽取…………………………………………………………48 3.8. 北方雜合分析 (Northern hybridization analysis) ……………………………..49 3.8.1. 將受試菌株培養後，採前述方法進行總RNA之萃取……………….49 3.8.2. RNA變性膠體電泳分析之步驟………………………………………...49 3.8.3. 膠體轉漬………………………………………………………………...50 3.8.4. 探針標定的流程………………………………………………………...50 3.8.5. 北方雜合反應…………………………………………………………...51 3.9. 隱球菌性狀表現型之分析……………………………………………………..51 3.9.1. 交配分析試驗 (Mating assay)………………………………………….51 3.9.2. 單核菌絲之分析試驗 (Monokaryotic fruiting assay)………………….52 3.9.3. 光線對黑色素產生之影響 (Melanin assay)…………………………....52 3.10. 親緣樹狀分析 (Phylogenetic analysis)……………………………………….52 第四章 結果……………………………………………………………………….53 4.1. 隱球菌Cwc1蛋白質之探討…………………………………………………….53 4.1.1. 隱球菌不同血清型Cwc1蛋白質胺基酸序列的比對分析……………..53 4.1.2. 隱球菌Cwc1蛋白質與其他真菌同源蛋白質保守性功能區域的比對 分析……………………………………………………………………….54 4.1.3. 隱球菌Cwc1蛋白質區域系列突變之建構及選殖…………………….57 4.1.4. 隱球菌Cwc1蛋白質區域系列突變過度表現菌株之交配反應與monokaryotic fruiting分析………………………………………………58 4.1.5. 隱球菌Cwc1蛋白質區域系列突變過度表現菌株之黑色素形成分析..60 4.2. 隱球菌Cwc2蛋白質之探討…………………………………………………….61 4.2.1. 隱球菌Cwc2蛋白質在不同血清型之胺基酸序列比對分析…………..61 4.2.2. 隱球菌Cwc2蛋白質與其他真菌同源基因蛋白質保守性功能區域 的比對分析………………………………………………………………..62 4.2.3. 隱球菌Cwc2蛋白質區域系列突變之建構及選殖…………………….64 4.2.4. 隱球菌Cwc2蛋白質區域系列突變之過度表現菌株之交配反應與 monokaryotic fruiting分析………………………………………………64 4.2.5. 隱球菌Cwc2蛋白質區域系列突變過度表現菌株之黑色素形成分 析…………………………………………………………………………..66 第五章 討論………………………………………………………………………..68 圖表…………………………………………………………………………………..75 參考文獻…………………………………………………………………………..119附錄………………………………………………………………………………….131 | |
| dc.language.iso | zh-TW | |
| dc.title | 隱球菌藍光調控因子Cwc1及Cwc2蛋白質功能區域之探討 | zh_TW |
| dc.title | Domain characterization of the blue light regulators Cwc1 and Cwc2 in Cryptococcus neoformans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉瑞芬,陳昭瑩,鄭貽生,廖秀娟 | |
| dc.subject.keyword | 隱球菌,藍光光反應,藍光接收器,紅色麵包黴,Cwc1,Cwc2,LOV功能區塊,PAS功能區塊,NLS序列, | zh_TW |
| dc.subject.keyword | Cryptococcus neoforman,blue light photoresponses,blue light regulators,Neurospora crassa,Cwc1,Cwc2,LOV domain,PAS domains,NLS, | en |
| dc.relation.page | 117 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-05-16 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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