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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 沈偉強(Wei-Chiang Shen) | |
dc.contributor.author | Yu-Sheng Lin | en |
dc.contributor.author | 林裕盛 | zh_TW |
dc.date.accessioned | 2021-06-13T01:32:37Z | - |
dc.date.available | 2009-07-19 | |
dc.date.copyright | 2007-07-19 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-17 | |
dc.identifier.citation | Aberg, J. A., and Powderly, W. G. 1997. Cryptococcal disease: importance of recent clinical trail on treatment and management. AIDS Clin Rev : 229-248.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30046 | - |
dc.description.abstract | 隱球菌為人體重要病原真菌。實驗室過去的研究發現,藍光抑制隱球菌生殖菌絲的形成。進一步利用同源序列的比對,在隱球菌基因體中,找到真菌保守性藍光調控基因CWC1及CWC2,並證明二者為隱球菌藍光反應不可或缺的因子(Lu et al., 2005)。為進一步探討隱球菌光反應Cwc複合體下游抑制菌絲形成的相關機制,本研究以農桿菌逢機突變轉殖技術,利用 CWC1基因過度表現株完全抑制生殖菌絲形成的特性,進行回復生殖菌絲生長突變株之篩選。利用此suppressor screening的策略,經篩選4132個農桿菌轉殖株,其中64株具有不同程度回復生殖菌絲生長之情形。其中一農桿菌轉殖株AZ5,T-DNA插入破壞CWC2基因之表現,印證了本篩選策略的有效性。另一轉殖株AY18,經實驗證明,T-DNA插入破壞隱球菌SSN8同源基因,並因此造成生殖菌絲回復生長之特徵。啤酒酵母菌SSN8基因,又稱為SRB11/UME3/CYCC/RYE2,為轉錄調控mediator complex的一員,參與糖類利用、減數分裂、細胞生長,以及轉錄反應等生理之調控。為瞭解SSN8基因在隱球菌中所扮演之角色,本研究亦建構野生型ssn8突變株、回復突變株,及過度表現株。SSN8基因突變造成的性狀包括,生殖菌絲的減少, haploid fruiting菌絲的大量生成、黑色素的明顯累積,而在營養充分的培養條件下,ssn8突變株細胞形態發生改變,並且在固體培養基表面可見侵入菌絲的形成等。相關菌株性狀分析的結果顯示,SSN8基因參與隱球菌許多生理及發生的過程,並且為負向調控之角色,而SSN8基因如何調控相關生理反應,以及其與Cwc複合體間的關連性,有待進一步的探討。 | zh_TW |
dc.description.abstract | Cryptococcus neoformans is an important human fungal pathogen. In our previous investigation, we demonstrated that blue light negatively regulates the sexual filamentous growth via the Cwc1/Cwc2 complex in C. neoformans. To further understand how light inhibits mating filamentation, we conducted a suppressor screening using Agrobacterium-mediated insertional mutagenesis to identify the components interacting with or downstream of the Cwc complex under the CWC1 overexpression strain background. Among 4132 nourseothricin-resistant transformans screened, we identified 64 strains restored different levels of mating filamentation. The sites of T-DNA integration among 21 transformans were recovered. One strain, AZ5, restored the highest level of filamentation was observed and that T-DNA was found to be inserted at the 5’-UTR region of the CWC2 gene. The finding of CWC2 gene in the screen validates our approach. In this study, we further characterized the transformant AY18 in which T-DNA integration affected the activity of a gene homologous to the Saccharomyces cerevisiae SSN8. In budding yeast, SSN8 gene, also named SRB11/UME3/CYCC/RYE2, has been demonstrated to play diverse roles in sugar utilization, meiosis, cell phase control, and transcription regulation. To reveal the roles of SSN8 gene in C. neoformans, we generated the ssn8 mtant, reconstituted strain, and overexpression strain under the wild-type background. The mutant phenotypes included slightly reduced mating filamentation, extensive haploid filamentation, increased melanin accumulation, changed cellular morphology and production of invasive hyphae under rich YPD growth condition. Our results suggest that C. neoformans SSN8 homologue plays critical roles in the diverse physiological and differentiation processes. This is the first demonstration of mediator component in C. neoformans. Its roles related to the Cwc complex remain to be further investigated. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T01:32:37Z (GMT). No. of bitstreams: 1 ntu-96-R93633017-1.pdf: 1805902 bytes, checksum: 672113bd103fe1971f0012053dd6583b (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 中文摘要 1
英文摘要 2 第一章 研究動機 4 第二章 前人研究 5 2.1隱球菌 (Cryptococcus neoformans) 5 2.2隱球菌生活史 6 2.3隱球菌致病因子 7 2.4 隱球菌光反應之研究 9 2.5 隱球菌轉殖方法之研究 11 2.6啤酒酵母菌SSN8同源基因之研究 13 2.6.1 SSN8同源基因的發現 14 2.6.2 UME gene family的發現 15 2.6.3 RYE gene family的研究 17 2.6.4 C-type cyclin之研究 18 2.6.5 The mediator complex 20 2.7 SSN3同源基因之研究 23 第三章 材料與方法 25 3.1 實驗菌株以及培養條件 25 3.2農桿菌基因轉殖流程 25 3.2.1 農桿菌以及隱球菌的培養 26 3.2.2 農桿菌的活化 26 3.2.3 ATMT感染試驗 26 3.2.4轉殖株的篩選 27 3.3 農桿菌轉植菌落的篩選 27 3.4 T-DNA插入位點目標基因之鑑定 28 3.5 SSN8同源基因突變質體之建構及SSN8同源基因突變株之篩選 29 3.6 SSN8基因回覆突變株質體的製備及回覆突變株的篩選 30 3.7 SSN8基因過度表現株的製備以及篩選 30 3.8基因槍轉殖技術 (biolistic transformation) 30 3.8.1 宿主細胞的培養 31 3.8.2宿主細胞的製備 31 3.8.3轉殖DNA的製備 32 3.8.4基因槍之操作流程 32 3.9隱球菌少量DNA的抽取 33 3.10隱球菌大量DNA之萃取 34 3.11隱球菌RNA的抽取 34 3.12南方雜合分析 35 3.13北方雜合分析 37 3.14菌株交配子代之分離實驗 38 3.15交配分析試驗(Mating assay) 39 3.16單核菌絲之分析實驗 ( Monokaryotic fruiting assay ) 39 3.17黑色素形成分析 (Melanin assay) 39 3.18莢膜表現性狀之分析 40 3.19親緣樹狀分析 (Phylogenetic analysis) 40 第四章 結果 41 4.1農桿菌轉殖菌株之篩選 41 4.2 T-DNA插入破壞目標基因之分析 42 4.3隱球菌農桿菌突變轉殖菌株AZ5之分析 43 4.4隱球菌農桿菌突變轉殖株AY18菌株表現型態及T-DNA插入分析 44 4.5隱球菌CWC1基因過度表現之ssn8突變株之建構 46 4.6隱球菌CWC1基因過度表現ssn8突變株之表現型態分析 46 4.7隱球菌SSN8同源基因之探討 46 4.8 ssn8野生型突變株、回復突變株及SSN8基因過度表現株之建構 48 4.8.1 ssn8野生型突變株之篩選 48 4.8.2 ssn8 + SSN8回覆突變株的建構 48 4.8.3 SSN8同源基因過度表現株的建構 49 4.9 ssn8突變株與SSN8基因過度表現菌株之表現型態分析 49 4.9.1生殖菌絲的形成分析 49 4.9.2 Haploid fruiting 單核菌絲的形成分析 50 4.9.3黑色素的形成分析 51 4.9.4 37℃生長測定 52 4.9.5菌株細胞型態之觀察 52 4.9.6侵入菌絲之形成觀察 52 4.10 ssn8 cwc1及ssn8 ste12α等雙突變株表現型態之觀察 53 第五章 討論 55 圖表 65 參考文獻 106 附錄 118 表目錄 表一、本實驗所使用之隱球菌菌株 66 表二、本實驗所使用之引子 69 表三、隱球菌農桿菌突變轉殖株 70 表四、定序確認插入位置之隱球菌農桿菌突變轉殖菌株 71 圖目錄 圖一、隱球菌農桿菌突變株生殖菌絲形成及南方雜合分析 73 圖二、隱球菌農桿菌突變轉殖株AZ5之生殖菌絲表現型態與T-DNA插入位置之分析 75 圖三、隱球菌農桿菌突變轉殖株AY18之表現型態分析 77 圖四、隱球菌農桿菌轉殖株AY18 T-DNA插入位置之確認 79 圖五、隱球菌農桿菌轉殖株AY18子代之基因型分析 81 圖六、隱球菌Ssn8與其他同源蛋白質胺基酸序列之比對及親源分析結果 83 圖七、隱球菌SSN8基因突變載體之建構及YKC38突變轉殖株之南方雜合分析結果 85 圖八、有性生殖子代PCR篩選確認各種基因型之ssn8突變株 87 圖九、隱球菌SSN8基因突變載體及各式ssn8突變株與回覆突變株之南方雜合分析結果 89 圖十、隱球菌ssn8突變株、ssn8 + SSN8回覆突變株,及SSN8基因過度表現株之生殖菌絲形成分析 91 圖十一、隱球菌ssn8突變株,ssn8 + SSN8回覆突變株,以及SSN8基因過度表現株monokaryotic fruiting菌絲之形成分析 93 圖十二、隱球菌ssn8突變株,ssn8 + SSN8回覆突變株,以及SSN8基因過度表現株之黑色素生成分析 95 圖十三、隱球菌ssn8突變株,ssn8 + SSN8回覆突變株,以及SSN8基因過度表現株之37℃生長測定 97 圖十四、隱球菌ssn8突變株,ssn8 + SSN8回覆突變株,以及SSN8基因過度表現株細胞形態之觀察99 圖十五、隱球菌ssn8突變株侵入菌絲之觀察101 圖十六、cwc1 ssn8與ste12α ssn8雙突變株生殖菌絲形成之觀察103 圖十七、cwc1 ssn8與ste12α ssn8雙突變株單核菌絲形成之觀察105 | |
dc.language.iso | zh-TW | |
dc.title | 隱球菌光反應之研究與隱球菌SSN8同源基因之探討 | zh_TW |
dc.title | Dissection of the Cryptococcus neoformans light response and characterization of Cryptococcus neoformans SSN8 homologue | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 藍忠昱,羅秀容,鄧述諄,劉瑞芬 | |
dc.subject.keyword | 隱球菌,藍光反應,CWC1,CWC2,農桿菌逢機突變篩選,SSN8同源基因,mediator complex, | zh_TW |
dc.subject.keyword | Cryptococcus neoformans,blue light response,CWC1,CWC2,Agrobacterium random insertional mutagenesis,suppressor screen,SSN8 homologue,mediator complex., | en |
dc.relation.page | 128 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-17 | |
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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