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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 沈偉強(Wei-Chiang Shen) | |
| dc.contributor.author | Kuang-Hung Liu | en |
| dc.contributor.author | 劉廣宏 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:44:50Z | - |
| dc.date.available | 2010-08-08 | |
| dc.date.copyright | 2008-08-08 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-30 | |
| dc.identifier.citation | Alspaugh, J. A., Davidson, R. C. and Heitman, J. (2000) Morphogenesis of Cryptococcus neoformans. Contrib Microbiol 5: 217-238.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40323 | - |
| dc.description.abstract | 隱球菌(Cryptococcus neoformans)，為一伺機性人類病原真菌，在分類地位上屬於擔子菌，具有酵母菌與菌絲體兩種形態。隱球菌存在MATa 與 MATα兩種交配型細胞，在氮素源缺乏的環境下，MATa 與 MATα 細胞產生接合管進行細胞融合，並產生雙核生殖菌絲，擔子柄及擔孢子完成有性生活史。本實驗室過去的研究發現，隱球菌藉由Cwc1與Cwc2兩蛋白質形成之複合體，在光照環境下，感應藍光抑制雙核生殖菌絲的生長。為了進一步瞭解藍光抑制雙核菌絲生長的機制，我們利用農桿菌轉殖系統(Agrobacterium-mediated insertional mutagenesis)，針對CWC1過度表現株進行逢機突變，並篩選於持續光照環境下，表現型由雙核生殖菌絲完全被抑制回復生長雙核生殖菌絲的突變株，藉以找出與藍光調控機制有關的下游基因。其中一株回覆突變株DJ22，進一步分析發現T-DNA插入破壞隱球菌CRK1基因。啤酒酵母菌(Saccharomyces cerevisiae) IME2基因及玉米黑穗病菌(Ustilago maydis) crk1基因為隱球菌CRK1之同源基因。Ime2 是一個減數分裂專一性蛋白質，具有高度保守性之Ser/Thr激酶區與TXY雙重磷酸化位置。在我們研究中發現，crk1突變株細胞融合效率提高，產生之雙核生殖菌絲多於野生株，且其雙核菌絲、擔子柄(basidia)、擔孢子(basidiaspore)的發生皆早於野生株。過度表現CRK1則會抑制雙核生殖菌絲的形成。另一方面，crk1突變株與CRK1過度表現株皆會抑制monokaryotic fruiting菌絲的發生。由本研究結果得知，CRK1在C. neoformans有性生殖過程中，扮演一負調控因子的角色。 | zh_TW |
| dc.description.abstract | Cryptococcus neoformans is a heterothallic basidiomycete and grows vegetatively as yeast. Under nitrogen limitation conditions, strains of opposite mating types, MATa and MATα, produce conjugation tubes and fuse to form sexual dikaryotic filaments with clamp connections. Our previous studies demonstrated that C. neoformans Cwc1 and Cwc2 are two central photoregulators which form complex to inhibit the production of sexual filaments upon light stimulation. To reveal the detailed regulatory mechanisms, Agrobacterium-mediated insertional mutagenesis screen was conducted to identify the components interacting with or functioning downstream of the Cwc1/Cwc2 complex in the pathway. Upon light irradiation, T-DNA mutants restoring different extents of filamentation under the CWC1 overexpression background were identified. In this study, one suppressor mutant, DJ22, was characterized and T-DNA was found to integrate at the C. neoformans CRK1 gene, a homologue of Saccharomyces cerevisiae IME2 and Ustilago maydis crk1. Ime2 is a meiosis-specific gene with the conserved Ser/Thr kinase domain and the significant TXY dual phosphorylation site. Consistent with the findings of other suppressors in our screen, C. neoformans Crk1 played negative role in the mating process. Mating efficiency was increased in the crk1 mutants. Dikaryotic filaments, basidia, and basidiospores produced earlier in the crosses involved the crk1 mutants. Elevation of the CRK1 mRNA level inhibited sexual differentiation. On the other hand, monokaryotic fruiting was defective both in the MATα crk1 mutant and CRK1 overexpression strains. Our study demonstrated that mating process was negatively regulated by the CRK1 gene in C. neoformans. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:44:50Z (GMT). No. of bitstreams: 1 ntu-97-R94633017-1.pdf: 1380019 bytes, checksum: 01a11f7f12fe84735329b2debaeb16b9 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 中文摘要…………………………………………………………………………ii
Abstract…………………………………………………………………………iii Chapter I Introduction………………………………………………………1 11 Meiotic program in Saccharomyces cerevisiae…………………………1 12 Signaling pathways and the crk1 gene in Ustilago maydis ………………4 13 Cryptococcus neoformans………………………………………………6 14 C neoformans blue light photoresponse…………………………………9 Chapter II Materials and Methods…………………………13 21 Strains, media and growth conditions…………………13 22 Identification of the T-DNA insertion site in the DJ22 suppressor strain14 23 Generation of the crk1 deletion mutant………………………………………14 24 Overexpression of the C neoformance CRK1 gene…………………15 25 Generation of the crk1+CRK1 reconstitution strains……………16 26 Southern blot analysis……………………………………………………17 27 Northern blot analysis………………………………………………………17 28 Cell fusion assay……………………………………………………………18 29 Melanin formation assay………………………………………………………19 210 Capsule formation assay……………………………19 211 Cell growth assay…………………………………………20 212 Slide mating…………………………………………………20 213 Phylogenetic analysis……………………………………………………21 Chapter III Results ………………………………………………22 31 Identification and characterization of the C neoformans CRK1 gene…22 32 Disruption of the C neoformans CRK1 gene…………………………………23 33 C neoformans crk1 mutants grew normally as the wild-type strains…24 34 Production of dikaryotic filaments and cell fusion efficiency were elevated in the crk1 mutants……………………………………………………………………25 35 Dikaryotic filaments, basidia, and basidiospores arouse earlier in the bilateral crk1 mutant cross……………………………………………………………………27 36 Overexpression of the CRK1 gene inhibited the filamentation in C neoformans …………………………………………………………………………………28 37 Monokaryotic filamentation was defective in the crk1 mutant…………………29 38 Overexpression of CRK1 in the MATα cells caused growth defect in C neoformans……………………………………………………………………30 39 CRK1 played roles in regulating capsule formation in C neoformans…………31 Chapter IV Results …………………………………………33 Reference……………………………………………………………………………61 List of Figures Fig 1 Phylogenetic analysis and amino acid sequence alignment of the conserve Ser/Thr kinase domain of Cryptococcus neoformans Crk1 and related homologues…44 Fig 2 Construction of the crk1::NAT disruption allele and Southern hybridization analysis of the wild-type, crk1, crk1+CRK1 reconstitution strains…………46 Fig 3 Deletion of CRK1 showed no growth defect…………………………………47 Fig 4 Mating filamentation was increased in the crk1 mutant………………………48 Fig 5 Cell fusion efficiency was increased in the crk1 mutant……………………49 Fig 6 Crosses involved the crk1 mutants produce dikaryotic filaments earlier than the wild-type cross………………………………………………………………51 Fig 7 The crk1 bilateral mutant cross produced basidia and basidiospores earlier than the wild-type or crk1 unilateral mutant cross………………………………53 Fig 8 Overexpression of the CRK1 gene inhibited the sexual filamentation………54 Fig 9 Monokaryotic filamentation was defective in the MATα crk1 mutant and CRK1 overexpression strains………………………………………………………55 Fig 10 Overexpression of the CRK1 gene resulted in large capsule size in the MATa but not MATα cells………………………………………………………………57 Fig 11 Overexpression of the CRK1 gene increased the melanization in the MATa but not MATα cells………………………………………………………………58 List of Tables Table 1 Cryptococcus neoformans strains used in this study…………………59 Table 2 Oligonucleotide primers used in this study…………………………60 | |
| dc.language.iso | en | |
| dc.title | 隱球菌CRK1基因功能特性之分析研究 | zh_TW |
| dc.title | Identification and characterization of the CRK1 gene in Cryptococcus neoformans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉瑞芬(Ruey-Fen Liou),鄧述諄(Shu-Chun Teng),藍忠昱(Chung-Yu Lan),呂俊毅(Jun-Yi Leu) | |
| dc.subject.keyword | 隱球菌,Cwc1,Cwc2,Agrobacterium-mediated insertional mutagenesis,CRK1,IME2, | zh_TW |
| dc.subject.keyword | Cryptococcus neoformans,Cwc1,Cwc2,Agrobacterium-mediated insertional mutagenesis,CRK1,IME2, | en |
| dc.relation.page | 72 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-08-01 | |
| 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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