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研究生:孫楷惠
研究生(外文):Kai-Hui Sun
論文名稱:隱球菌藍光訊息傳導分子機制之探討
論文名稱(外文):Dissection of the molecular mechanisms regulating the blue light photoresponses in Cryptococcus neoformans
指導教授:沈偉強
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:86
中文關鍵詞:隱球菌藍光訊息傳導
外文關鍵詞:Cryptococcus neoformansblue lightCWCTUP1
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自然界中,包括真菌在內的許多物種,其生理現象與型態發生之過程皆會受到光的影響與調控。隱球菌 (Cryptococcus neoformans) 乃一人體伺機性病原真菌,為異宗交配型 (heterothallic) 之擔子菌。在一般培養狀態下,主要以酵母菌的型態存在;於氮素源缺乏的環境下,兩種交配型 (MATa和MATα) 的細胞會進行細胞融合,產生雙核菌絲,最終形成擔子孢子。本實驗室過去的研究發現,隱球菌可感受藍光,其雙核菌絲之形成會受光線抑制。為了探討隱球菌藍光反應之分子機制,之前的研究已由隱球菌的基因體中,選殖出紅色麵包黴 (Neurospora crassa) wc-1的同源基因CWC1。根據功能性分析的結果,已知CWC1為參與隱球菌藍光訊息傳導的重要因子;此外,因Cwc1具有保守性之LOV功能區塊,故被推測為隱球菌的藍光接受器。在本研究中,吾人進一步從隱球菌基因體中,選殖出紅色麵包黴wc-2的同源基因CWC2。透過分析Cwc2的胺基酸序得知,Cwc2具有PAS和GATA-type zinc finger兩種重要的保守性功能區塊。吾人後續的研究結果發現,cwc2突變株和cwc1突變株一樣,均會失去光的敏感性,從而證明CWC2亦為隱球菌藍光訊息傳導途徑中,另一不可或缺的調控因子。再者,分析CWC2基因表現的結果顯示,CWC2的表現量會於光照後而有些微增加的情況,並且光亦為活化Cwc2功能與其調控路徑、進而抑制生殖菌絲的必要因子。綜合CWC1和CWC2相關的研究結果,吾人提出兩種模式,以解釋藍光如何透過Cwc1和Cwc2來調控隱球菌的生理反應。為了尋找光反應抑制生殖菌絲之可能下游抑制因子,吾人於是鎖定隱球菌已報導的抑制因子Tup1,來進行遺傳學的分析。研究結果顯示,Tup1可能為Cwc下游抑制隱球菌生殖菌絲形成的因子之一;除此以外,可能尚有其他抑制因子參與其中。為了進一步尋找隱球菌藍光反應之其他下游基因,吾人也進行了subtractive PCR;然而,截至目前為止,尚未找到可能的抑制因子,唯發現部分基因具有被CWC1調控之情形。這些受CWC1調控的基因,尚需後續的探討與分析,始能了解其於隱球菌藍光反應中的角色。
Light affects growth and development of various organisms, including fungi. Cryptococcus neoformans, an opportunistic human fungal pathogen, is a heterothallic basidiomyceteous fungus that grows vegetatively as a yeast under normal conditions. Under nitrogen limitation conditions, cells of opposite mating type, MATa and MATα, fuse to form dikaryotic filaments, and subsequently produce the meiotic basidiospores. Our prior studies have shown that C. neoformans can only sense blue light and its sexual filamentation is negatively regulated by light. To dissect the molecular mechanisms of light signaling in C. neoformans, the CWC1 gene, a homologue of the Neurospora crassa wc-1, has been identified and characterized. Inhibition of sexual filamentation by CWC1 overexpression requires light activation suggested that the putative LOV domain-containing Cwc1 protein may function as the blue light photoreceptor in C. neoformans. In this study, through a candidate gene approach, we report the identification and characterization of C. neoformans CWC2 gene, a homologue of the N. crassa wc-2. Analysis of Cwc2 amino acid sequence revealed that the putative PAS domain and GATA-type zinc finger DNA-binding domain are present. Deletion of the CWC2 gene similarly results in loss of light inhibition of the sexual filamentation as the cwc1 mutants, suggesting that CWC2 is also crucial in the blue light response pathway in C. neoformans. In addition, gene expression analysis showed that the CWC2 gene is slightly up-regulated in response to light, and light dependent activation of the Cwc2 protein is also required for its proper function. Based on the results, we propose two models in which Cwc1 functions as a blue light photoreceptor to mediate photoresponses through complexing with the conserved DNA-binding domain containing Cwc2 via the conserved protein dimerization PAS domains. Furthermore, a putative repressor Tup1 homologue potentially downstream of the Cwc complex was identified. To determine the role of C. neoformans TUP1 gene, we conducted epistasis analysis and created the cwc mutant strains under tup1 mutant background. Preliminary results suggested that Tup1 may function downstream of the Cwc complex; however, additional repressor(s) may be present and coordinately regulate C. neoformans genes involved in the sexual filamentation. To identify genes under direct control of Cwc complex and also involved in light-regulated filamentation or other biological processes in C. neoformans, subtractive screen approaches were also employed. Although no putative repressor has been identified so far, some genes of interest have been isolated. Further characterization is required to reveal their functions.
Abstract…………………………………………………………………..6
中文摘要……………………………………………………………….....8
Chapter I…………...………………………………………………….......10
Introduction…………...………………………………………………........10
1.1 Blue-light photoresponses in N. crassa………………………………........11
1.1.1 Identification and characterization of wc-1 and wc-2…..………........11
1.1.2 Localization and light-dependent phosphorylation of WC-1 and
WC-2………………………………………………………………..12
1.1.3 WC-1 is a blue-light photoreceptor………………………………….14
1.1.4 WC-1 and WC-2, two PAS-domain-containing transcription factors,
are essential for blue-light input pathway in N. crassa……………..15
1.2 Cryptococcus neoformans…………………………………………………16
1.2.1 Life cycle of C. neoformans…………………………………………16
1.2.2 Infectious cycle and virulence factors……………………………….18
1.2.3 Blue light negatively regulates the sexual filamentation in C.
neoformans via the Cwc1 protein…………………………………...21
1.2.4 The CWC1 gene is expressed at a very low level, and inhibition of
the filamentation by CWC1 requires light activation……..………...23
1.2.5 BWC1 is required for UV resistance and regulates virulence in a
mammalian host…………………………………………………….24
1.2.6 Proposed functional roles of Cwc1 in the blue-light signaling
pathway of C. neoformans……………….…………………………24
Chapter II……………………………………………………...…………27
Material and Methods……………………………...……………………27
2.1 Strains and media…………………………….……………………………27
2.2 Identification, disruption, and reintroduction of the C. neoformans CWC2
gene…………………………………………………………………...….27
2.3 Overexpression of the C. neoformans CWC2 gene……………………….29
2.4 Overexpression of the C. neoformans TUP1 gene………………………29
2.5 Generation of the cwc1 tup1 and cwc2 tup1 double mutant strains……….30
2.6 Isolation of genomic DNA and Southern blot analysis………………….30
2.7 Isolation of RNA and Northern blot analysis……………………………31
2.8 Real-time PCR analysis ………………………………………………...31
2.9 Mating assay……………………………………………………………...32
2.10 Capsule formation assay………………………………………………...32
2.11 Subtractive-PCR procedures……………………………………………..32
Chapter III……………………………………………………………....35
Results………………………………………………………………………35
3.1 Identification and characterization of the C. neoformans CWC2
gene………………………………………………………………………..35
3.2 Disruption of the CWC2 gene results in loss of inhibition of sexual
filamentation by light……………………………………………………...36
3.3 The CWC2 gene is expressed at a very low level and slightly up-regulated
in response to light………………………………………………………...38
3.4 Inhibition of the filamentation by overexpression of CWC2 requires light
activation………………………………………………………………….39
3.5 Cwc1 and Cwc2 function interdependently to regulate the light response..40
3.6 Tup1 may be one of the repressors downstream of the Cwc complex to
negatively regulate genes involved in the sexual filamentation…………...41
3.7 Expression analyses of C. neoformans TUP1, CWC1, CWC2 genes……...43
3.8 Subtractive PCR approaches were preformed to isolate the downstream
genes of the Cwc1/Cwc2 complex……………………………………….44
3.9 Deletion of either CWC1 or CWC2 gene slightly reduces the size of
capsules………..…………………………………………………………..45
Chapter IV………………………………………………………………..47
Discussion…………………………………………………………………...47
References………………………………………………………………...74
Appendix………………………...………………………………………...82
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