(3.236.231.61) 您好!臺灣時間:2021/05/15 22:37
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:盧映谷
研究生(外文):Ying-Ku Lu
論文名稱:藍光透過Cwc1蛋白抑制隱球菌生殖菌絲之形成
論文名稱(外文):Blue light negatively regulates the sexual filamentation via the Cwc1 protein 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:2004
畢業學年度:92
語文別:中文
論文頁數:74
中文關鍵詞:隱球菌
外文關鍵詞:filamentationCryptococcus neoformanslight
相關次數:
  • 被引用被引用:1
  • 點閱點閱:140
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
自然界中,許多物種會受光的調控而表現不同的生理現象與形態發生,包括趨光性或背光性、誘導器官發生、生物週期、色素的生合成及特定生殖構造的發育。生物並非可感受每一種波長的光線,大部分生物對光的反應是受紅光、遠紅光、UV-A、UV-B及藍光的調控。而大部分真菌受藍光及UV-A之調控,如Neurospora crassa,藍光會調控其色素的形成,生物週期性,無性及有性生殖構造的誘導,產孢構造之向光性等。隱球菌(Cryptococcus neoformans)是人體伺機性病原性真菌,主要感染免疫不全的病人,若不加以治療,將造成極高之致死率。隱球菌為異宗交配型 (heterothallic)之擔子菌,在一般培養狀態,以酵母菌的形態存在,當兩種交配型細胞混和在合適的環境下,會進行細胞融合產生雙核菌絲,最終形成擔孢子。研究隱球菌之有性生殖,發現雙核菌絲之產生會受光照影響 (Shen et al., 2002)。在本研究中,確認C. neoformans雙核菌絲之生成受光抑制,並證實C. neoformans可感受藍光,光譜中之藍光抑制雙核菌絲之形成。為進一步探討隱球菌光反應的分子調控機制,以紅色麵包黴為模式,利用其藍光光受器WC-1氨基酸序列,在隱球菌的基因體中找到一同源基因,並命名為CWC1。Cwc1蛋白序列保守功能區之分析顯示,其亦具有與chromophore結合之LOV domain。為了進一步探討CWC1基因在隱球菌上扮演的功能,吾人進行cwc1突變株之建構及其性狀分析。由交配菌絲生長分析結果顯示,CWC1的確參與隱球菌光反應之調控,cwc1突變株生殖菌絲之形成,不再受光之抑制。在基因表現分析實驗中顯示,CWC1轉錄不受光照活化,但光照為活化Cwc1蛋白質功能之必要因子。綜合各項實驗結果,推測Cwc1為隱球菌之藍光接受器。
Abstract
Living organisms are regulated by light in the course of their development and physiology including phototropism, apheliotropism, organs induction, circadian clock, pigment biosynthesis and the growth of some developmental structures. Organisms usually are not capable of perceiving light spanning the whole range of radiant energy spectrum, and they sense red, far-red, UV-A, UV-B, and blue light instead. Most fungi perceive light in the blue/UV range, and blue light involves in regulating the pigmentation, circadian rhythm, the formation of asexual and sexual fruiting structure, and the phototropism. Cryptococcus neoformans, an opportunistic human fungal pathogen, causes the life-threatening meningoencephalitis mainly in individuals with compromised immune functions. C. neoformans is a heterothallic basidiomycete and grows vegetatively as yeast. Under appropriate environmental conditions, cells of opposite mating type fuse and form dikaryotic filaments with typical clamp connections, and subsequently four chains of the basidiospores eventually are produced on the top of basidia. Studying the mating response in C. neoformans, we found that the formation of dikaryotic filaments was influenced by light. In this study we demonstrated the filamentation process associated with sexual life cycle is inhibited by blue light. To dissect the molecular mechanisms regulating the blue light photoresponses in C. neoformans, we have identified a gene homologous to the N. crassa blue light photoreceptor WC-1 and named CWC1 (Cryptococcus WC-1). Analysis of Cwc1 amino acid sequence revealed that the putative LOV domain involving the binding of chromophore is also present. To elucidate its role in the light response, the C. neoformans cwc1 mutant strains were created and characterized. The phenotypic observation that mating filamentation in the bilateral cross of cwc1 MATa and MAT?strains was not sensitive to light indicated Cwc1 is a central regulator of light response in C. neoformans. Gene expression analysis showed CWC1 gene is not transcriptionally activated by light. However, light dependent activation of Cwc1 protein is required for its proper function. Taken together, our findings suggest Cwc1 possibly also functions as the blue light photoreceptor in C. neoformans.
中文摘要…………………………………………………………………………3 Abstract………………………………………………………………………………..4
壹、序言…………………………………………………………………………..…..6
貳、前人研究……………………………………………………………………..…..7
一、阿拉伯芥 (Arabidopsis thaliana)的藍光反應………………………..………7
二、紅色麵包黴 (Neurospora crassa)的藍光反應…………………………….12
三、隱球菌 (Cryptococcus neoformans)………….……………………..….….16
參、材料與方法……………………………………………………………………..19
一、實驗材料來源及培養條件……………………………………………….….19
二、隱球菌CWC1基因之選殖. …………………………………………………19
三、質體之建構………………………………………………………………..….19
(一) 隱球菌CWC1基因突變載體之建構…………………………………..…19
(二) 隱球菌cwc1突變株回復 (Reconstitute)載體之建構……………...……20
(三) CWC1大量表現質體之建構……………………………………………20
四、轉形方法 (Transformation) …………………………………………………21
(一) 隱球菌之基因槍轉形流程—Biolistic transformation……………...…21
菌株之培養………………………………………………………………......21
微攜帶子及DNA之製備……………………………………………………20
基因槍轉殖操作流程………………………………………………….…….22
(二) cwc1 MATα突變株之篩選及CWC1突變株的回復轉殖菌株…………..23
五、隱球菌之性狀表型分析…………..………………………………………24
(一) 交配反應試驗 (Mating assay)……………………………………….…..24
(二) Haploid fruiting assay…………………………………………………...…24
(三) 光線對黑色素產生之影響 (Melanin assay)……..………………..……24
六、隱球菌基因體DNA之抽取………………..……………………………24
(一) 隱球菌基因體DNA之小量抽取方法……..…………………………24
(二) 基因體DNA之大量抽取方法……………………………………………25
七、Cryptococcus neoformans總RNA之抽取方法……………………….……26
八、北方雜合分析 (Northern analysis) …………………………………………27
(一) RNA變性電泳………………………………………………………….…27
(二) 膠體轉漬………………………………………………………….………28
(三) 探針標定 (Probe labeling) ………………………………………………28
(四) 北方式雜合反應 (Northern hybridization) ……………………………...28
九、南方雜合分析 (Southern analysis) …………………………………………29
十、Real-time PCR相對定量分析…………………………………………….…29
肆、結果…………………………………………………………………………..…31
一、藍光抑制Cryptococcus neoformans生殖菌絲的生成…………………..…31
二、隱球菌CWC1 基因之選殖及分析………………………………….…32
三、cwc1突變株之篩選與確認……………………………………...………34
四、cwc1變異株之光反應表型……………………………………….……34
五、CWC1基因表現分析………………………………………………….……..36
六、CWC1大量表現對隱球菌的影響…………………………………..….36
七、光照時期及光照長度對CWC1大量表現菌株抑制生殖菌絲的影響…...37
八、光照對cwc1突變株及CWC1大量表現菌株黑色素形成影響之分析…..37
伍、討論………………………………………………………………………………38
陸、圖表………………………………………………………………………………44
柒、參考文獻…………………………………………………………………………64
捌、附錄………………………………………………………………………………72
Ahmad, M., and Cashmore, A.R. (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366: 162-166.
Ahmad, M., Jarillo, J.A., and Cashmore, A.R. (1998) Chimeric proteins between cry1 and cry2 Arabidopsis blue light photoreceptors indicate overlapping functions and varying protein stability. Plant Cell 10: 197-207.
Alspaugh, J.A., and Perfect, J.R., and Heitman, J. (1998) Signal transduction pathways regulating differentiation and pathogenicity of Cryptococcus neoformans. Fungal Genet Biol 25: 1-14.
Ballario, P., and Macino, G. (1997) White collar proteins: PASsing the light signal in Neurospora crassa. Trends Microbiol 5: 458-462.
Ballario, P., Talora, C., Galli, D., Linden, H., and Macino, G. (1998) Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins. Mol Microbiol 29: 719-729.
Ballario, P., Vittorioso, P., Magrelli, A., Talora, C., Cabibbo, A., and Macino, G. (1996) White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. EMBO J 15: 1650-1657.
Baum, G., Long, J.C., Jenkins, G.I., and Trewavas, A.J. (1999) Stimulation of the blue light phototropic receptor NPH1 causes a transient increase in cytosolic Ca2+. Proc Natl Acad Sci USA 96: 13554-13559.
Borthwick, H.A., Hendricks, S.B., Toole, E.H., Toole, V.K. (1954) Action of light on lettuce seed germination. Bot Gaz 115: 205-225.
Buder J (1918) Die Inversion des Phototropismus bei Phycomyces. Ber Dtsch Bot Ges 36: 104-105.
Casadevall, A., and Perfect, J.R. (1998) Cryptococcus neoformans. American Society for Microbiology. Washington, USA.
Casal, J.J. (2000) Phytochromes, cryptochromes, phototropin: photoreceptor interactions in plants. Photochem Photobiol 71: 1-11.
Chen, S., Sorrell, T., Nimmo, G., Speed, B., Currie, B., Ellis, D., Marriott, D., Pfeiffer, T., Parr, D., and Byth, K. (2000) Epidemiology and host- and variety- dependent characteristics of infection due to Crypptococcus neoformans in Australia and New Zealand. Clin Infect Dis 31: 499-508.
Christie, J.M., Reymond, P., Powell, G.K., Bernasconi, P., Raibekas, A.A., Liscum, E., and Briggs, W.R. (1998) Arabidopsis NPH1: A Flavoprotein with the Properties of a Photoreceptor for Phototropism. Science 282: 1698-1701.
Christie, J.M., Salomon, M., Nozue, K., Wada, M., and Briggs, W.R. (1999) LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): Binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci USA 96: 8779-8783.
Crosson, S., and Moffat, K. (2001) Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction. Proc Natl Acad Sci U S A. 98: 2995-3000.
Crosson, S., Rajagopal, S., and Moffat, K. (2003) The LOV domain family: photoresponsive signaling modules coupled to diverse output domains. Biochemistry 42: 2-10.
Doering, T.L. (2000) How does Cryptococcus get its coat? Trends Microbiol 8: 547-553.
Dykstra, M.A., Friedman, L., and Murphy, J.W. (1977) Capsule size of Cryptococcus neoformans: control and relationship to virulence. Infect Immun 16: 129-135.
Edman, J.C., and Kwon-Chung, K.J. (1990) Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for trandformation. Mol Cell Biol 10: 4538-4544.
Emmons, C.W. (1955) Saprophytic sources of Cryptococcus neoformans associated with the pigeon (Columbia livia). Am J Hyg 62: 227-252.
Emmons, C.W. (1960) Prevalence of Cryptococcus neoformans in pigeon habitats. Public Health Rep 75: 362-364.
Evans, E.E. (1950) The antigenic composition of Cryptococcus neoformans. I. A serologic classification by means of the capsular and agglutination reactions. J Immunol 64: 423-430.
Griffith, G.W., Jenkins, G.I., Milner-White, E.J., and Clutterbuck, A.J. (1994) Homology at the amino acid level between plant phytochromes and a regulator of asexual sporulation in Emericella (= Aspergillus) nidulans. Photochem Photobiol 59: 252-256.
Guo, H., Duong, H., Ma, N., and Lin, C. (1999) The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light-dependent post-transcriptional mechanism. Plant J 19: 279-287.
Guo, H., Yang, H., Mockler, T.C., and Lin, C. (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279: 1360-1363.
Harding, R.W., and Melles, S. (1983) Genetic analysis of phototropism of Neurospora crassa perithecial beaks using white collar and albino mutants. Plant Physiol 72: 996-1000.
Harding, R.W., and Turner, R.V. (1981) Photoregulation of the carotenoid biosynthetic pathway in albino and white collar mutants of Neurospora crassa. Plant Physiol 68: 745-749.
He, Q., Cheng, P., Yang, Y., Wang, L., Gardner, K.H., and Liu, Y. (2002) White collar-1, a DNA binding transcription factor and a light sensor. Science 297: 840-843.
Huala, E., Oeller, P.W., Liscum, E., Han, I-S., Larsen, E., and Briggs, W.R. (1997) Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278: 2120-2130.
Hull, C.M., Dacidson, R.C., and Heitman, J. (2002) Cell identity and sexual development in Cryptococcus neoformans are controlled by the mating-type- specific homeodomain protein Sxl1alpha. Genes Dev 16: 3046-3060.
Ito, S. (1993) Biochemistry and physiology of melanin. In: N Levine, ed. Pigmentation and Pigmentory Disorders. Ann Arbor, MI: CRC Press, pp 33-59.
John, M., and Winslow, R.B. (2001) Blue light sensing in higher plants. J Biol Chem 276: 11457-11460.
Kagawa, T., and Wada, M. (2002) Blue light-induced chloroplast relocation. Plant Cell Physiol 43: 367-71.
Kwon-Chung, K.J. (1975) A new genus, Filobasidiella, the perfect state of Cryptococcus neoformans. Mycologia 67: 1197-1200.
Kwon-Chung, K.J. (1976a) Morphogenesis of Filobasidiella neoformans, the sexual state of Cryptococcus neoformans. Mycologia 68: 821-833.
Kwon-Chung, K.J. (1976b) A new species of Filobasidiella, the sexual state of Cryptococcus neoformans B and C serotypes. Mycologia 68: 943-946.
Kwon-Chung, K.J., Edman, J.C., and Wickes, B.L. (1992) Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 60: 602-605.
Lewis, Z.A., Correa, A., Schwerdtfeger, C., Link, K.L., Xie, X., Gomer, R.H., Thomas, T., Ebbole, D.J., and Bell-Pedersen, D. (2002) Overexpression of White Collar-1 (WC-1) activates circadian clock-associated genes, but is not sufficient to induce most light-regulated gene expression in Neurospora crassa. Mol Microbiol 45: 917-931.
Lin, C., Ahmad, M., Chan, J., and Cashmore, A.R. (1996) CRY2: A second member of the Arabidopsis cryptochrome gene family. (Accession No. U43397) (PGR96-001). Plant Physiol 110: 1047.
Lin, C., Yang, H., Guo, H., Mockler, T., Chen, J., and Cashmore, A.R. (1998) Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc Natl Acad Sci USA 95: 2686-2690.
Linden, H. (2002) A white collar protein senses blue light. Science 297: 777-778.
Linden, H., Ballario, P., and Macino, G.. (1997) Blue light regulation in Neurospora crassa. Fungal Genet Biol 22: 141-150.
Linden, H., and Macino, G. (1997) White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa. EMBO J 16: 98-109.
Liscum, E., and Briggs, W.R. (1995) Mutations in the NPH1 Locus of Arabidopsis Disrupt the Perception of Phototropic Stimuli. Plant Cell 7: 473-485.
Liu, Y., He, Q., and Cheng, P. (2003) Photoreception in Neurospora: a tale of two White Collar proteins. Cell Mol Life Sci 60: 2131-2138
Millar, A.J., Carr, I.A., Strayer, C.A., Chua, N.H., and Kay, S.A. (1995) Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science 267: 1161-1163.
Mooney, J.L., Hassett, D.E., and Yager, L.N. (1990) Genetic analysis of suppressors of the veA1 mutation Aspergillus nidulans. Genetics 126: 869-874.
Mooney, J.I., and Yager, L.N. (1990) Light is required for conidiation in Aspergillus nidulans. Genes Dev 4: 1473-1482.
Moore, T.D.E., and Edman, J.C. (1993) The α-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol 13: 1962-1970.
Parks, B.M., Cho, M.H., and Spalding, E.P. (1998) Two genetically separable phases of growth inhibition induced by blue light in Arabidopsis seedlings. Plant Physiol 118: 609-15.
Payen, A. (rapporteur) (1843) Extrait d’un rapport adresse´ a` M. Le Mare´chal Duc de Dalmatie, Ministre de la Guerre, Pre´sident du Conseil, sur une alte´ration extraordinaire du pain de munition. Ann Chim Phys 9: 5–21.
Peters, J.L., Széll, M., and Kendrick, R.E. (1998) The Expression of Light-Regulated Genes in the High-Pigment-1 Mutant of Tomato. Plant Physiol 117: 797–807.
Pitkin, J.W., Panaccione, D.G., and Walton, J.D. (1996) A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum. Microbiology 142: 1557-1565.
Rose, M.D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Sakai, T., Kagawa, T., Kasahara, M., Swartz, T.E., Christie, J.M., Briggs, W.R., Wada, M., and Okada, K. (2001) Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc Natl Acad Sci USA 98: 6969-6974.
Salomon, M., Christie, J.M., Knieb, E., Lempert, U., and Briggs, W.R. (2000) Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry 39: 9401-9410.
Sanford, J.C., Smith, F.D. and Russell, J.A. (1993) Optimization of the biolistic process for different biological applications. Methods of Enzymology 217: 483-509.
Sargent, M.L., and Briggs, W.R. (1967) The effects of light on a circadian rhythm of conidiation in Neurospora. Plant Physiol 42: 1504-1510.
Schopfer, P., and Plachy, C. (1993) Photoinhibition of radish (Raphanus sativus L.) seed germination: control of growth potential by cell-wall yielding in the embryo. Plant Cell Environ 16: 223-229.
Schwerdtfeger, C., and Linden, H. (2000) Localization and light-dependent phosphorylation of white collar 1 and 2, the two central components of blue light signaling in Neurospora crassa. Eur J Biochem 267: 414-422.
Shen, W.C., Davidson, R.C., Cox, G.M., and Heitman, J. (2002) Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans. Eukaryot Cell 1: 366-377.
Starostzik, C., and Marwan, W. (1995) Functional mapping of the branched signal transduction pathway that controls sporulation in Physarum polycephalum. Photochem Photobiol 62: 930-933.
Talora, C., Franchi, L., Linden, H., Ballario, P., and Macino, G. (1999) Role of a white collar-1-white collar-2 complex in blue-light signal transduction. EMBO J 18: 4961-4968.
Toffaletti, D.L., Rude, T.H., Johnston, S.A., Durack, D.T., and Perfect, J.R. (1993) Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175: 1405-1411.
Wang, P., Perfect, J.R., and Heitman, J. (2000) The G-protein b subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans. Mol Cell Biol 20: 352–362.
Weidman, F.D., and Freeman, W. (1924) India ink in the microscopic study of yeast cells. JAMA 83: 1163-1164.
Wickes, B.L., Mayorga, M.E., Edman, U., and Edman, J.C. (1996) Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type. Proc Natl Acad Sci USA 93: 7327-31.
Wilson, D.E., Bennett, J.E., and Bailey, J.W. (1968) Serologic grouping of Cryptococcus neoformans. Proc Soc Exp Biol Med 127: 820-823.
Yang, H.Q., Wu, Y.J., Hang, T.R., Liu, D., Liu, Y., and Cashmore, A.R. (2000) The C termini of Arabidopsis cryptochromes mediate a constitutive light response. Cell 103: 815-27.
Zimmer, B.L., and Roberts, G.D. (1979) Rapid selective urease test for presumptive identification of Cryptococcus neoformans. J Clin Microbiol 10: 380-381.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top