Blue light negatively regulates the sexual filamentation via the Cwc1 protein in Cryptococcus neoformans

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………………………………………………………………………………7