沈偉強Shen, Wei-Chiang臺灣大學:植物病理與微生物學研究所呂佳政Lu, Jia-ZhengJia-ZhengLu2010-05-112018-06-292010-05-112018-06-292009U0001-1901200916181700http://ntur.lib.ntu.edu.tw//handle/246246/181947常見的含硫胺基酸包括半胱胺酸（cysteine）與甲硫胺酸（methionine），此者對於生物細胞的正常生理活動有重要貢獻。硫酸鹽同化途徑（sulfatessimilation pathway，SAP），包含有一系列的還原步驟，將無機氧化態硫素進行化，並合成含硫胺基酸。在真菌界的各物種中，SAP 的主要步驟相當類似，參其中基因的蛋白質序列，也有很高的保守性。以啤酒酵母菌（Saccharomyceserevisiae）為例，環境中的硫酸鹽類被 Sul1p 與 Sul2p（sulfate transporter）運送細胞內，經 Met3p（ATP sulfurylase）催化成 5’-adenylylsulfate（APS）。接，經 Met14p（APS kinase）作用，加上一個磷酸根，後成為 3’-phospho-5’-denylylsulfate （ PAPS ） ， 再經 Met16p （ PAPS reductase ） 轉為亞硫酸鹽sulfite）。最後，由 Met10p 與 Met5p（sulfite reductase α and β subunit）組成的素複合體，將亞硫酸鹽還原成負二價硫離子（sulfide），硫離子即可繼續作為成含硫胺基酸或其他含硫化合物之用。本研究之目的，為探討 MET5 基因於隱菌之生理角色，首先利用 in vitro transposition 反應製備得突變載體，再以基因轉殖技術，將其送入隱球菌野生菌株內。經南方雜合分析及細胞內硫離子含量定，確認隱球菌 met5 突變株。met5 突變株造成半胱胺酸營養缺陷型、生長速緩慢、生殖菌絲減少、無黑色素形成，以及於替代性昆蟲宿主體內之致病力大下降等情形。上述 met5 突變株的缺陷，在重新轉殖 MET5 基因至突變株後，復至野生株之性狀。本研究之結果與前人研究一致，證實硫酸鹽同化途徑，對隱球菌的生理作用有極大重要性，後續之深入研究，有潛力發展出抗真菌藥劑可能標的。Sulfur-containing amino acids such as cysteine and methionine are important forellular physiology. Sulfate assimilation pathway (SAP) is a reduction sequencenvolved in the biosynthesis of these amino acids from the inorganic oxidized sulfurource. In Fungi, sequential steps of SAP are similar, and enzymes involved in thisathway are also highly conserved. In Saccharomyces cerevisiae, exogenous sulfate isransported from the environment into yeast cells by sulfate transporter, Sul1p andul2p, and catalyzed by ATP sulfurylase, Met3p, to form 5’-adenylylsulfate (APS).PS is subsequently phosphorylated by Met14p, APS kinase, to produce 3’-phospho-’-adenylylsulfate (PAPS), and then reduced by Met16p (PAPS reductase) to generateulfite. Finally, the sulfite reductase enzyme complex consisted of α subunit Met10pnd β subunit Met5p further reduces sulfite to sulfide. The reduced sulfur ion can thene incorporated into sulfur-containing amino acids or compounds. In this report, wetudy the roles of the MET5 homologue in the human fungal pathogen Cryptococcuseoformans. The gene disruption construct was created by in vitro transposition andelivered into the wild-type strain by biolistic transformation. Gene disrupted mutantsere verified and characterized. C. neoformans met5 mutants were auxotrophic forysteine, reduced the growth rate, severely attenuated for mating differentiation, failedo produce melanin in vitro, and lost virulence in the alternative insect host model. Allhe defects were reverted to the wild-type by reintroduction of the intact copy MET5ene. Consistent with previous reports, our results showed that the components of SAPlay important roles in the physiological processes of C. neoformans and maybe serves potential targets for antifungal therapy.誌謝..............................................................................................................................i要.............................................................................................................................iibstract......................................................................................................................iii一章 前言.............................................................................................................. 1.1 硫酸鹽同化途徑（sulfate assimilation pathway，SAP） .................................. 1.2 啤酒酵母菌於 SAP 的相關研究........................................................................ 2.3 隱球菌（Cryptococcus neoformans） ................................................................ 3.4 隱球菌的生命週期............................................................................................. 4.5 隱球菌的致病因子............................................................................................. 5.6 隱球菌於 SAP 的相關研究................................................................................ 6二章 材料與方法.................................................................................................. 9.1 實驗菌株及培養條件......................................................................................... 9.2 隱球菌 MET5 同源基因之選殖........................................................................ 10.3 突變質體 pmet5-NAT 之建構及隱球菌 met5 突變株之篩選.......................... 10.4 隱球菌 MET5R 重建株之建構......................................................................... 11.5 基因槍轉殖技術（biolistic transformation）................................................... 11.6 隱球菌少量基因體 DNA 之抽取..................................................................... 12.7 隱球菌大量基因體 DNA 之抽取..................................................................... 12.8 南方雜合分析（Southern hybridization analysis） .......................................... 13.9 隱球菌 RNA 之抽取........................................................................................ 14.10 北方雜合分析（Northern hybridization analysis） ........................................ 14.11 同步定量 PCR（real-time quantitative PCR） ............................................... 15.12 隱球菌菌體內硫離子生成之測定.................................................................. 15.13 隱球菌生長速率之測定................................................................................. 16.14 隱球菌致病因子之測定................................................................................. 16.15 Scanning EM 標本之製備.............................................................................. 16.16 隱球菌之毒性試驗......................................................................................... 17三章 結果............................................................................................................ 18.1 隱球菌硫酸鹽同化途徑................................................................................... 18.2 隱球菌 MET5 同源基因................................................................................... 18.3 隱球菌 met5 突變株與回復突變株之建構與確認........................................... 19.4 隱球菌 met5 突變株生長速率較慢.................................................................. 20.5 隱球菌 met5 突變株為半胱胺酸（cysteine）營養缺陷型.............................. 20.6 隱球菌 met5 突變株無法正常進行有性生殖................................................... 21.7 隱球菌 met5 突變株能正常地合成莢膜.......................................................... 22.8 隱球菌 me5 突變株可能具有細胞壁的缺陷.................................................... 22.9 隱球菌 met5 突變株無法形成黑色素.............................................................. 23.10 隱球菌 met5 突變株失去致病性.................................................................... 24四章 討論............................................................................................................ 26............................................................................................................................... 31............................................................................................................................... 43考文獻.................................................................................................................. 46錄........................................................................................................................... 51application/pdf7569786 bytesapplication/pdfen-US硫酸鹽同化途徑隱球菌MET5半胱胺酸甲硫胺酸啤酒酵母菌Sulfate assimilation pathwayCryptococcus neoformansCysteineMethionineSaccharomyces cerevisiaehttp://ntur.lib.ntu.edu.tw/bitstream/246246/181947/1/ntu-98-R95633021-1.pdf