沈偉強臺灣大學：植物病理與微生物學研究所劉啟宏Liou, Ci-HongCi-HongLiou2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/58063隱球菌 (Cryptococcus neoformans) 為一伺機性人體重要病原真菌，對於免疫系統具有缺陷的患者如AIDS、器官移殖或癌症化療病患等，會造成致命的感染；由於隱球菌基因體計畫的完成，再加上其基因轉殖系統和病原性動物試驗系統的完善建立，隱球菌已成為研究病原真菌致病機制及真菌生理的重要模式系統。隱球菌屬於異宗交配型 (heterothallic) 的擔子菌，在實驗室一般培養條件下，以酵母菌的形式存在，當不同交配型 (mating type) 的MATa和MATα菌株混合培養在氮素源缺乏的環境下，細胞會進行融合產生雙核菌絲，進行有性生殖。本實驗室過去的研究證明，隱球菌透過真菌保守性藍光反應調控因子Cwc1與Cwc2，共同調控生殖菌絲受光抑制的藍光反應。為進一步探究光反應之調控機制，亦利用農桿菌轉殖技術 (Agrobacterium-mediated transformation)，針對CWC1過度表現菌株進行隨機突變，並篩選於照光的條件下，表現型由生殖菌絲完全抑制回復成有菌絲生成的菌株，以找出參與藍光反應抑制生殖菌絲生長的重要下游基因。在篩選得到4132株農桿菌轉殖菌株中，其中64株在照光條件下具有不同程度生殖菌絲生成的情形，經由進一步實驗分析發現，其中一轉殖株AS31，其T-DNA破壞之基因為histone-lysine N-methyltransferase，亦即啤酒酵母菌 (Saccharomyces cerevisiae) 之SET2同源基因。在本研究中，除了確認AS31農桿菌轉殖株，表型回復成有菌絲生成的現象，完全肇因於SET2基因受T-DNA的插入破壞所導致，另外亦建構set2突變株及SET2過度表現株，以探究SET2於隱球菌中所扮演之角色。根據交配反應及haploid fruiting等試驗結果顯示，SET2基因在不同交配型細胞扮演不同之角色，交配型α的set2突變株及SET2過度表現株，不論在照光或黑暗的培養下，菌絲生成皆有受抑制的情形，而交配型a的set2突變株及SET2過度表現株，其表型則皆與野生型菌株相當；此外，在cwc1 set2二基因突變株的性狀分析試驗中，發現生殖菌絲的生成及haploid fruiting菌絲之生長，可能擁有不同的調控機制。Cryptococcus neoformans is an opportunistic human fungal pathogen which primarily infects immunocompromised patients, such as AIDS, organ transplant, and cancer chemotherapy. Due to its available completed genome sequences, well-developed transformation systems, and robust animal models, C. neoformans has become an excellent model system for studying the virulence and physiological processes in fungi. C. ceoformans is a heterothallic basidiomycete, and exists predominantly as a haploid yeast under routinely laboratory culture condition. When opposite mating types strains, MATa and MATα, are co-cultured under nitrogen starvation condition, the conjugation tubes are formed and fused to poduce the sexual dikaryotic filaments. Our prior studies have shown that blue light inhibits the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans. To reveal the molecular mechanisms of blue light photoresponses, genome-wide random insertional mutagenesis by Agrobacterium-mediated transformation was employed to identify genes involved in the light inhibition of filamentation. We have identified 64 strains from 4132 transformants, of which restore the filamentation phenotype of the CWC1 overexpression strain under constant light condition. One of these strains, AS31 strain, was found that T-DNA was integrated at the coding region of Saccharomyces cerevisiae SET2 homologue. In this study, we demonstrated that the suppression of light-dependent phenotype of the CWC1 overexpression strain was caused by disruption of SET2 function in the AS31 strain. Furthermore, overexpression and disruption of SET2 gene in the wild-type strain background were conducted. Overexpression or disruption of SET2 in the MATα strain both displayed reduced sexual filamentation and haploid fruiting under light or dark conditions; however, the filamentation of the MATa set2 mutant and MATa SET2 overexpression strain were all comparable to the MATa wild-type strain. Additionally, the discrepancies between the sexual and haploid filamentation were also observed in the MATα cwc1 set2 double mutants. Taken together, our results suggest that SET2 gene functions to regulate the filamentation pathway, however, further study is needed to address its roles in different mating type strain.目錄 中文摘要 ..……………………………………………………………… 1 Abstract .....………………………………………………………………. 3 壹、序言 .………………………………………………………………. 5 貳、前人研究 ………………………………………………………….. 7 一、隱球菌 (Cryptococcus neoformans) …………………………. 7 二、隱球菌光反應之研究 ……………………………………….. 11 三、農桿菌轉殖系統 (ATMT) …………………………………… 13 四、啤酒酵母菌SET2基因的相關研究 ………………………... 15 參、材料與方法 ……………………………………………………..… 21 一、實驗材料及培養條件 ………………………..……………… 21 二、AS31農桿菌轉殖株之確認以及隱球菌SET2同源基因之 選殖 ………………………………………………………….. 21 三、質體之建構及隱球菌基因轉殖株之篩選 ……….…………. 22 （一）隱球菌SET2過度表現載體之建構及過度表現株之 篩選 …………………………………………………….. 22 （二）隱球菌SET2突變載體之建構及突變株之篩選 …….. 24 （三）隱球菌set2突變株回復載體之建構及回復株之 篩選 …………………………………………………...... 27 四、基因槍轉殖技術 (Biolistic transformation) ……………...… 29 （一）欲轉殖菌株之培養及準備 …………………………..... 29 （二）轉殖金粉 (Gold particle；microcarrier) 及DNA之 製備 …………………………………………………..... 30 （三）基因槍之操作流程 …………………………………… 31 五、隱球菌基因體DNA之抽取 ………………………………… 33 （一）隱球菌少量基因體DNA之抽取 ………………….….. 33 （二）隱球菌大量基因體DNA之抽取 ……………….…….. 34 六、隱球菌總RNA之抽取 …………………………………….... 35 七、南方雜合分析 ……………………………………………..… 37 八、北方雜合分析 ………………………………………………. 40 九、隱球菌之性狀表型分析 …………………………………….. 42 （一）交配分析試驗 (Mating assay) ………………………… 42 （二） Haploid fruiting assay ………………………………… 43 肆、結果 ……………………………………………………….……… 44 一、AS31農桿菌轉殖株之確認 ……………………….………. 44 二、隱球菌SET2基因之分析 ……………………….…………. 48 三、隱球菌SET2過度表現株之表型分析 …………………….. 50 四、隱球菌set2突變株之表型分析 …………………………… 52 五、隱球菌cwc1 set2二基因突變株之表型分析………………. 54 伍、討論 ……………………………………….……………………… 55 陸、圖表 ………………………….…………………………………… 66 柒、參考文獻 ………………….……………………………………… 82 捌、附錄 ……………………….……………………………………… 943916674 bytesapplication/pdfen-US隱球菌農桿菌轉殖技術組蛋白甲基化Cryptococcus neoformansSET2Agrobacterium-mediated transformationhistone-lysine N-methyltransferase[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58063/1/ntu-95-R91633005-1.pdf