臺灣博碩士論文加值系統

過去本實驗室在酵母菌多樣性調查研究中，分離出458株酵母菌，其中部份菌株經傳統形態及生理生化鑑定，同時比對LSU rRNA基因的D1/D2區域序列，仍無法確認其菌種學名。本實驗挑選這類模糊菌株，經隨機擴增多型性DNA (RAPD)、核糖體RNA基因序列 (LSU rRNA基因之D1/D2區域、ITS基因序列及SSU rRNA基因)比對及染色體核型等分子生物技術進行菌種鑑定，並以鄰聚法 (Neighbor- joining)分析實驗菌株與相近菌種的親源關係。實驗結果說明了，不同之分子生物技術在鑑定研究上有其應用範圍與限制，其中隨機擴增多型性DNA能提供大量酵母菌株快速而方便的區分，但是因其結果的重複性差，無法作為鑑定特徵；核糖體RNA基因序列則能作為較明確的鑑定依據，然而只比對單一序列有時會產生較大的誤差，再經由親源關係分析能了解各菌種的相互關係；染色體核型則能提供菌種鑑定上明確的證據，然而相近的染色體核型也會有不易判斷的狀況。本實驗所有菌株之鑑定結果顯示，子囊菌中Candida sp.(1)、Candida sp.(2)、Candida sp.(3)、Candida sp.(4)、Kazachstania wufongensis、Lachancea dasiensis、Candida sp.(6)、Wickerhamomyces sp.(1)、Wickerhamomyces sp.(2)及Candida sp.(8)等10個菌種，擔子菌中Cryptococcus sp.(1)、Cryptococcus sp.(2)、Cryptococcus sp.(3)、Cryptococcus sp.(4)、Cryptococcus sp.(5)、Cryptococcus sp.(6)及Rhodotorula sp.(1)等7個菌種，與現有菌種呈現明顯差異，為未被紀錄之分類項，得進一步進行新種描述。另其餘5種中，Candida sp.(5)鑑定為Issatchenkia hanoiensis之無性世代。Candida sp.(7)與Yamadazyma scolityy在各項特徵都極為相似，應為現有種之無性世代。Cryptococcus arengaensis與Cryptococcus heimaeyensis於各項特徵比對均有明顯差異，其中SJ11L06與其他二者在各特徵也呈現差異，應屬於不同之分類地位，而Tetrapisispora sp.(1)及Torulaspora sp.(1)此2個菌種，在各項特徵比對中均與現有種呈現些微差異，但不足以證明為不同之分類項，須進一步研究藉由其他方法探究其分類地位。

Four hundred and fifty-eight yeast strains were isolated from soil and leaves in middle and northern mountains of Taiwan in the previous study in our lab. Some strains could not be clearly identified with traditional approaches and analysis of the D1/D2 domain of the LSU rRNA gene. These strains are examined and species delimited by random amplified polymorphism DNA (RAPD), sequence analysis of ribosomal RNA genes, such as D1/D2 domain of the LSU rRNA gene, ITS sequence and the SSU rRNA gene, and electrophoretic karyotyping. As the results showed, random amplified polymorphism DNA is rapid and convenient in distinguishing among many strains of yeasts, but the technique can not be used for identification of yeasts owing to reiterating difficultly. Yeast identification could stand on sequence analysis of ribosomal RNA genes, but the identification could be still ambiguous when the sequence is 3-10 nt different from closely related species. Electrophoretic karyotypes are valuable characteristics in yeast declineation, according to the size and number of chromosomal DNAs of the examined strains. The results of identification showed that the ascomycetous species, such as Candida sp.(1), Candida sp.(2), Candida sp.(3), Candida sp.(4), Kazachstania wufongensis, Lachancea dasiensis, Candida sp.(6), Wickerhamomyces sp.(1), Wickerhamomyces sp.(2) and Candida sp.(8), and the basidiomycetous species, such as Cryptococcus sp.(1), Cryptococcus sp.(2), Cryptococcus sp.(3), Cryptococcus sp.(4), Cryptococcus sp.(5), Cryptococcus sp.(6) and Rhodotorula sp.(1), are different from currently recognized species., These strains could be described as new species based on characteristics examined in this study. Candida sp.(5) was identified as Issatchenkia hanoiensis. Candida sp.(7) was quietly similar to Yamadazyma scolity in all characteristics, the species could be regarded as the anamorph of the latter species. Cryptococcus arengaensis was different from Cryptococcus heimaeyensis by all characteristics, and the strain SJ11L06 seemed to be distinguished from the other two strains. Tetrapisispora sp.(1) and Torulaspora sp.(1) should be examined by further identification approaches.

中文摘要 I
英文摘要 II
目錄 IV
表格目錄 V
圖目錄 VI
壹、前言 1
一、酵母菌的分類與鑑定 1
二、分子生物技術在酵母菌分類鑑定的應用 1
三、實驗標準菌種之現行分類體系與特徵 4
貳、材料與方法 12
一、藥品與培養基 12
二、器材 15
三、儀器 15
四、本實驗所使用之菌株 16
五、菌株之分離、培養與保存 21
六、實驗菌株之傳統鑑定 21
七、實驗菌株之分子特徵 22
参、結果與討論 31
一、形態與生理生化試驗 31
二、分子生物特徵 51
三、分類研究 97
四、分子生物技術評估 103
肆、結論 105
伍、參考文獻 106
附錄 115

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