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研究生:郭大榮
論文名稱:白色念株菌之EFG1和/或CPH1下游基因的分類與確認
論文名稱(外文):Categorization and identification of the genes downstream of EFG1 and/or CPH1 in Candida albicans
指導教授:楊昀良
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:69
中文關鍵詞:白色念株菌相減雜交技術
外文關鍵詞:Candida albicanssuppression subtractive hybridization
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白色念珠菌是一種伺機型真菌類病源菌,和人類共生,在免疫系統不健全的人體中,可引起致命性的傷害。而大部份的抗真菌藥物都具有副作用,且在使用頻繁的情況下,抗藥性問題也日益嚴重。野生種白色念珠菌是屬於雙形態生物,會形成菌絲或以單細胞酵母形態存在。一般認為菌絲的形成是造成宿主細胞致病的重要步驟(43)。過去的研究發現在缺乏轉錄因子Efg1和Cph1的突變種在含有羊血清的培養液中,失去菌絲形成的能力,且對小鼠失去致病力(32)。因此推測在這兩個轉錄因子調控的下游中包括了形態變化與致病能力的基因,這些基因將是新藥物目標的研究方向。
實驗室先前運用相減雜交技術(suppression subtractive hybridization,SSH),得到在野生種(SC5314)基因表現相對於EFG1和CPH1雙突變種(HLC54)基因表現的差異,如果在野生種中表現量較大,即表示這類基因受Efg1或Cph1正向調控,其中包含影響形態或致病之基因。我們共得到991個選殖體,但EFG1出現的比例過高,所以用限制脢圖析(restriction mapping)的方式,期望將有相同片段數量及大小的選殖體分為同一組,以減少定序的數量,接著將定序結果在Stanford's Current Assembly of Candida albicans Sequence及NCBI中做比對,把所得到的基因依照功能分類,共得到五十二個已知基因及四十八個未知基因,再經由北方墨點法(northern blot)對野生種及突變種的mRNA層次做進一步的確認,共挑選十個基因,分別為ASH1、HEM13、HWP1、KEL2、RCK2、TCA2、TPS2、UBR1、URA3、YMR128,發現HWP1、URA3、HEM13、KEL2、YMR128五個基因符合野生種的表現量較大。在其它基因晶片未問世的物種,可以使用此方法找出特定基因。

Candida albicans is commonly found as a commensal in the human population, causing infection in immunocompromised patients. Many of the antifungal drugs have undesirable side effects and their widespread usages have led to the development of drug-resistant isolates. C.albicans is dimorphic and capable of growth in filamentous form and in yeast form. Filamentous growth is believed to be an important step in the invasion and pathogenesis process(43). A mutant strain lacking the transcription factors Efg1 and Cph1 is extremely defective in filamentous growth in YPD plus goat serum and also avirulence in a mouse model(32). Hence, they are involved in both morphology and virulence pathways. Genes regulated by Efg1 and Cph1 may be new antifungal drug targets of C.albicans.
Previous research in the laboratory has used the method of suppression subtractive hybridization(SSH) for identifying virulence genes by screening for difference in the expression between the wild type strain(SC5314) and the cph1/cph1 efg1/efg1 double mutant strain(HLC54) in C.albicans. If a gene has higher expression level in the wild-type strain than in the cph1/cph1 efg1/efg1 double mutant strain, this gene is downstream of the CPH1 and EFG1 and is positively regulated by them. The gene may involve in filamentous growth and may even be a virulence gene. 991 clones have come out of the SSH screening. EFG1 is the most abundant one. Restriction mapping was used to sort those clones into groups to reduce the number of samples for sequencing. After comparing those sequences with the databases of Stanford's Current Assembly of Candida albicans Sequence and NCBI, the sequences are classified by functions. Totally, there are 52 known genes and 48 unknown genes. I pick 10 samples, including ASH1、HEM13、HWP1、KEL2、RCK2、TCA2、TPS2、UBR1、URA3 and YMR128, to determine the expression level of mRNA. Five of them, HWP1、URA3、HEM13、KEL2 and YMR128, match the expected results. Their gene expression levels are higher in the wild type than in the cph1/cph1 efg1/efg1 double mutant. The SSH can be used for other organisms whose gene chips are not yet available.

頁次
中文摘要………………………………………………………………….i
英文摘要…………………………………………………………………ii
誌謝……………………………………………………………………...iii
目錄………………………………………………………………………iv
圖表目錄………………………………………………………………..vi
壹、 緒論………………………………………………………………...1
1.1研究緣由……………………………………………………………..1
1.2致病因子……………………………………………………………..2
1.3形態與致病……………………………………………………….….3
1.4實驗室前人研究…………………………………………………….4
1.5研究內容簡介……………………………………………………….6
1.5.1基因分類…………………………………………………………..6
1.5.2基因表現確認……………………………………………………..6
貳、實驗材料……………………………………………………………8
2.1菌種…………………………………………………………………..8
2.2質體…………………………………………………………………..8
2.3白色念珠菌野生種(SC5314)相對於
突變種(HLC54)之特有基因群……………………………………8
2.4引子…………………………………………………………………..9
2.5藥品試劑……………………………………………………………..9
2.6溶液及緩衝液………………………………………………………11
2.7培養基與培養液……………………………………………...……12
2.8設備………………………………………………………………….12
參、 方法………………………………………………………….……13
3.1質體DNA粹取(Plasmid DNA extraction)……………………..13
3.2大腸桿菌轉形(E.coli. Transformation)…………………………13
3.3 RNA離析(Total RNA Isolation)…………………………………13
3.4膠體粹取(Gel extraction)………………………………………..14
3.5 DNA標記(DNA labeling)……………………………………….14
3.6北方墨點(Northern bloting)……………………………………..15
3.7聚合脢連鎖反應(Polymerase Chain Reaction)………………..16
3.8 Clones的分組……………………………………………………..16
3.9基因序列比對過程………………………………………………..16
肆、 結果……………………………………………………………….18
4.1限制脢圖析分類…………………………………………………..18
4.2 定序比對…………………………………………………………..18
4.3 功能分類…………………………………………………………..19
4.4 北方墨點法………………………………………………………..19
4.4.1 片段確認及定量………………………………………………..19
4.4.2 各基因相關特性簡介及表現量結果…………………………19
(1) HWP1及RBT1……………………………………………………..19
(2) TPS2…………………………………………………………………20
(3) URA3………………………………………………………………..21
(4) ASH1………………………………………………………………...21
(5) HEM13………………………………………………………………21
(6) KEL2………………………………………………………………...22
(7) RCK2………………………………………………………………..22
(8) TCA1………………………………………………………………...23
(9) UBR1…………………………………………………….………….23
(10) YMR128……………………………………………………………24
4.4.3 綜合結果……………………………………………………...…24
伍、 討論……………………………………………………………….47
5.1 目標基因的比對……………………………………………….…47
5.2 資料庫比對之結果……………………………………………….47
5.3 Suppression subtractive hybridization方法的正確度………..48
5.4 致病基因定義…………………………………………….……….49
5.5 依功能分類篩選結果…………………………………………….50
5.6 基因表現分析……………………………………………………..51
陸、 參考文獻………………………………………………………….57
圖表目錄
頁次
圖一、基因序列比對過程………………………………….…….…..25
圖二、HaeⅢ restriction mapping……………………………………26
圖三、SauⅢAⅠ restriction mapping…………….…………………27
圖四、Ribosomal RNAs在電泳膠上的相對位置………………….28
圖五、HWP1……………………………………………………………29
圖六、TPS2…………………………………………….…………….…30
圖七、URA3……………………………………………….……………31
圖八、ASH1…………………………………………………………….32
圖九、HEM13…………………………….…………………………….33
圖十、KEL2…………………………………………………………….34
圖十一、RCK2………………………………………………………....35
圖十二、TCA2………………………………………………………….36
圖十三、UBR1………………………………………………………….37
圖十四、YMR128………………………………………………………38
表一、Restriction mapping分類及各組定序比對的結果………..39
表二、直接定序比對的結果……………………………….………..40
表三、未知功能基因分組的結果..……………………………….…42
表四、對已知知基因依功能分類…………………………………...44
附圖一、S. cerevisiae因外在環境所引發的MAP kinase
pathway……………………………………………….……..64
附圖二、C.albicans形態變化的調控途徑…………………………65
附表一、已知為毒性因子的代謝基因………….………….…….…66
附表二、已知為毒性因子的細胞壁基因…………………….…..…67
附表三、已知為毒性因子的訊號傳遞基因與轉錄因子……….….68
附表四、已知為毒性因子的分泌因子與其它真菌決定因子…….69

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