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研究生:林啟陽
研究生(外文):LIN CHI YANG
論文名稱:白色念珠菌抗毒性相關基因之研究
論文名稱(外文):Study of Genes Associated with Anti-virulence Activity
指導教授:楊昀良
指導教授(外文):Yang Yun-Liang
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:59
中文關鍵詞:白色念珠菌
外文關鍵詞:Candida albicans
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中文摘要
白色念珠菌為伺機型真菌性病菌,所以應有特殊的調控基因,平時抑制白色念珠菌之致病活性,而與人體能相安無事。前人藉由使用抑制刪除雜交法,找到長菌絲(SC5314)與不長菌絲(HLC54)兩者之間表現不同的基因片段,得到可能抑制菌絲生長者有340 cDNA clones。同時,不長菌絲者亦明顯降低致病能力(Lo,1997)。因此這些會抑制菌絲生長的基因,能抑制白色念珠菌酵母菌狀轉變成菌絲狀的途徑,同時,此一型態轉換途徑也是伴隨著致病力產生的途徑(目前對於型態、致病兩者間關係並不清楚)。所以可以將抑制菌絲生長及影響致病力的基因同時分離出來。本論文之目的在分析整理所分組之負調控因子,並採樣以北方墨點法確認抑制刪除雜交法的結果。為減少定序340 cDNA clones數量,先以restriction mapping將之分成266組。再將定序結果透過Stanford白色念珠菌基因資料庫及NCBI資料庫,依功能分類得到220種基因,其中Protein synthesis佔了15 %、Metabolism佔了5 %、Structure佔了 6 %、Others佔了 6 %、Unknown function佔了 55 %其餘則為Undetermined。依定序結果比對資料庫後,選定上述經分類10個基因,作北方墨點實驗,以檢定抑制刪除雜交法之結果。其中3個符合HLC54 strain抑制菌絲生長的mRNA表現量大於SC5314 strain菌絲生長的mRNA表現量,與抑制刪除雜交法結果一致。但與期待相反之基因有1個,即此基因反而在SC5314 strain菌絲生長的mRNA表現量多。其餘則沒有差別。

ABSTRACT
Candida albicans is an opportunistic fungal pathogen. It can colonize in the healthy bodies of humans without doing harms to them. Supposedly, there are genes that can regulate the pathogenic activity of Candida albicans in our healthy everyday life. Previously, researchers have screened for the genes repressing hyphal formation and virulence using the method of Suppressive Subtracted Hybridization (SSH). They have obtained 340 cDNA clones that are associated with repression of hyphal formation. It has been shown that HLC54 strain (cph1/cph1 efg1/efg1) repressed hyphal formation and dropped down pathogenicity in mouse model dramatically (Lo, 1997). Therefore, those genes, which repress the hyphal formation, can block the pathway of switching from yeast form to hyphal form in Candida albicans, and the switch may come with the pathogenicity (the relationship between morphology and pathogenicity is not clear.). As a conclusion, researchers can obtain the genes that affect the pathogenesis and repress the hyphal formation at the same time. The objectives of this research are (1) to analyze those clones of potential negative regulators of filamentous growth and virulence, and (2) to assess the result of SSH by northern blotting. First, I divided the samples into 266 groups by restriction mapping in order to reduce the number of samples undergoing sequence analysis. Then, I sorted the results of sequence analyses into 220 categories and further classified them according to gene function using the information of Candida albicans in the NCBI and Stanford databases. Totally, 15% of the genes are used for protein synthesis, 5% for metabolism, 6% for structure, 6% for others, 55% for unknown functions, and the remaining are undetermined. Following the sequence analyses and the databases search, I chose 10 genes to determine the gene expression by northern blotting. And then, I found that 3 genes expressed much more mRNA in the HLC54 strain than in the SC5314 strain, which is consist with the result of SSH. But there was one gene expressed more mRNA in the SC5314 strain, which forms hypha, and is in opposite of the result of SSH. The rest showed no difference in the two strains.

目錄
頁數
中文摘要………………………………………………………………………….......i
英文摘要(Abstract).... …….................................................…...............................…..ii
誌謝..................................................................................…..................................…...iii
目錄................................................................................…....................................…...iv
圖表目錄..........................................................................................................…….... vi
壹、緒言………………………………………………………………………………1
1.1 前言...…………………………………………………………………………..1
1.2 白色念珠菌毒性因子………………………………………………………….1
1.2.1 白色念珠菌的黏附作用(Adhesin)………………………………………..2
1.2.2 環境改變時白色念珠菌的反應…………………………………………..2
1.2.3白色念珠菌分解酶…………………………………………………………3
1.3 研究計劃設計及原理簡介…………………………………………………….4
1.3.1抑制刪除雜交法(Suppression Subtractive Hybridization) ……………….4
1.3.2選殖株之分類……………………………………………………………...5
1.3.3資料分析…………………………………………………………………...5
1.3.4北方墨點法確認基因活性………………………………………………...6
1.3.5快速破壞突變法(Rapid disruption) ……………………………………….6
1.4 實驗目標……………………………………………………………………….6
貳、材料………………………………………………………………………………7
2.1 菌種…………………………………………………………………………….7
2.2 質體…………………………………………………………………………….7
2.3 引子…………………………………………………………………………….7
2.4 藥品試劑……………………………………………………………………….8
2.5 緩衝溶液及溶劑……………………………………………………………….9
2.6 儀器設備………………………………………………..…………………….10
參、方法………………………………………………………………………………12
3.1 RNA之萃取…………………………………………………………………...12
3.2 北方墨點(Northern blot analysis).……………………………………………12
3.2.1 DNA標記…………………………………………………………………12
3.2.2 RNA轉漬..………………………………………………………………..13
3.2.3 雜交反應…………………………………………………………………14
3.2.4 免疫偵測…………………………………………………………………14
3.3基因之破壞……………………………………………………………………14
3.3.1含篩選標誌(selection marker)及欲突變基因兩旁序列之置換片段……15
3.3.2白色念珠菌轉形………………………………………………………….15
3.3.3確定突變位置…………………………………………………………….16
3.4質體DNA之萃取……………………………………………………………...16
3.5 PCR產物之純化………………………………………………………………17
3.6洋菜膠內DNA之萃取………………………………………………………..17
3.7 選殖株之分類………………………………………………………………...17
3.8 資料分析……………………………………………………………………...17
肆、結果………………………………………………………………………………19
4.1選殖株之分類結果……………………………………………………………19
4.2定序資料分析…………………………………………………………………19
4.3北方墨點法確認基因活性……………………………………………………20
4.3.1 TSL1北方墨點法結果……………………………………………………20
4.3.2 PUT3北方墨點法結果…………………………………………………...21
4.3.3 LRT3北方墨點法結果……………………………………………………21
4.3.4 ASC1北方墨點法結果…………………………………………………...22
4.3.5 ITR2北方墨點法結果……………………………………………………22
4.3.6 CaNIK1北方墨點法結果………………………………………………...23
4.3.7 Clone no.53北方墨點法結果…………………………………………….23
4.3.8 Clone no.78北方墨點法結果…………………………………………….23
4.3.9 Clone no.146北方墨點法結果…………………………………………...24
4.3.10 Clone no.161北方墨點法結果………………………………………….24
4.4快速破壞突變法………………………………………………………………24
伍、討論………………………………………………………………………………26
5.1選殖株之分類…………………………………………………………………26
5.2定序資料分析…………………………………………………………………27
5.3北方墨點法確認基因活性……………………………………………………27
5.3.1 TSL1北方墨點法…………………………………………………………28
5.3.2 PUT3北方墨點法………………………………………………………...28
5.3.3 LRT3北方墨點法…………………………………………………………28
5.3.4 ASC1北方墨點法………………………………………………………...28
5.3.5 ITR2北方墨點法…………………………………………………………29
5.3.6 CaNIK1北方墨點法……………………………………………………...29
5.3.7 Clone no.53北方墨點法………………………………………………….29
5.3.8 Clone no.78北方墨點法………………………………………………….29
5.3.9 Clone no.146北方墨點法………………………………………………...30
5.3.10 Clone no.161北方墨點法……………………………………………….30
5.3.11 SSH結果之探討………………………………………………………...30
5.4快速破壞突變法………………………………………………………………30
參考文獻……………………………………………………………………………..32
圖表目錄
頁數
表一、限制酵素切割cDNA clones 之分類…………………………………………37
表二、cDNA clones依資料比對結果之功能性分類………………………………..38
圖一、限制酵素切割cDNA clones…………………………………………………..45
圖二、TSL1北方墨點………………………………………………………………...46
圖三、PUT3北方墨點…...…………………………………………………………..47
圖四、LRT3北方墨點……...………………………………………………………...48
圖五、ASC1北方墨點……………………………………………………………….49
圖六、ITR2北方墨點………………………………………………………………...50
圖七、Ca NIK1北方墨點……………………………………………………………51
圖八、Clone no.53 北方墨點………………………………………………………..52
圖九、Clone no.78 北方墨點………………………………………………………..53
圖十、Clone no.146 北方墨點………………………………………………………54
圖十一、Clone no.161 北方墨點……………………………………………………55
圖十二、確認PUT3之突變…………………………………………………………56
圖十三、確認URA3轉形至BWP17……………………………………………….57
附圖A、pCRⅡ vector ……………………………………………………………..58
附表A、glutamate對長菌絲之影響 ………………………………………………59

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