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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27210完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩 | |
| dc.contributor.author | Ru-Yin Huang | en |
| dc.contributor.author | 黃儒音 | zh_TW |
| dc.date.accessioned | 2021-06-12T17:58:02Z | - |
| dc.date.available | 2013-02-01 | |
| dc.date.copyright | 2008-02-01 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-01-30 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27210 | - |
| dc.description.abstract | 本實驗室由百合根圈分離到一株臘狀芽孢桿菌(Bacillus cereus) C1L菌株，對多種植物病原真菌具有拮抗作用。由於目前對臘狀芽孢桿菌C1L菌株是否會產生已知的抗生物質，或是亦會產生其他抗生物質瞭解甚少，因此擬由轉位子誘變方式篩選失去對真菌拮抗作用之C1L誘變株，以進一步獲得與臘狀芽孢桿菌C1L菌株產生抗生物質有關的基因。本研究以紅黴素誘導Tn917ac1轉位子於C1L菌株中轉位，獲得850個誘變株，並將誘變株與十字花科蔬菜黑斑病菌（Alternaria brassicicola）對峙培養，篩選出35株拮抗能力減弱的誘變株；接著再以煙草赤星病菌（Alternaria longipes）進行篩選，獲得5株抗真菌能力顯著減弱的誘變株。進行南方雜合分析確定Tn917ac1插入這5個誘變株的全基因體中，其中只有1個誘變株（M-902）帶有單個Tn917ac1。當與四個病原真菌（A. brassicicola, A. longipes, Botrytis cinerea, B. elliptica）對峙培養時，誘變株M-902均表現減弱之抑菌能力，培養上清液對此四種真菌孢子之發芽亦表現減弱之抑菌能力。Tn917ac1週邊序列分析指出Tn917ac1插入一質體上，南方雜合分析亦顯示，誘變株M-902的質體在電泳膠體上有位移的現象，且Tn917ac1插入一開放解讀框架-orf1及其預測啓動子間。orf1緊鄰另一開放解讀框架-orf2，可能為同一個操縱組（operon）中的結構基因；以A. brassicicola做為測菌株進行互補試驗，結果顯示，包含預測之啟動子，orf1及orf2的載體，或者包含單一orf2 (含啓動子)在對孢子發芽之抑菌試驗中，可使誘變株M-902部分回復對孢子發芽之抑菌功能，以單一orf1 (含啓動子)無法使誘變株M-902回復對A. brassicicola之抑菌功能，由此結果推測orf2可能與C1L菌株抗生物質的產生相關。 | zh_TW |
| dc.description.abstract | Bacillus cereus C1L, an antagonistic bacterium, was originally isolated from the rhizosphere of Fomorsa lily. In order to identify the genes related to the antagonistic ability of B. cereus C1L, a transponson insertion mutant library was constructed. Totally, 850 mutants were obtained. Alternaria brassicicola and Alternaria longipes were used in mutant screening on potato dextrose agar and a mutant, M-902, with significant decrease on antifungal activity was selected. M-902 loses its inhibitory activity against mycelial growth of A. brassicicola, A. longipes, Botrytis cinerea, and Botrytis elliptica ; in addition, the culture supernatant of M-902 decreases spore germination rate. Sequence analysis revealed that the Tn917ac1 was inserted into a plasmid pC1L8 of B. cereus C1L wild-type strain. Southern blot analysis showed a band shift of pC1L8 of B. cereus M-902. The Tn917ac1 insertion site was located downstream a putative promoter of open reading frames-orf1 and orf2。These two ORFs may constitute a two-gene operon. In a complementay test using A. brassicicola as the test fungus, the vector containing predicted promoter, orf1 and orf2, or orf2 singly showed a partial recovery of the inhibition on conidial germination, but orf1 singly could not recover the inhibition on conidial germination. Thus, orf 2 related to antibiotic production of strain C1L was presumed. | en |
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| dc.description.tableofcontents | 壹、中文摘要 iv
貳、英文摘要 v 參、前 言 1 肆、前人研究 3 伍、材料與方法 8 一、供試菌株與質體 8 二、利用轉位子Tn917ac1建構C1L菌株之突變庫 8 三、誘變株篩選 8 四、確認轉位子插入C1L誘變株染色體之南方雜合分析 9 4.1 C1L誘變株染色體DNA之抽取 9 4.2 南方雜合分析 10 五、確認誘變株抗真菌能力減弱 11 5.1 對峙培養 11 5.2 孢子發芽試驗 11 六、生長曲線 11 七、確認轉位子插入位置 11 7.1 將帶有轉位子的染色體片段送進大腸桿菌 11 7.2 大腸桿菌勝任細胞之製備及轉形 12 7.3 大腸桿菌質體抽取、限制酵素切割分析及聚合酶連鎖反應 12 7.4 定序及電腦序列分析 13 7.5 臘狀芽孢桿菌質體抽取及其南方雜合分析 13 八、互補試驗 14 8.1 質體的構築 14 8.2 穿梭質體之構築 15 8.3 電穿孔轉形 15 8.4 確認轉形株 17 8.5 孢子發芽試驗 17 九、野生株及誘變株內孢子形成能力測試 17 陸、結 果 19 一、誘變株的篩選 19 二、誘變株的抑制真菌能力 19 三、生長曲線 19 四、Tn917ac1插入誘變株的位置及序列比對 20 五、互補株試驗 21 六、野生株及誘變株內孢子形成測試 23 柒、討 論 24 捌、圖表集 28 表一、供試菌株 29 表二、供試質體 30 表三、臘狀芽孢桿菌誘變株抑制真菌能力之等級 31 表四、臘狀芽孢桿菌誘變株之抑菌能力等級 32 表五、臘狀芽孢桿菌C1L野生菌株及誘變株M-902之抑制真菌的能力比較 34 表六、pC1L8之4076∼1878 bp預測啟動子及其相關序列 35 表七、引子 36 圖一、以南方雜合分析偵測不同臘狀芽孢桿菌誘變株其內含Tn917ac1的數目 37 圖二、臘狀芽孢桿菌C1L野生菌株及誘變株M-902對真菌的拮抗能力 38 圖三、臘狀芽孢桿菌C1L野生菌株及誘變株M-902上清濾液對四種菌孢子發芽之抑制率 39 圖四、臘狀芽孢桿菌C1L野生菌株及誘變株M-902之生長曲線 40 圖五、pM902E及pM902B 之限制酶切割分析 41 圖六、以反向聚合酶連鎖反應確定pM902E具有Sp6及T7引子 42 圖七、Tn917ac1插入位置之分析 43 圖八、質體pC1L8之之序列分析(4076∼1878 bp) 45 圖九、臘狀芽孢桿菌C1L野生型菌株及誘變株M-902之質體pC1L8的 南方雜合分析 46 圖十、pC1L8之ORF1與Sorangium cellulosum 'So ce 56' 假設性分泌蛋白質之胺基酸序列比對 47 圖十一、pC1L8之ORF2與Sorangium cellulosum 'So ce 56' 假設性膜蛋白質之胺基酸序列比對 48 圖十二、ORF1及ORF2之transmembrane helices預測 49 圖十三、以專一性引子308及309對臘狀芽孢桿菌C1L野生菌株染色體及質體進行聚合酶連鎖反應之電泳圖 50 圖十四、pGLKO1O2及pGLKO1之質體構築方式 51 圖十五、pGLKO2之質體構築方式 52 圖十六、pGLK、pGLKO1、pGLKO2及pGLKO1O2之質體圖譜 53 圖十七、pGLK、pGLKO1、pGLKO2及pGLKO1O2之限制酶切割分析 54 圖十八、以聚合酶連鎖反應確認pGLKO1、pGLKO2及pGLKO1O2轉 形入誘變株M-902 55 圖十九、互補試驗 56 圖二十、臘狀芽孢桿菌C1L野生菌株及誘變株M-902內孢子產生之比較 57 玖、參考文獻 58 | |
| dc.language.iso | zh-TW | |
| dc.title | 臘狀芽孢桿菌C1L菌株之轉位子誘變及抗真菌相關基因選殖 | zh_TW |
| dc.title | Transposon mutagenesis of antagonistic bacterium
Bacillus cereus C1L and the cloning of genes related to antifungal activity | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 潘銘正,劉世東,曾顯雄 | |
| dc.subject.keyword | 轉位子誘變, 臘狀芽孢桿菌, 抗真菌活性, 生物防治菌, 分子選殖, | zh_TW |
| dc.subject.keyword | transposon mutagenesis, Bacillus cereus, antifungal activity, biocontrol bacterium, molecular cloning, | en |
| dc.relation.page | 66 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-01-30 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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