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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩 | |
| dc.contributor.author | Yi-Hung Liu | en |
| dc.contributor.author | 劉益宏 | zh_TW |
| dc.date.accessioned | 2021-06-08T07:02:33Z | - |
| dc.date.copyright | 2009-02-10 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2009-02-02 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26194 | - |
| dc.description.abstract | 已知施用臘狀芽孢桿菌C1L菌株之菌液於葵百合及臺灣百合根圈，可有效地降低灰黴病之發病嚴重度，本論文研究C1L菌株誘導百合之抗病機制，並推測可能之訊息傳遞路徑。首先利用抑制性扣減雜交（suppression subtractive hybridization, SSH）技術，於臺灣百合上鑑定出預先處理C1L菌株而有差異性表現的基因LfGRP1、LfMT1、LfPsbR等。其中LfMT1為正調控表現，LfGRP1及LfPsbR呈負調控表現；在葵百合上，同源性基因之表現亦有有相同趨勢，推測C1L菌株由改變百合之基因表現，影響百合之生理功能，致使百合對灰黴病菌具有抵抗性，其作用機制與水楊酸誘導表現的抗病機制不同。其次，觀察到C1L菌株可促使葵百合抑制由灰黴病菌感染所造成之過氧化氫累積及百合細胞過度氧化及細胞死亡的情形。由活性氧(reactive oxygen species, ROS)抑制劑之作用及北方雜合分析之結果推測LfGRP1及LfPsbR基因的負調控表現與ROS代謝途徑有關。再者，利用乙烯及甲基茉莉酸處理葵百合時，LfGRP1及LfPsbR基因有增量表現，但預先處理離層酸時，此二基因之表現則會受到抑制，指出C1L菌株誘導百合對灰黴病產生抗病性，可能參與離層酸訊息傳遞路徑。另一方面，於葵百合葉片上單獨接種灰黴病菌24小時，與對照組比較之下，以Evan blue染色可以偵測到高含量的細胞死亡，而預先處理C1L菌株再接種灰黴病菌 24小時，可抑制百合葉片過氧化氫累積及細胞死亡。此外，以C1L菌株處理百合時，百合葉之過氧化酶活性，於處理後48小時達到最高，單一接種灰黴病菌 24小時，過氧化酶活性最強，但48小時即呈現負調控；預先處理C1L菌株24小時後再接種灰黴病菌時，過氧化酶活性並不會增加。另一方面，分析β-1,3-葡聚糖酶基因之表現，預先處理C1L菌株24小時再接種灰黴病菌，可提早於接種後48小時偵測到β-1,3-葡聚糖酶基因之表現，可能與百合葉抗灰黴病菌感染的能力有關。為進一步分析C1L菌株誘導百合抗灰黴病之作用機制，利用cDNA-AFLP技術在不同處理中共獲得葵百合之104個cDNA片段，以半定量反轉錄聚合酶鏈鎖反應偵測基因之表現，發現C1L菌株處理百合根系，可經由鈣離子的訊息傳遞，啟動抗病反應，而此過程會帶動與離層酸或似離層酸訊息路徑有關之清除活性氧物質的抗氧化反應。本研究並發現C1L菌株可誘導麩醯胺酸合成酵素基因之表現，其可能減輕灰黴病菌感染對百合細胞的毒化作用，因而降低灰黴病之嚴重度。 | zh_TW |
| dc.description.abstract | Application of Bacillus cereus C1L suspension in the rhizosphere of Lilium formosanum and lily cv. Star Gazer to reduce gray mold severity has been demonstrated. In this dissertation, the mechanism of induced systemic resistance (ISR) was approached to figure out the signaling pathway involved. Firstly, differentially expressed genes in strain C1L-treated L. formosanum were identified to be LfGRP1, LfMT1, and LfPsbR, by the use of suppression subtractive hybridization method. When L. formosanum was treated with strain C1L, LfMT1 was up-regulated. However, LfGRP1 and LfPsbR were down-regulated. The expression patterns of the 3 homologues in oriental lily cv. Star Gazer were identical to those of LfGRP1, LfMT1 and LfPsbR. When using ethylene and methyl jasmonate to treat lily cv. Star Gazer, LsGRP1 and LsPsbR were up-regulated. On the contrary, these genes were down-regulated by the treatment of abscissic acid (ABA), indicating that ABA signaling pathway is involved in the systemic resistance induced by strain C1L. When ‘Stra Gazer’ lily plants were treated with strain C1L 24 hours before inoculation with B. elliptica, accumulation of hydrogen peroxide and cell death in the leaves was suppressed as compared to those exhibited in the plant singly inoculated with B. elliptica. The peroxidase activity was not increased in the leaves inoculated with B. elliptica post strain C1L treatment, unlike that inoculated with B. elliptica singly. On the other hand, strain C1L induced early expression of LpGlu1 (β-1,3-glucanase) in response to B. elliptica infection post strain C1L treatment as compared to that inoculated with B. elliptica singly. In order to dissect the mechanism of ISR activated by strain C1L, cDNA-AFLP was analyzed and 104 cDNA clones representative of differentially expressed genes of lily were obtained. Expressions of selected genes were analyzed by semi-quantitative reverse-transcription polymerase chain reaction. The results showed that strain C1L could induce systemic resistance via calcium ion signaling pathway and ABA signaling pathway, followed by anti-oxidative response. Additionally, strain C1L induced expression of glutamate synthase of lily which might decrease cellular toxicity caused by B. elliptica and therefore reduced the disease severity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T07:02:33Z (GMT). No. of bitstreams: 1 ntu-97-D93633004-1.pdf: 15947206 bytes, checksum: 901723eab96694f8b51430a8d49888a6 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 中文摘要..................................................5
英文摘要..................................................7 前 人 研 究............................................9 壹、應用根圈細菌誘導植物抗病性之研究....................9 貳、活性氧與植物抗病之關係.............................14 參、植物抗氧化反應.....................................16 肆、百合灰黴病.........................................18 第一章 臘狀芽孢桿菌誘導百合系統抗病性之驗証及基因表現分析.......................................................21 壹、中文摘要...........................................21 貳、英文摘要...........................................22 參、前 言...........................................23 肆、材料與方法.........................................25 一、供試植物與菌株...................................25 二、百合灰黴病罹病度之計量...........................25 三、臘狀芽孢桿菌 C1L菌株群聚於百合根系試驗...........26 四、3-(3,4-dichlorophenyl)-1,1-dimethylurea ( DCMU)處理對百合之影響.............................................26 五、臺灣百合GRP基因之選殖............................26 六、百合葉片全RNA之萃取..............................27 七、百合葉片訊息RNA之萃取............................27 八、臘狀芽孢桿菌C1L菌株處理百合之SSH cDNA基因庫之製備.......................................................28 九、DNA 黏接反應.....................................30 十、大腸桿菌勝任細胞製備.............................31 十一、大腸桿菌細胞轉形反應...........................31 十二、菌落PCR........................................31 十三、百合cDNA探針製備...............................32 十四、以雜合點漬分析篩選差異性表現之cDNA.............32 十五、序列分析.......................................33 十六、核酸探針製備...................................33 十七、北方雜合分析...................................33 伍、結 果...........................................35 一、臘狀芽孢桿菌C1L菌株之生物防治潛力................35 二、選殖臘狀芽孢桿菌 C1L菌株誘導之百合差異性表現基因.......................................................35 三、LfGRP1、LfMT1及LfPsbR基因分析....................36 四、百合防禦相關基因之表現...........................37 五、DCMU抑制百合灰黴病之抗性及影響基因之表現.........38 陸、討 論...........................................39 柒、圖 表 集.........................................42 表一、臘狀芽孢桿菌C1L菌株於葵百合根系減少灰黴病之發生.......................................................43 表二、臘狀芽孢桿菌C1L菌株誘導臺灣百合基因之差異性表現.......................................................44 圖一、臘狀芽孢桿菌C1L菌株抑制臺灣百合灰黴病之發生....45 圖二、臘狀芽孢桿菌C1L群聚臺灣百合根系之族群量........46 圖三、電泳分析SSH之cDNA產物..........................47 圖四、LfGRP1之核苷酸及預測胺基酸序列.................48 圖五、LfMT1之核苷酸及預測胺基酸序列..................49 圖六、葵百合LsMT1及臺灣百合LfMT1之核苷酸序列比對.....50 圖七、葵百合LsPsbR及臺灣百合LfPsbR之核苷酸序列比對...51 圖八、百合葉LfGRP1及LsGRP1基因表現之北方雜合分析.....52 圖九、百合葉MT1及PsbR基因表現之北方雜合分析..........53 圖十、施用DCMU抑制灰黴病在葵百合葉造成之病斑數目.....54 圖十一、DCMU處理葵百合之全RNA北方雜合分析............55 第二章 臘狀芽孢桿菌誘導百合葉抗灰黴病之分子機制探討......56 壹、中文摘要...........................................56 貳、英文摘要...........................................57 參、前 言.............................................58 肆、材料與方法.........................................61 一、供試植物及菌株...................................61 二、灰黴病菌對過氧化氫之感受性試驗...................61 三、植物抑制劑處理...................................61 四、植物荷爾蒙誘導百合抗病性試驗.....................62 五、百合葉組織化學試驗...............................62 六、過氧化氫之萃取與定量.............................64 七、過氧化酶之萃取與定量.............................64 八、葵百合葉全RNA之萃取..............................64 九、北方雜合分析.....................................65 伍、結 果...........................................67 一、觀察灰黴病菌誘發百合細胞死亡之情形...............67 二、灰黴病菌對過氧化氫之耐受性試驗...................67 三、臘狀芽孢桿菌C1L菌株抑制灰黴病菌感染葵百合造成之過氧化氫累積.................................................68 四、臘狀芽孢桿菌C1L菌株抑制LsGRP1及LsPsbR基因之表現.......................................................68 五、百合中過氧化氫參與LsGRP1及LsPsbR基因之表現.......69 六、臘狀芽孢桿菌C1L菌株誘導百合抗病反應參與著離層酸訊息傳遞路徑.................................................70 七、臘狀芽孢桿菌C1L菌株抑制百合細胞死亡..............70 八、臘狀芽孢桿菌C1L菌株誘發百合葉過氧化酶活性增加....71 九、臘狀芽孢桿菌C1L菌株誘發百合抗灰黴病與促使β-1,3-葡聚糖酶提早表現有關.........................................71 陸、討 論...........................................72 柒、圖 表 集.........................................77 圖一、觀察灰黴病菌誘發葵百合細胞死亡之情形...........78 圖二、過氧化氫對灰黴病菌孢子發芽及菌絲生長影響.......79 圖三、葵百合葉過氧化氫之累積.........................80 圖四、於不同時間點分析不同處理之葵百合葉過氧化氫含量.......................................................81 圖五、葵百合葉LsGRP1及LsPsbR基因表現之時間模式分析...82 圖六、DPI及CAT處理之葵百合葉LsGRP1及LsPsbR基因表現之北方雜合分析...............................................83 圖七、DPI及CAT處理之葵百合葉過氧化氫含量.............84 圖八、ROS抑制劑及C1L菌株處理降低葵百合灰黴病之發病程度.......................................................85 圖九、ACC、MeJA及ABA影響葵百合葉LsGRP1及LsPsbR基因表現之時間模式分析...........................................86 圖十、葵百合葉之酚化物累積觀察.......................87 圖十一、葵百合葉細胞死亡計量.........................88 圖十二、葵百合葉過氧化酶活性之時間模式分析...........89 圖十三、葵百合葉β-1,3-glucanase基因表現分析..........90 第三章 臘狀芽孢桿菌誘導百合葉抗氧化反應以抗灰黴病菌之感染.......................................................91 壹、中文摘要...........................................91 貳、英文摘要...........................................92 參、前 言...........................................93 肆、材料及方法.........................................95 一、供試植物及菌株...................................95 二、 cDNA-AFLP分析之植物處理.........................95 三、化學物質處理.....................................95 四、葵百合葉全RNA之萃取..............................96 五、葵百合葉訊息RNA之萃取............................97 六、構築cDNA-AFLP（Amplified Fragment Length Polymorphism）基因庫.....................................97 七、cDNA-AFLP電泳分析................................98 八、半定量反轉錄聚合酵素連鎖反應（Semi-quantitative reverse transcription polymerase chain reaction, sqRT-PCR）....................................................99 九、葵百合核酸探針製備..............................100 十、北方雜合分析....................................100 伍、結 果............................................101 一、 葵百合cDNA-AFLP及分子選殖......................102 二、 葵百合差異性表現cDNAs驗証......................103 三、 葵百合差異性表現cDNAs之分群....................103 四、 以sqRT-PCR分析葵百合基因之差異性表現...........104 五、 化學物質對百合受灰黴病菌感染及其麩醯胺酸合成酶基因表現之影響..............................................105 陸、討 論..........................................105 柒、圖 表 集........................................112 表一、cDNA-AFLP定序分析.............................112 表一（續）、cDNA-AFLP定序分析.......................114 表一 （續）、cDNA-AFLP定序分析......................116 表二、DPI , ABA, CaCl2及LaCl3對灰黴病菌感染百合之影響......................................................120 圖一、cDNA-AFLP之cDNAs選殖..........................121 圖二、以cDNA-AFLP技術分析不同處理之葵百合葉片基因表現......................................................122 圖三、cDNA-AFLP之cDNA選殖株之電泳分析...............123 圖四、驗証cDNA-AFLP之cDNA選殖株的基因差異性表現.....124 圖五、不同處理下之葵百合葉差異性表現基因之分群......125 圖六、以sqRT-PCR分析不同處理後之葵百合葉基因表現....126 圖七、以sqRT-PCR分析不同處理後葵百合葉麩醯胺酸合成酵素(GS)基因之表現..........................................127 圖八、臘狀芽孢桿菌誘導百合抗病路徑模式圖............128 參考文獻................................................129 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細胞死亡 | zh_TW |
| dc.subject | 根圈細菌 | zh_TW |
| dc.subject | 抗氧化作用 | zh_TW |
| dc.subject | 離層酸 | zh_TW |
| dc.subject | 差異性表現 | zh_TW |
| dc.subject | 過氧化氫 | zh_TW |
| dc.subject | 誘導系統性抗病 | zh_TW |
| dc.subject | plant cell death | en |
| dc.subject | abscisic acid | en |
| dc.subject | hydrogen peroxide | en |
| dc.subject | induced systemic resistance | en |
| dc.subject | antioxidation | en |
| dc.subject | Rhizobacteria | en |
| dc.title | 臘狀芽孢桿菌C1L菌株誘導百合系統性抗灰黴病菌之研究 | zh_TW |
| dc.title | Studies on Bacillus cereus C1L-induced systemic resistance against Botrytis elliptica in Lilium | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 葉瑩,曾德賜,許元勳,王肇芬,洪挺軒 | |
| dc.subject.keyword | 根圈細菌,誘導系統性抗病,過氧化氫,細胞死亡,差異性表現,抗氧化作用,離層酸, | zh_TW |
| dc.subject.keyword | Rhizobacteria,induced systemic resistance,hydrogen peroxide,plant cell death,antioxidation,abscisic acid, | en |
| dc.relation.page | 151 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2009-02-02 | |
| 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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