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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 陳昭瑩 | |
dc.contributor.author | Ying-Chieh Pan | en |
dc.contributor.author | 潘映潔 | zh_TW |
dc.date.accessioned | 2021-06-15T13:44:46Z | - |
dc.date.available | 2017-12-11 | |
dc.date.copyright | 2015-12-11 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-12-03 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51690 | - |
dc.description.abstract | 植物演化出多種防禦機制,如產生抗菌物質以抵禦病原菌感染。本研究室以水楊酸誘發葵百合對灰黴病菌 (Botrytis elliptica) 之抗病性,並觀察到一解碼為富含甘胺酸蛋白之基因LsGRP1 (Lilium ‘Star Gazer’ glycine-rich protein 1) 表現量增加。生體外抑菌試驗指出LsGRP1C (LsGRP1 C端富含半胱胺酸區段) 合成胜肽能夠引起多種真菌的細胞膜透化、活性氧物質 (reactive oxygen species, ROS) 累積以及染色質凝集 (chromatin condensation) 之現象。據此,本研究推測B. elliptica感染過程中,葵百合可能透過LsGRP1C誘發B. elliptica之計畫性細胞死亡 (programmed cell death, PCD) 以增進百合對B. elliptica的抗病性。利用LsGRP1衍生性合成胜肽與大腸桿菌表現之融合蛋白於生體外處理B. elliptica的孢子,確認LsGRP1C具有抑制B. elliptica孢子發芽的活性;續以大腸桿菌蛋白表現系統生產LsGRP1、LsGRP1C與LsGRP1ΔC融合蛋白,處理B. elliptica之接種結果則指出, LsGRP1C不僅能夠抑制B. elliptica孢子發芽,亦可阻止百合上的病斑擴展。使用4',6-diamidino-2-phenylindole (DAPI) 與terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) 染色分析的結果指出,含有LsGRP1C的融合蛋白會於生體外誘發B. elliptica染色質凝集、細胞核DNA斷裂等PCD現象。經水楊酸預處理之百合,有較多的LsGRP1累積與較少的B. elliptica感染所造成之植物細胞死亡,且B. elliptica的PCD提早發生及發生率增加,暗示植物體上真菌PCD現象與LsGRP1的作用有關。進一步將LsGRP1衍生性融合蛋白分別注入葵百合葉片後接種B. elliptica,以trypan blue 染色和即時定量聚合酶連鎖反應 (real-time quantitative polymerase chain reaction) 進行檢測的結果,含有LsGRP1C之融合蛋白的葉片中病原菌族群量較低,且B. elliptica的PCD發生時間點較早,持續時間較久且發生率較高。因此,LsGPR1之C端區段應可透過誘發真菌PCD以減少B. elliptica對百合的侵染,此為百合上未曾被報導之防禦機制。 | zh_TW |
dc.description.abstract | Plants evolve various defense mechanisms against pathogen infection, such as production of antimicrobial compounds. Our previous studies demonstrated that salicylic acid pretreatment was able to induce disease resistance against pathogenic fungus Botrytis elliptica, accompanying with an enhanced expression of LsGRP1, a glycine-rich protein encoding gene. The C’-terminal region of LsGRP1, LsGRP1C, was proved to inhibit several plant fungal pathogens in vitro via a mechanism involving permeabilization of fungal membrane, reactive oxygen species accumulation and chromatin condensation. Accordingly, function of LsGRP1 was proposed to increase lily resistance to B. elliptica via inducing fungal programmed cell death (PCD) in planta, through the antimicrobial activity offered by LsGRP1C. In this study, in vitro antifungal assay of the synthetic peptides and Escherichia coli-expressed fusion proteins derived from LsGRP1 confirmed that LsGRP1C exhibited inhibition effect on spore germination of B. elliptica. Furthermore, coinoculation of B. elliptica with E. coli-expressed LsGRP1, LsGRP1C and LsGRP1ΔC fusion proteins conducted reduction of spore germination and lesion development in lily. Using 4',6-diamidino-2-phenylindole staining (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL), LsGRP1C-containing fusion proteins were proved to cause PCD phenomena, including chromatin condensation and DNA strand breaks in B. elliptica. The PCD phenomena of B. elliptica occurred earlier in lily leaves pre-treated with salicylic acid, in which the amount of LsGRP1 increased and plant cell death became less, suggesting the phenomena of fungal PCD in planta were related to the action of LsGRP1. In planta assay showed that less B. elliptica biomass was present in lily leaves pre-infiltrated with LsGRP1C-containing fusion proteins as investigated by trypan blue staining and real-time quantitative polymerase chain reaction; meanwhile, the PCD phenomena of B. elliptica occurred earlier and lasted longer at a higher level. Thus, the C-terminal region of LsGRP1 capable of protecting lily from B. elliptica infection via inducing fungal PCD was presumed, which is a defense mechanism firstly reported in lily. | en |
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dc.description.tableofcontents | 壹、 中文摘要I
貳、 英文摘要III 參、 前言1 肆、 前人研究3 一、 百合與百合灰黴病3 二、 植物富含甘胺酸蛋白 (glycine-rich proteins, GRPs) 分類和功能3 三、 葵百合上富含甘胺酸蛋白中的水楊酸誘導性防禦相關蛋白LsGRP14 四、 植物抗菌胜肽 (antimicrobial peptides, AMPs) 特徵和理化性質5 五、 植物抗菌胜肽對病原菌的抗菌機制6 六、 LsGRP1C (cysteine-rich region of LsGRP1) 之抗菌潛力7 七、 計畫性細胞死亡之分類8 八、 絲狀真菌細胞凋亡之生理現象9 九、 計畫性細胞死亡參與植物與病原菌交互作用10 伍、 材料與方法12 一、 供試植物與種植條件12 二、 供試菌株之培養與保存12 1. 菌株培養與接種源製備12 2. 菌株保存12 三、 LsGRP1衍生性胜肽合成12 四、 大腸桿菌表現LsGRP1衍生性融合蛋白13 1. 大腸桿菌表現蛋白菌株與菌株保存13 2. 大腸桿菌菌株培養、蛋白誘導與蛋白粗萃抽取13 3. 蛋白質純化與透析14 4. 蛋白質定量14 5. 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳與西方墨點分析15 五、 LsGRP1衍生性合成胜肽與融合蛋白之體外抑菌試驗16 六、 LsGRP1衍生性融合在百合上的蛋白抑病能力測試17 七、 LsGRP1衍生性融合蛋白生體外誘導B. elliptica計畫性細胞死亡 17 八、 評估水楊酸誘導之百合葉片LsGRP1相對累積量18 九、 水楊酸誘導抗性下B. elliptica感染百合情形18 十、 百合上注射LsGRP1衍生性融合蛋白之抑菌效果19 1. 注射大腸桿菌LsGRP1衍生性融合蛋白於百合葉片19 2. 石蠟切片與免疫組織染色19 3. 以錐蟲藍染色觀察百合葉片中真菌族群20 4. 定量百合葉片中真菌族群20 十一、 LsGRP1衍生性融合蛋白或水楊酸處理於百合葉片誘導B. elliptica發生計畫性細胞死亡之時間與比例22 陸、 結果23 一、 LsGRP1衍生性合成胜肽在生體外抑制B. elliptica孢子發芽之IC5023 二、 大腸桿菌表現之LsGRP1衍生性融合蛋白在生體外抑制B. elliptica孢子發芽之IC5023 三、 含有C端區段的融合蛋白對百合灰黴病之抑病效果24 四、 含有C端區段的融合蛋白能夠誘發B. elliptica發生細胞核DNA斷裂和染色質凝集24 五、 水楊酸誘導下B. elliptica的感染病程延緩 25 六、 B. elliptica感染百合葉片過程中發生計畫性細胞死亡之現象26 七、 水楊酸誘導百合累積LsGRP1後B. elliptica於感染過程發生計畫性細胞死亡之現象26 八、 LsGRP1衍生性融合蛋白注射至百合葉片之分布情形27 九、 含有C端區段的融合蛋白注射至百合葉片中對B. elliptica孢子發芽、菌絲生長與族群累積之抑制作用27 十、 LsGRP1衍生性融合蛋白注射至百合葉片對B. elliptica感染過程發生計畫性細胞死亡之影響27 柒、 討論29 捌、 參考文獻34 玖、 圖表集42 表一、LsGRP1衍生性合成胜肽抑制百合灰黴病菌孢子發芽之IC50值43 表二、大腸桿菌表現之LsGRP1衍生性融合蛋白抑制百合灰黴病菌孢子發芽之IC50值44 圖一、含有LsGRP1 C端區段之合成胜肽對百合灰黴病菌孢子發芽抑制效果45 圖二、以十二烷基硫酸鈉聚丙烯醯胺膠體電泳分析大腸桿菌表現之LsGRP1衍生性融合蛋白46 圖三、含有LsGRP1 C端區段之融合蛋白對百合灰黴病菌的抑病效果47 圖四、SUMO CK蛋白不會誘發百合灰黴病菌細胞核DNA斷裂和染色質凝集48 圖五、LsGRP1C融合蛋白誘發百合灰黴病菌細胞核DNA斷裂和染色質凝集 49 圖六、含有LsGRP1 C端區段之融合蛋白誘發百合灰黴病菌細胞核DNA斷裂和染色質凝集50 圖七、LsGRP1衍生性融合蛋白誘發百合灰黴病菌計畫性細胞死亡之比例51 圖八、水楊酸誘導百合葉片之LsGRP1累積量52 圖九、水楊酸處理後百合灰黴病菌於葉片所造成之病斑大小53 圖十、水楊酸處理後葉片接種百合灰黴病菌造成之葉片死亡面積大小54 圖十一、感染葉片之百合灰黴病菌發生計畫性細胞死亡時間點 55 圖十二、水楊酸處理後感染葉片之百合灰黴病菌發生計畫性細胞死亡之時間點56 圖十三、水楊酸處理後感染葉片之百合灰黴病菌發生計畫性細胞死亡之比例57 圖十四、LsGRP1衍生性融合蛋白注射於百合葉片之分布58 圖十五、含有LsGRP1 C端區段之融合蛋白可抑制葉片上百合灰黴病菌之孢子發芽和菌絲生長 59 圖十六、含有LsGRP1 C端區段之融合蛋白可降低葉片中百合灰黴病菌之生物量60 圖十七、葉片注射SUMO CK蛋白後感染葉片之百合灰黴病菌發生計畫性細胞死亡之時間點61 圖十八、葉片注射LsGRP1C融合蛋白後感染葉片之百合灰黴病菌發生計畫性細胞死亡之時間點 62 圖十九、葉片注射LsGRP1融合蛋白後感染葉片之百合灰黴病菌發生計畫性細胞死亡之時間點 63 圖二十、葉片上注射LsGRP1衍生性融合蛋白之百合灰黴病菌發生計畫性細胞死亡之比例64 拾、附錄65 圖一、LsGRP1衍生性融合蛋白66 | |
dc.language.iso | zh-TW | |
dc.title | LsGRP1C誘導百合灰黴病菌之計畫性細胞死亡現象 | zh_TW |
dc.title | LsGRP1C inducing programmed cell death of Botrytis elliptica in lily | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃秀珍,廖秀娟,王智立,陳穎練 | |
dc.subject.keyword | LsGRP1C,百合灰黴病菌,計畫性細胞死亡, | zh_TW |
dc.subject.keyword | LsGRP1C,Botrytis elliptica,programmed cell death, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-12-03 | |
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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