臺灣博碩士論文加值系統

百合為無皮鱗莖類球根花卉，以灰黴病為其主要真菌性病害，病原菌為Botrytis elliptica。本實驗室發現以水楊酸誘導處理可以有效地增加東方型葵百合之抗病性，進而由差異性表現分析篩選出一cDNA片段，其全長cDNA (LsGRP1) 解碼一富含甘胺酸蛋白質；北方雜合分析顯示LsGRP1可於水楊酸誘導下增量表現。本論文擬於水楊酸誘導下，利用農桿菌注入法 (agroinfiltration)， 使LsGRP1轉基因於葵百合葉片短暫表現 (transient expression) 及病原菌接種試驗，探討LsGRP1與水楊酸誘導抗病之相關性。首先以北方雜合分析觀察LsGRP1正股轉基因在農桿菌注入葵百合葉片6-120小時之表現，結果指出LsGRP1轉基因可於農桿菌注入後 72 小時有增強之偵測訊號，於 96 小時有明顯之表現。當以水楊酸澆灌處理葵百合根圈同時於葉片注入農桿菌菌株，三天後接種灰黴病菌，表現LsGRP1正股、反股及髮夾構型轉基因之葉片，LsGRP1訊息核酸量均較對照組少，病斑數目則較對照組多，顯示此三種構築對原有LsGRP1之誘導表現有基因靜默之作用，使葵百合對灰黴病之抗性減弱，故認為LsGRP1在水楊酸誘導之抗病性中扮演一重要的角色。

Lilium is a bulbous crop, severely affected by a major fungal disease, gray mold, caused by Botrytis elliptica. As know, development of necrotic lesions in the leaves of Oriental lily cultivar Star Gazer could be suppressed by the application of salicylic acid (SA). A cDNA sequence, named LsGRP1, coding for a 138-amino acid protein has been identified via suppression subtractive hybridization and differential screening. When the lily plants were treated with SA solution, the amount of LsGRP1 transcript analyzed by Northern blot hybridization increased. In the study of the relatedness between LsGRP1 and salicylic acid-induced disease resistance, agroinfiltration was applied for analysis of LsGRP1 transient expression in the leaves of Lilium cv. Star Gazer. In Northern blot hybridization, LsGRP1 transcript increased 72 hours after agroinfiltration of sense strand LsGRP1–expressing Agrobacterium tumefaciens EHA101 strain and more obviously 96 hours after agroinfiltraion. When the lily plants were treated with SA solution, the amount of LsGRP1 mRNA decreased in ‘Star Gazer’ leaves infiltrated with A. tumefaciens EHA101 strains expressing sense, anti-sense and hairpin LsGRP1, but the lesion numbers increased as compared to that in the control. These results showed that a gene silencing reaction was induced by introducing sense, anti-sense or hairpin LsGRP1, that abated the resistance of ‘Star Gazer’ leaves to gray mold disease. Thus, LsGRP1 playing an important role in salicylic acid-induced disease resistance of lily wasconcluded.

壹、中文摘要 3
貳、英文摘要 4
參、前言 5
肆、前人研究 7
一、百合 7
二、百合灰黴病 7
三、水楊酸誘導抗病性 8
四、同源性富含甘胺酸蛋白質及 LsGRP1 8
五、基因靜默 9
六、農桿菌注入法（Agroinfiltration） 11
七、單子葉植物之農桿菌介導轉化(Agrobacterium-mediated transformation) 12
伍、材料與方法 13
一、百合植株栽培 13
二、灰黴病菌接種原之製備 13
三、農桿菌載體構築 13
3-1 正股與反股LsGRP1載體構築 13
3-2髮夾型LsGRP1載體構築 14
3-3 β-Glucuronidase ( GUS ) 表現載體構築 14
四、大腸桿菌勝任細胞製備 15
五、大腸桿菌細胞轉形反應 15
六、聚合酵素連鎖反應 16
七、農桿菌電穿孔法（Agrobacterium electroporation） 16
八、農桿菌質體抽取 17
九、農桿菌注入法 17
十、葵百合葉片全RNA之萃取 18
十一、核酸探針之製備 19
十二、北方雜合分析（Northern blot hybridization） 19
十三、β-Glucuronidase ( GUS ) 活性於植物葉片上之檢測 20
十四、葵百合癒傷組織誘導及分化試驗 21
十五、葵百合基因轉殖試驗 21
陸、結果 22
一、LsGRP1農桿菌載體構築 22
1-1 正股與反股LsGRP1 (ssLsGRP1、asLsGRP1)表現載體 22
1-2 Hairpin LsGRP1 (hpLsGRP1) 表現載體 22
1-3 β-Glucuronidase 表現載體構築 22
二、葵百合短暫表現之β-Glucuronidase活性測試 23
三、A. tumefaciens EHA101 轉形株注入葵百合葉片之 LsGRP1 表現時間點測試 23
四、A. tumefaciens EHA101 轉形株注入之葵百合葉片 LsGRP1 表現分析 23
五、A. tumefaciens EHA101 轉形株注入葵百合葉片罹病度分析 24
六、葵百合癒傷組織及細胞分化誘導 24
七、葵百合 LsGRP1 轉型株之建構 25
柒、討 論 26
捌、參考文獻 29
玖、圖表集 35
表一、引子 36
表二、菌株與質體 37
圖一、 p35SNOS-sGRP1, p35SNOS-aGRP1大腸桿菌轉形株質體限制酶切分析 38
圖二、以pBS(35SNOS)表現正股或反股LsGRP1序列之質體 39
圖三、以pCAMBIA1300建構含LsGRP1序列之質體 40
圖四、A. tumefaciens轉形株之PCR產物 41
圖五、LsGRP1-intron之構築策略 42
圖六、建構LsGRP1-intron之PCR產物 43
圖七、pCAMBIA1300上髮夾型LsGRP1之構築 44
圖八、髮夾型LsGRP1之農桿菌轉形株質體檢測 45
圖九、p1300sGRP1及p1300aGRP1之LsGRP1序列 47
圖十、A. tumefaciens EHA101(pCAMBIA1300-GUS)之GUS表現分析 48
圖十一、農桿菌注入葵百合葉片之GUS表現分析 49
圖十二、農桿菌注入葵百合葉片LsGRP1訊息核酸之偵測 50
圖十三、水楊酸處理下A. tumefaciens EHA101 轉形株注入之葵百合葉片LsGRP1表現分析 51
圖十四、水楊酸處理下A. tumefaciens 轉形株注入葵百合葉片之罹病程度 52
圖十五、水楊酸處理下A. tumefaciens 轉形株注入葵百合葉片之罹病反應 53
圖十六、A. tumefaciens 轉形株注入葵百合葉片之抗病性分析 54
圖十七、葵百合癒傷組織及細胞分化誘導 55
拾、附錄 56

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