利用農桿菌注入法探討葵百合LsGRP1與水楊酸誘導抗病之相關性

Abstract

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.