N-gene介導植物的抗病性 植物生理學實驗 10 N-gene mediated resistance to plant virus 授課教授：傅士峯老師 助教：呂學宇、張凱雅 值日組：G7 104/11/11
今日上課流程 課程回饋 (實驗6-觀察根尖與莖頂分生組織) 開始上課 菌液注射
問題討論 NbCMT3-2 基因主要在分生組織大量表現，試由基因表現位置推論其功能
實驗目的 藉由觀察植物的感染程度和病死斑，了解植物防禦病害的現象和機制 了解農桿菌介導的基因轉殖法
實驗原理
過敏反應 Hypersensitive reaction, HR 植物在生物逆境下的感知和訊息傳遞機制 活化病源性相關蛋白 (Pathogenesis-related proteins, PR proteins): proteinase inhibitors, phytoalexins (secondary produces), cell wall protect substance (lignin, callose, and suberin) 誘發程序性細胞死亡（病原體附近細胞的壞死，限制其擴散）
植物如何辨識病原菌與 啟動防禦機制？ Gene-for-gene hypothesis 病原體攜帶非致病基因 (avirulence genes, avr genes) 寄主植物攜帶對應的抗性基因 (resistance genes, R genes) 受體與誘導原結合後引發過敏反應 病原菌與寄主植物具有專一性 誘導原 (elicitor) 受體 (可辨識誘導原) nn NN
TMV Tobacco mosaic virus
TMV-GFP NN: N gene Lesion
系統性後天抗性 Systemic acquired resistance, SAR 由局部的過敏反應，引發系統性的免疫能力 增加抗病性 避免植物再受其他病原體感染 水楊酸 (Salicylic acid) 扮演訊息傳遞的角色 HR: Hypersensitive reaction MSA: Methylsalicylic acid SA: salicylic acid
1 - Plant R protein (receptor) and pathogen Avr protein (ligand) join 2 - signal transduction pathway is initiated 3 - HR response: cells seal off pathogens and then undergo apoptosis 4 - dying cells release a chemical signal (salicylate) 5 - salicylate is transported to the other parts of the plant 6 - signal transduction pathways are initiated in distant parts of the plant 7 - SAR is achieved when phytoalexins and PR proteins are produced in those remote tissues.
SAR (Systematic acquired resistance) responses Tobacco NN genotypes
SAR (Systematic acquired resistance) responses Tobacco NN genotypes HR (Lesion) SAR (Healthy)
SAR (Systematic acquired resistance) responses Tobacco NN genotypes
Gene-for-Gene model Tobacco Genotypes: nn NN Resistance gene: X N gene Symptoms (Local): No HR lesion Symptoms (Upper): mosaic No
SAR (Systematic acquired resistance) responses Tobacco NN genotypes
Gene-for-Gene model nn NN
Tobacco nn genotypes
Tobacco nn genotypes
Tobacco nn and NN genotypes
Agrobacterium tumefaciens transformation
實驗步驟 培養農桿菌 每管 50ml tube 加入：(1)10ml LB (2)5µl kanamycin (3)5ml 菌液 28℃/150rpm/24hr 菌液離心 10min/3000rpm 取 5ml infiltration medium 回溶沉澱 Shaker:65rpm/3hr 農桿菌 (TMV-GFP) 感染 infiltration medium (50ml): 1ml MES (0.5M)1 0.5ml MgCl2 (1M) 50µl AS 將菌液注射入植物葉片
Any question?
問題討論
報告繳交 報告格式：實驗目的、原理、結果、問題討論、補充資料（自行整理）、心得 繳交期限：下週 (11/18) 課程結束前