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標題: 桃褐腐病菌果膠分解酵素基因之選殖與特性分析
Cloning and characterization of Monilinia fructicola polygalacturonase genes
作者: 周建銘
Chou, Chien-Ming
關鍵字: Monilinia fructicola;桃褐腐病;endopolygalacturonase;cloning;functional analysis;果膠分解酵素;選殖;功能性分析
出版社: 植物病理學系所
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由Moninilina fructicola 所引起的桃褐腐病,會造成桃果實在病害發展過程中產生軟腐的病徵,此病徵通常被認為是由果膠分解酵素所引起。我們先前已將M. fructicola endopolygalacturonase基因 (MfPG1)分離解序,並證實其在感染果實及花辦初期就有表現的情形。欲了解MfPG1在M. fructicola感染桃果樹過程中扮演的角色,在本研究中修改先前已建立之原生質體轉殖系統。利用修改之轉殖系統搭配Split marker策略造成M. fructicola MfPG1基因的缺失,經五次實驗後共獲得4個MfPG1基因缺失的轉型株,南方雜合法顯示所篩選之轉型株僅部分細胞核MfPG1基因被打斷。分析轉型株特性,顯示其孢子發芽率與附著器形成率與野生型無明顯差異。將轉型株與野生型菌株接種於桃、櫻花與玫瑰花瓣及桃果實顯示MfPG1部分基因缺失轉型株接種可促進褐腐病病徵擴展。且MfPG1基因表現在富含於抗病時期桃果表的抗氧化物caffeic acid處理時可促進其基因表現,而處理可造成植物細胞死亡而促進壞疽型病原菌感染的氧化物H2O2時則表現量下降。綜合上述實驗結果顯示MfPG1在M. fructicola與桃果樹的交互作用中可能扮演的是elicitor的角色,且caffeic acid之抗病機制可能還包括誘導MfPG1 基因表現以激發植物之抗病反應。為了瞭解endopolygalacturonase在M. fructicola感染過程中扮演的角色,我們進一步分離了三個MfPGs基因稱為MfPG2、MfPG3、MfPG5。經由反向PCR (Inverse PCR)可分離出此三個MFPG基因上游序列。分析目前所有的MfPGs基因上游序列,我們可以發現其上包含許多與碳氮素源及pH有關之轉錄調控子結合位,而在所有已分離的MfPGs上游序列上皆可找到如AP-1及Sp1等與氧化還原調控有關之轉錄調控子結合位,也顯示說caffeic acid可能會調控已分離之MfPGs基因表現以降低褐腐病病害發展。

Monilinia fructicola, the causal agent of peach brown rot disease, causes severe soft rot symptom during disease development, which was thought to be resulted from the reactions of cell wall-degrading enzymes. In our lab, an M. fructicola endopolygalacturonase gene MfPG1 was isolated and shown to be expressed in the early stage of infection on peach petals and on infected fruits. To understand the role of MfPG1 during peach brown rot disease development, here I modifieded the M. fructicola protoplast transformation protocol from a previous study and performed MfPG1 gene disruption with the modified protoplast transformation protocol combined with split marker strategy. Four transformants with gene replacement were obtained from 5 separate transformations. Southern blot analysis revealed that the MfPG1 gene was partialy disruptied in the generated transformants. Three of the resulted transformants did not show significant differences on conidium germination and appressorium formation when compared with wild type. Pathogenicity assay of the transformants on peach, cherry and rose petals and peach fruits, a significant promotion on lesion development was observed. Moreover, the expression of MfPG1 was up-regulated by the antioxidant caffeic acid, which is abundant in the brown rot resistant stage of peach fruits, and down-regulated by the prooxidant H2O2, which can induce plant cell death to promote the infection of a necrotrophic pathogen. Taken together, MfPG1 may play a role in eliciting plant defense responses during M. fructicola infection. To fully understand the role of polygalacturonase in M. fructicola infection process, here I also isolated three additional endopolygalacturonase genes, named MfPG2, MfPG3 and MfPG5. The flanking regions of MfPGs were further isolated by inverse PCR and the analysis of the putative upsteam flanking regions in silico indicated that MfPG genes might be regulated by different carbon and nitrogen sources and pH. The redox regulated transcription factor, AP-1 and Sp1, binding sites were found on all isolated MfPGs promoter regions, which suggests that caffeic acid and H2O2 may also regulate the three MfPGs to modulate M. fructicola endopolygalacturonase expression and therefore brown rot disease development.
其他識別: U0005-2308201016291400
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