软腐欧氏杆菌Ⅲ型分泌系统对梨火疫欧文氏杆菌harpin蛋白识别及分泌.pdf

摘 要 本文研究软腐欧氏杆菌(Erwinia carotovora)分泌致病蛋白的 III 型分泌系统的组分是 否能识别梨火疫欧氏杆菌(Erwinia amylovora)存在于mRNA 上的分泌识别信号 用PCR 的 方法 将带有上游75bp 可以在其 mRNA 的5 端形成一个典型的作为 III 型分泌识别信号 的茎环结构的梨火疫欧氏杆菌的诱导植物过敏反应的 harpin 的基因hrpN 从带有 hrp 基 因簇的质粒 pCPP430 上克隆到 pGEM-T 载体上 获得重组质粒 pWGF1 经化学法转化 到hrpN 基因的转座突变体 DH5(pCPP430hrpN ) 中 同时将pWGF1 电击转化到胡萝卜 软腐欧氏杆菌Se9R 中 转化子的无细胞抽提物 CFEP 经Western-blot 检测到harpin 蛋白已被表达 带有双重质粒的 DH5 (pCPP430hrpN /pWGF1)在番茄植物上可以引起过 敏反应 Se9R pWGF1 在大白菜上和马铃薯上的致病力明显低于 Se9R 初步表明一 种植物病菌的 harpin 蛋白可被另外一种病菌的 III 型分泌系统所识别 分泌并保持生物活 性 i Abstract Bacterial pathogens of both animals and plants use type III secretion machines to secret virulence proteins. The signal that leads to the type III secretion is encoded as stem loop structure in the messenger RNA. Harpin, produced by Erwinia amylovora is secreted via type III secretion machinery. Erwinia carotovora Se9R and Erwinia amylovora belong to the same genus and they use type III secretion machines to secret virulence proteins. We used PCR to amplify hrpN coding region with 75bp upstream segment from pCPP430 which harbors hrp N gene cluster, and cloned it to pGEM-T vector to get pWGF1, which was then chemically transformed into DH5(pCPP430hrpN ) and electroporated into Se9R. DH5 (pCPP430hrpN /pWGF1) induced hypersensitive response on tomato leaf and Se9R(pWGF1) showed significantly reduced virulence than Se9R on Chinese cabbage leaf and potato tube . Western-blotting analysis of the CFEP of Se9R (pWGF1) showed production of harpin protein. These results suggest the type III secretion machine in Se9R can recognize secretion sig nal in the gene of harpin in Erwinia amylovora and secret it as a biologically native form. 4