软腐欧氏杆菌的Ⅲ型分泌系统对harpin蛋白的识别与分泌  

Type Ⅲ Secretion System in Se9R Can Recognize and Secret Harpin in Erwinia amylovora

作　　者：魏桂芳[1] 赵淑清[2] 赵立平[1] 

机构地区：[1]上海交通大学生命科学技术学院,上海200240 [2]山西大学生物工程实验室,太原030006

出　　处：《微生物学报》2002年第4期465-470,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金资助项目 ( 39970 1 5 7)~~

摘　　要：本文研究软腐欧氏杆菌分泌致病蛋白的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pCPP43 0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DH5α(pCPP43 0hrpN- )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DH5α(pCPP43 0hrpN- pWGF1 )在番茄植物上可以引起过敏反应 ,Se9R(pWGF1 )在大白菜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。Bacterial pathogens of both animals and plants use type Ⅲ secretion machines to secret virulence proteins. The signal that leads to tye type Ⅲ secretion is encoded as stem loop structure in the messenger RNA. Harpin, produced by \%Erwinia anylovora\% is secreted via type Ⅲ secretion. \%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 \%hrp\%N from pCPP430 which harbors hrp N gene cluster, and cloned it to pGEM\|T vector to get pWGF1, which was then chemically transformed into DH5α(pCPP430 hrp N\+-) and electrotransformed into Se9R. DH5α(pCPP430 hrp N\+-/pWGF1) induced hypersensitive response on tomato leaf and Se9R(pWGF1) showed significantly reduced virulence than Se9R on Chinese cabbage leaf. 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 singal in the gene of harpin in \%Erwinia amylovora\% and secret it as a biologically native form.

关 键 词：软腐欧氏杆菌 Ⅲ型分泌系统 HARPIN蛋白 识别 分泌 

分 类 号：Q933[生物学—微生物学]