寄生疫霉parA1基因的克隆及在大肠杆菌中的表达  

Cloning of the ParA1 Gene from Phytophthora parasitica and Its Expression in Escherichia coli

作　　者：梁元存[1] 刘爱新[1] 董汉松[2] 王金生[2] 张天宇[1] 

机构地区：[1]山东农业大学植物病理学系,泰安271018 [2]南京农业大学植物病理学系,南京210095

出　　处：《微生物学报》2004年第2期202-205,共4页Acta Microbiologica Sinica

基　　金：教育部博士点基金 (B2 0 0 10 6) ;山东省自然科学基金重点资助项目 (Z99D0 2 )~~

摘　　要：根据已报道的寄生疫霉 (Phytophthoraparasitica)parA1基因的序列设计引物 ,从 4株寄生疫霉中国菌株 (3株来自烟草 ,1株来自刺槐 )中克隆到此基因并进行了重组表达。序列分析表明 4株寄生疫霉parA1基因序列高度保守。对表达载体pET 30a(+)双酶切 ,构建表达Parasiticein蛋白的表达载体pET eli,用CaCl2 法转化大肠杆菌(Escherichiacoli)BL2 1,通过诱导在大肠杆菌中进行非融合表达 ,表达产物在烟草上引起过敏性反应。性质测定表明 ,表达产物有一定的耐热性 ,并对蛋白酶K敏感。Elicitins are a family of small proteins secreted by species of the genus Phytophthora and Pythium. They can cause a hypersensitive response (HR) and induce systemic acquired resistance (SAR) in tobacco. Here we report cloning and characterization of parA1 homologs from 4 strains of Phytophthora parasitica isolated from tobacco and locust in China. Amplification by PCR (polymerase chain reaction) of DNA from the 4 strains, using primers specific to a parA1 reported previously, produced 4 products. Their nucleotide sequences are highly conserved and identical to the parA1 gene previously cloned. Therefore,the parA1 genes were successfully cloned from the Chinese strains of P. parasitica. They were cloned into the vector pET-30a (+) and transformed into Escherichia coli BL21. The expressed product was biologically active, as it caused HR in tobacco leaf panels after direct infiltration; the protein was thermostable and sensitive to protease K. We have established a recombinant E. coli that produces the elicitin. This is the first report of cloning of parA1 genes from locust isolate of the pathogen.

关 键 词：寄生疫霉 parA1基因 克隆 大肠杆菌 表达 耐热性 蛋白酶K 序列分析 烟草 黑胫病 

分 类 号：Q786[生物学—分子生物学]