EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

Identification and Expression of hrpN Gene from Strain EcbCSL101 and the Activity of Purified Harpin_(EcbCSL101) Protein

胡萝卜软腐欧文氏菌甜菜亚种(Erwinia carotovora subsp.betavasculorum)EcbCSL101菌株具有很强胞外酶分泌活性,接种非寄主植物烟草引起过敏反应。Southern blotting结果表明EcbCSL101菌株中含有hrpN基因。PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a(+)中。核苷酸序列分析表明,EcbCSL101菌株的hrpN基因的ORF为1113bp,编码36.65 kD HarpinEcbCSL101蛋白(GenBank,DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性。将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应。

Erwinia carotovora subsp, betavasculorum （Ecb） strain EcbCSL101 produces high levels of extracellular enzymes including pectate lyase （Pel）, polygalacturonase （Peh）, protease （Prt） and cellulase （Cel） and elicits hypersensitive response （HR） in tobacco leaves. Southern blot analysis using Ecc strain CSDS001 hrpN DNA as probe revealed that EcbCSL101 possesses hrpN gene. A DNA fragment containing entire ORF of EcbCSL101 hrpN was PCR amplified and cloned into vector pET28a（＋）. Nucleotide sequence analysis reveals that EcbCSL101 hrpN possesses a 1113 bp ORF which encodes a 36.65 kD HarpinEcbCSL101 protein. The deduced amino acid sequence of EcbCSL101 hrpN shares high homology with Harpin proteins of several other Erwinia carotovora strains （GenBank number DQ355519）. HarpinFcbCSL101 protein was expressed in E. coli strain JM109 （DE3） by IPTG induction. The purified HarpinEcbCSL101 effectively elicits the HR in tobacco leaves.