Ecc36菌株hrpN基因的克隆、表达及HrpN_(EccPR)蛋白的生物学活性

Identification and Expression of hrpN Gene from Strain Ecc36 and the Activity of Purified HrpN_(EccPR) Protein

为了揭示hrp基因表达的Harpins蛋白能够广泛地诱导植物对植物病虫害防卫反应的分子机制,通过琼脂固体平板法,发现胡萝卜软腐欧文氏菌Ecc36菌株具有很高的胞外酶分泌活性,该菌接种非寄主植物烟草,能够引起过敏反应(HR),结果表明,Ecc36携带hrp基因。用地高辛标记的hrp N基因作探针,通过Southern杂交证明了Ecc36菌株中含有hrp N基因,命名为hrp NEcc PR基因;核苷酸序列分析表明,hrp NEcc PR基因的开放阅读框ORF为1 254 bp,编码的Harpin蛋白(命名为HrpN_(EccPR)蛋白)由417个氨基酸残基组成,其分子量为45.27 ku,等电点为5.73,与其他几种软腐欧文氏菌Harpin蛋白有较高的同源性。此外,将hrp NEcc PR基因克隆到表达载体p ET28a(+)上,将构建的hrp NEcc PR基因表达载体(p ET30a-Ecc PR)转化到大肠杆菌BL21(DE3)中,经IPTG(异丙基硫代-β-D半乳糖苷)诱导HrpN_(EccPR)蛋白表达,纯化后的HrpN_(EccPR)能诱导烟草发生过敏反应,表明HrpN_(EccPR)蛋白具有激发植物免疫反应的生物活性,可为进一步研究HrpN_(EccPR)蛋白诱导植物防卫反应的机制奠定基础。

Harpins protein expressed by hypersensitive response and pathogenity gene(hrp)can induce the defense response of plant to diseases and insect pests of plant.By the way of agar solid plate,Erwinia carotovora pv.carotovora strain Ecc36 can produce high levels of extracellular enzymes.The Ecc36 strain could elicit the hypersensitive response(HR)in tobacco leaves.These results showed that some kind of hrp was in Ecc36 strain.Southern Blot analysis of Ecc36 with a hrpN gene probe labeled by DIG-dUTP showed that the Ecc36 strain possesses hrpN gene,named hrpN EccPR.Through nucleotide sequence analysis,a 1 254 bp open reading frame(ORF)of Ecc36 hrpN was revealed.A Harpin protein(named HrpN EccPR protein)encoded by hrpN EccPR comprise 417 amino acid residues with 45.27 ku and pI 5.73 approximately.The deduced amino acid sequence of Ecc36 hrpN shares high homology with Harpin proteins of several other E.carotovora strains.The hrpN EccPR gene was cloned into vector pET28a(+)for expression,and then transferred into the cells of E.coli strain BL21(DE3).The HrpN EccPR protein was overexpressed in reconstructed BL21(DE3)when the strain was induced by IPTG.The purified HrpN EccPR effectively elicits the HR in tobacco leaves.Those show HrpN EccPR protein has the biological activity of stimulating the immune response of plants,and provide for further study on the mechanism of the defense response induced by HrpN EccPR protein in plant.