东方百合(Lilium)杂交品种索邦病程相关蛋白(PR1)基因的克隆与原核表达

病程相关蛋白（PR）广泛参与植物对生物和非生物胁迫的应答。采用cDNA末端快速克隆技术，从东方百合（Lilium）杂交品种索邦中克隆了PR1基因，并命名为LhSorPR1。LhSorPR1基因cDNA全长870 bp，5’非翻译区和3’非翻译区的长度分别为47 bp和274 bp。该基因的开放阅读框为549 bp，编码182个氨基酸。成熟的LhSorPR1蛋白（不含信号肽）分子量为18.19 kDa，等电点为5.69。采用同源建模方式构建的LhSorPR1蛋白三维结构与其模板番茄PR1蛋白p14a具有较高的相似性，说明百合PR1蛋白在结构和功能上与其他植物中的PR1蛋白具有较强的保守性。构建pCold Ⅱ-LhSorPR1原核表达，经IPTG诱导后，目的蛋白在大肠杆菌中以包涵体形式成功表达。使用免疫印迹方法对表达的LhSorPR1重组蛋白进行了验证，并对带有His标签的重组蛋白成功进行了纯化。

Cloning and Prokaryotic Expression of Pathogenesis-related protein 1 Gene from Oriental Hybrid Lily Sorbonne

Pathogenesis-related (PR) proteins are widely involved in plant defense against both biotic and abiotic stresses. In the present study, we characterized a PR1 protein from the oriental hyrid lily cultivar Sorbonne via rapid amplification of cDNA ends (RACE), and designated as LhSorPR1. The cDNA full-length of LhSorPR1 isolated was 870 bp, consisting a 5' untranslate region (UTR) of 47 bp and a 3' UTR of 274 bp. The predicted open reading frame (ORF) of LhSorPR1 was 549 bp, encoded a protein of 182 amino acids. The mature LhSorPR1 (devoid of a signal peptide) had a theoretical isoelectric point of 5.69 and a calculated molecular weight of 18.19 kDa. The three dimentional (3D) model of LhSorPR1 was bulit by homology modelling, and the bulit model showed high similarity to its template p14a. The similarity in 3D strucutures indicated the conservation of protein function of PR1 proteins in plants. The pCold Ⅱ-LhSorPR1 recombinant vector was constructed to express LhSorPR1 in E.coli, and the His₆-tagged recombinant protein was expressed as inclusion bodies in the insoluble fraction under IPTG induction. The His₆-labeled recombinant LhSorPR1 protein was verified by immunoblot, and purified by chromatography.