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【方法、目的】利用PCR方法从丁香假单胞菌大豆致病变种(Pseudomonas syringaepv.glycinea)Psg12菌株中克隆到1026 bp的hrp基因。将其定向插入到表达载体pGEX-4T-1上,并转化宿主菌BL21,IPTG诱导表达后,SDS-PAGE显示其表达产物为分子量为61 kDa的融合蛋白质。【结果】该蛋白质在性质与功能上类似于已发现的harpins,即富含甘氨酸、不含半胱氨酸,热稳定以及对蛋白酶K敏感,能够在烟草上引起典型的过敏性反应,过敏性反应还可被真核生物代谢抑制剂抑制。序列比较发现该基因与日本的Psg r0菌的hrpZ相似性为79%,与GenBank已公布的其它hrpZ的相似性为79%～99%,与其他革兰氏阴性植物病原细菌不存在相似性。【结论】本实验从丁香假单胞菌大豆致病变种(P.syringaepv.glycinea)Psg12菌株中克隆到新的hrpZ基因,并成功表达,这是国内首次从P.syringaepv.glycinea菌株中克隆到hrpZ基因。
【Method】 The objective of this study was to clone 1026 bp hrp gene from Psg12 strain of Pseudomonas syringaepv. Glycinate by PCR. The recombinant plasmid pGEX-4T-1 was inserted into pGEX-4T-1 and transformed into host strain BL21. After induced by IPTG, SDS-PAGE showed that its expressed product was a fusion protein with a molecular weight of 61 kDa. 【Result】 The protein was similar in nature and function to the harpins that have been found, that is, glycine-rich, cysteine-free, thermostable and sensitive to protease K, which can cause typical allergic reactions and allergic reactions on tobacco The reaction can also be inhibited by eukaryotic metabolic inhibitors. Sequence comparison showed that this gene shared 79% to 99% homology with other strains of hrpZ in Japan and 79% ~ 99% similarity with other strains of hrpZ in GenBank. There was no similarity to other gram-negative plant pathogenic bacteria. 【Conclusion】 In this study, a new hrpZ gene was cloned from P. syringaepv.glycinea strain Psg12 and successfully expressed. This is the first time that it was cloned from P. syringaepv.glycinea strain hrpZ gene.