大豆细菌性斑点病菌harpin编码基因的克隆与表达

摘要：【方法、目的】利用PCR方法从丁香假单胞菌大豆致病变种（Pseudomonas syringae pv. glycinea）Psg12菌株中克隆到1026bp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

Cloning and expressing of a harpin-encoding gene from Pseudomonas syringae pv. Glycinea

Methods, Objective] We amplified the 1026 bp hrp ( hypersensitive response and pathogenicity) gene from Pseudomonas syringae pv. glycinea isolate Psg12 genomic DNA by PCR technique,and then constructed expression vector pGEX-hrpZ_(Psg12) with regular molecular cloning operation. The recombinant plasmid was transformed into BL21 ( DE3 ) . Recombinant protein was induced by Isopropylthio-β-D-Galacgoside ( IPTG) . Results] The molecular mass of the fusion protein is 61kDa analyzed by SDS-PAGE. The protein,similar to the other known harpins,was heat-stable, which contained abundant glycine( G) , but had no cysteine. Furthermore, this protein was sensitive to protease K and able to trigger hypersensitive response ( HR) in common tobacco. The HR elicitation by the protein in tobacco was inhibited by eukayotic metabolic inhibitors, NH_4VO_3 and LaCl_3 . The hrpZ gene showed 79% identity to hrpZ_(Psg) which cloned from P . syringae pv. glycinea(Psg rO) in Japan and 79-99% identity to other hrpZ in GenBank. However, it did not show any sequence identity with those of other genus of gram-negative plant pathogenic bacteria.Conclusion] In summary,hrpZ_(Psg12) was a novel gene that was cloned by us from P . syringae pv. glycinea, and this is the first report to express hrpZ_(Psg12) gene in BL21.