大豆细菌性斑点病菌harpin编码基因的克隆与表达

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

摘要:

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

Abstract:

Abstract: [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-hrpZPsg12 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, NH4VO3 and LaCl3. The hrpZ gene showed 79% identity to hrpZPsg which cloned from P. syringae pv. glycinea(Psg r0) 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, hrpZPsg12 was a novel gene that was cloned by us from P. syringae pv. glycinea, and this is the first report to express hrpZPsg12 gene in BL21.