Jiang Zhaoyuan, Zou Xiaowei, Gao Jie, Bai Qingrong, Zhang Jiahuan
College of Agronomy, Jilin Agricultural University, Changchun 130118, China.
Wei Sheng Wu Xue Bao. 2009 Oct;49(10):1403-7.
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-beta-D-Galacgoside (IPTG).
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, NH4 VO3 and LaCl3. The hrpZ gene showed 79% identity to hrpZ(Psg) 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.
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.
方法、目的：我们通过PCR技术从丁香假单胞菌大豆致病变种分离株Psg12的基因组DNA中扩增出1026 bp的hrp（过敏反应和致病性）基因，然后通过常规分子克隆操作构建表达载体pGEX-hrpZ(Psg12)。将重组质粒转化到BL21(DE3)中。用异丙基硫代-β-D-半乳糖苷（IPTG）诱导重组蛋白表达。
通过SDS-PAGE分析，融合蛋白的分子量为61 kDa。该蛋白与其他已知的harpin蛋白相似，具有热稳定性，含有丰富的甘氨酸（G），但不含半胱氨酸。此外，该蛋白对蛋白酶K敏感，能够在普通烟草中引发过敏反应（HR）。烟草中该蛋白引发的HR受到真核生物代谢抑制剂NH4VO3和LaCl3的抑制。hrpZ基因与从日本大豆丁香假单胞菌（Psg r0）克隆的hrpZ(Psg)具有79%的同一性，与GenBank中的其他hrpZ具有79%-99%的同一性。然而，它与革兰氏阴性植物病原菌其他属的序列没有任何同一性。
综上所述，hrpZ(Psg12)是我们从大豆丁香假单胞菌中克隆的一个新基因，这是首次在BL21中表达hrpZ(Psg12)基因的报道。
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