[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-beta-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, 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.


CONCLUSIONS
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.
Jiang
Zhaoyuan
Z
College of Agronomy, Jilin Agricultural University, Changchun 130118, China. jzy_80@163.com
Zou
Xiaowei
X
Gao
Jie
J
Bai
Qingrong
Q
Zhang
Jiahuan
J
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D015870:Gene Expression; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology; D055550:Protein Stability; D044224:Pseudomonas syringae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000187:drug effects / Q000276:immunology / Q000382:microbiology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C075462:harpin protein, Erwinia amylovora