| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 98 === The harpin proteins of phytobacterial pathogenes are secreted via Type III secretion system (TTSS),which are heat stable and are components of translocon. The previous studies indicated that harpin proteins trigger the hypersensitive response (HR) and systemic acquire resistance (SAR). The harpin proteins, HopAK1 and HrpZ1 of Pseudmonas syringae pv. averrhoi HL1 are components of translocon, To investigate the application of harpin protein on bacterial disease control and the interaction with other translocon proteins, we constructed the HopAK1 and HrpZ1 fused with 6X His short peptide in C terminus which could be purified by using Ni-NTA affinity column. Results indicated the purified HopAK1-His6 and HrpZ1-His6 fusion protein could elicit the HR, but the purified HopAK1-His6 fusion protein were aggregated and precipitated. The results of gel filtration experment showed that HopAK1 was congregated into a complex of 2000 kDa in size. According to the prediction of bioinformatics software, the ratio of α-helix in amino acid sequence of HopAK1 is different from other harpins; nevertheless, it still has harpin activity. The HopAK1 in non-soluble fraction was restored by using buffer containing 8M Urea, then replaced the original buffer by dialysis. Then the precipitation of HopAK1 did not occurr, but molecular weight shown in SDS-PAGE analysis was shiffed to be larger molecular weight, this result revealed that the renatured HopAK1 protein might change the charge, and still could elicit the HR. The threshold concentration of HR elicitation was about 50 ng/μl. In the congo red binding assay, HrpZ1-His6 fusion protein showed the blue shift in peak of absorbance, while the HopAK1-His6 fusion protein had no change in absorbance. In the experiment for the controlling bacterial spot diease of tomato Xanthomonas vesicatoria 1 or 4 days after spraying the purified HopAK1-His6 and HrpZ1-His6, the result indicated that the disease severity of bacterial spot decreased 4 days and the expression of systemic acquired resistance related gene PR-1a was induced after harpin treatment than that without treatment. The western blotting probed with HopAK1 antibody showed that the anti-HopAK1 polyclonal antibodies could detect the purified HopAK1 under concentration 1pg/μl and also detect the secretion of HopAK1 of Pav HL1 grown on Hrp minimal medium, and the co-immunpreciptation assay indicated that HopAK1could interact with HrpZ1.
|
|---|
