Studies on expression and characteristics of PelE pectate lyase of bacterial soft rot pathogen of Phalaenopsis, Erwinia chrysanthemi

• Main Authors: Chien-ming Lee, 李健銘
• Other Authors: Yung-An Lee
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/09830818852858374774

碩士 === 輔仁大學 === 生命科學系碩士班 === 95 === Phalaenopsis soft rot bacterium, Erwinia chrysanthemi produces several pectic enzymes. Pectate lyases (Pels) the main E. chrysanthemi enzyme, depolymerize pectic substances of plant cell walls. The degradation products, unsaturated oligogalacturonates, have been proposed to induce plant defence reactions. If healthy plants are treated with Pels or oligosaccharides to make them produce defence reactions or resist soft rot pathogen. Constructed E. coli BL21 (DE3) and E. coli Rosetta (DE3) pLysS E.coli strains could express PelE and PelZ. Purified 47.4 kDa PelE and 48.5 kDa PelZ were used to produce PelE and PelZ antibodies. Soft rot leaves filtering solution had Pel activity. PelE and PelZ antibodies were used in Western blot for detecting soft rot leaves filtering solution. There had detected PelE and PelZ expression, the results indicated that PelE and PelZ express in soft rot pathogenesis. E. coli BL21 (DE3) (pET29a-pelE) and E. coli Rosetta (DE3) pLysS (pET29a-pelE) could secrete PelE protein out of the cell and was also able to degrade polygalacturonic acid. Its molecular weight of 40 kDa was smaller than we calculated. The N-terminal amino acid sequence of PelE had a signal peptide and its cleavage site based on sequence analysis and N-terminal amino acid sequencing of PelE. Signal peptide DNA sequence amplification by PCR was used to construct a new expression vector for other protein expression. PelE enzyme was purified from culture supernatant. PelE enzyme activity decreased significantly after seven days at room temperature. A complete removal of PelE enzyme activity required 100 ℃ for 30 minutes. The Km of PelE enzyme was 0.46 and the Vmax of PelE enzyme was 1.75. Analysis the products of PelE degraded polygalacturonic acid by HPLC. A maximum absorption was found at 230 nm, and the HPLC separation test showed a clear peak at 5.3 minute after sample injection. The time was similar to digalacturonic acid showed a clear peak at 5.2 minute after sample injection. The E.coli strains which could secrete PelE protein were inoculated into tobacco and triggered hypersensitive reaction but not seen in phalaenopsis. Purified active PelE enzyme was inoculated into tobacco and phalaenopsis both triggered hypersensitive reaction. The products of PelE degraded polygalacturonic acid were inoculated into tobacco and phalaenopsis both could not trigger hypersensitive reaction.