Cloning and transcriptional regulation of pelA gene in Xanthomonas campestris pv. campestris

• Main Authors: Chen-hung Wang, 王振宏
• Other Authors: Yi-Min Hsiao
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/87178193922040783959

碩士 === 朝陽科技大學 === 生物技術研究所 === 95 === Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease. The pathogenic determinants of Xcc including the exopolysaccharide (xanthan) and extracellular enzymes such as protease, cellulase, and pectinase. It is only known that the global transcription factor Clp (cAMP receptor protein-like protein) regulates the biosynthesis of these virulence factors. This study was to characterize the pelA gene encoding pectate lyase in Xcc. First, the pelA mutant PL17 was constructed by insertional inactivation. The extracellular pectate lyase activity was analyzed by plate assay using polygalacturonic acid (PGA) as substrate. The result was suggested that PelA is the major pectate lyase in Xcc. Second, PelA protein was induced and expressed by pET system. Third, the upstream region of pelA was characterized by promoter probing. Results of report assays indicated that both PGA and CaCl2 were required for pelA expression. It was aslo shown that the pelA promoter activity was reduced in clp mutant, suggested Clp positively regulates the transcription of this gene. Fourth, gel retardation assay indicated that Clp and upstream region of pelA form a DNA-protein complex, suggested Clp regulates pelA transcription directly.