Molecular cloning, characterization, expression analysis, and fungal inhibitory effect of an endochitinase cDNA from the entomopathogenic fungus, Paecilomyces javanicus

• Main Authors: Chien-Cheng Chen, 陳鍵政
• Other Authors: 葉開溫
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/59051550756720860446

碩士 === 國立臺灣大學 === 植物科學研究所 === 93 === Paecilomyces javanicus is an entomopathogenic fungi naturally parasitizing on Diptera and Lepidopteran. Based on the analysis of SDS-PAGE gel activity assay, a strong chitinase activity was discovered in artificial cultured mycelium. The full-length cDNA, designated PjCHi-1, was subsequently cloned from mycelium by using both degenerated primer/RT-PCR amplication and 5’-/3’-RACE extension. This cDNA gene, comprising 1,180 bp, coveres a 1035-bp open reading frame and encodes a 345-amino acid polypeptide. A conserved motif for chitinase activity ,–F82DGIDIDWE90- ,was present in deduced amino acid sequence. It implicated that it is belonged to class V, family 18 chitinase. Recombinant PjCHi-1 protein, expressed from Eschaerichia coli, was purified and its endochitinase activity toward 4-metilumbelliferilβ-D-N,N,Ntriacetilchitotrioside(4MU-(GlcNAc)3) was verified. Southern blot analysis showed that a single copy of PjCHi-1 gene was contained in haploid genome. Both of RT-PCR and Northern gel analysis revealed that the expression of chitinase gene was constitutive but in low-level, only enhanced to high-level expression in chitin-containing medium. This demonstrated that the physiological function of PjCHi-1 (family 18) in P. javanicus plays an invasive role in insect parasitism, and evolutionarily different from those chitinase genes in plants. To study the effect of pH on chitinase activity, buffers of different pH were used. The results showed that the enzyme was highly active at acidic pH with the highest activity at pH 5. An in vitro assay showed that the purified PjCHi-1 protein could inhibit mycelia growth and sclerotial body germination of Sclerotium rolfsii, a major fungal pathogen of plants.