Title：Molecular cloning, characterization, and gene expression of
endochitinase cDNA from the entomopathogenic fungi, Paecilomyces
Javanicus
M.S. Student：Chien-Cheng Chen（R92B42012）
Advisor：Dr. Kai-Wun Yeh
Abstract：
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 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 in P. javanicus plays an invasive role in insect
parasitism, and evolutionarily differs 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 high 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.