Cloning and Expression Analysis of a Chitinase Gene Crchil from the Mycoparasitic Fungus Clonostachys rosea (syn. Gliocladium roseum)

摘要

Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchil was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchil ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchil encodes 426 amino acid residues and shares a high degree of similarity with other chi-tinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchil in response to carbon (5'-SYGGRG-3') and nitrogen (5'-GATA-3') were identified in the upstream of Crchil. Expression of Crchil gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchil expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.