Study On Chitinase Gene Aachit From Verticillium Lecanii

Verticillium lecanii which is important entomopathogenic fungus plays a significant role in the bio-control against pests (mainly against whitefly, scale and aphid). Many researches indicated that chitinase may be an important toxic factor of entomopathogenic fungi, which play specific roles in the process of fungal infection against insect, for example, it could act synergistically with proteases to hydrolyze insect cuticle. Compared with Metarhizium anisopliae and Beauveria bassiana, the study on Verticillium lecanii is scarce and there have been few reports on cloning of infection related gene in this fungus. In this research, we cloned an endochitinase gene from V. lecanii, analyzed its nucleic acid sequence and deduced amino acid sequence with bioinformatic methods and expressed the chitinase gene in Escherichia coli.A 1567-bp full length cDNA sequence (Genbank accession number: DQ412945) was produced by RACE reaction. Chintinase gene Aachit (Genbank accession number: DQ412945) was obtained by the analysis of amplified sequences and RT-PCR. The Aachit gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa) and three extrons, but also is interrupted by three short introns. Bioinformatic analysis of deduced amino acid sequence reveals that precursor protein has a predicted molecular weight of 45．9 kDa and is considered to be a stable acid protein with a theoretical pI of 5．55 and an instability index of 26．89．The active domains of the family 18 glycosyl hydrolases were indentified in the deduced amino acid sequence of Aachit. Protein Aachit harbours a 22-aa signal peptide and a coiled coil region may exist between aa 150 and 200．There is no leucine zipper structure in the whole amino acid sequence indicating that Aachit is a simple protein. It forms 19．56% helix, 25．78% sheet, and 54．67% loop in secondary structure of Aachit chitinase; the whole Aachit protein appears as compact, as a globular domain in tertiary structure. A homology modelling of Aachit protein shows that the chitinase Aachit have a (α/β)₈ TIM barrel structure. The phylogeny of chitinase genes of entomopathogenic fungi were extensively examined in comparison with chitinases derived from bacteria, archaea, fungi, ameba, nematodes, insects, spider, fishes, amphibians, mammals and viruses. The phylogeny analysis shows that the fungi chitinases clusters within a monophyletic clade, and the chitinase from entomopathogenic fungi clusters within a monophyletic clade. The topology within the entomopathogenic fungi shows that Verticillium and Beauveria chitinases are closely related. The chitinases of Firmicutes appears to be more closely related to those of entomopathogenic fungi, indicating that these bacterial and fungal chitinases has a close origin.The ORF of Aachit was cloned into the expression vector pMAL-c2X to study the properties of Aachit protein. Preliminary experiment indicates that 4 h is optimal induction time. Solubility analysis reveals that the recombinant protein is dissoluble. The recombinant protein was purified by affinity chromatography using amylose resin. And chintinase activity assay suggests that protein Aachit has endochitinase activity.