Cloning And Expression Of Some CDNAS Encoding Lepidopteran Chitinase

Several insect chitinase specific cDNAs have been isolated and integrated into transgenic plants to enhance their defence mechanisms, and inserted into insect pathogens to increase their deleterious effects on insects. We reported here the cloning and expression of four insect chitinase cDNAs from the beet armyworm, Spodoptera exigua (Hübner), Oriential armyworm, Mythimna sepatata( Walker), Asian corn borer, Ostrinia furnacalis (Guenee) and rice stem borer, Chilo suppressalis (Walker). A detailed understanding of these chitinase molecular characteristics was necessary for the development of insect chitinase-based insecticides.Four chitinase cDNAs were isolated from the prepupae of S. exigua, M. sepatata, O. furnacalis and C. suppressalis, respectively. The S. exigua chitinase cDNA, 2838 base pairs in length, contained an open reading frame of 1674 base pairs and it encoded a polypeptide of 557 amino acid residues with a predicted molecular weight of 62.6 kDa. The M. sepatata chitinase cDNA, 2810 base pairs in length, contained an open reading frame of 1677 base pairs and it encoded a polypeptide of 558 amino acid residues with a predicted molecular weight of 62.8 kDa. The O. furnacalis chitinase cDNA, 3343 base pairs in length, contained an open reading frame of 1662 base pairs and it encoded a polypeptide of 553 amino acid residues with a predicted molecular weight of 61.8 kDa. The C. suppressalis chitinase cDNA, 2237 base pairs in length, contained an open reading frame of 1659 base pairs and it encoded a polypeptide of 552 amino acid residues with a predicted molecular weight of 61.4 kDa. Two conserved domains were revealed in anyone of the deduced amino acid sequences. One was the highly conserved catalytic domain at the N-terminus, and the other was chitin-binding type 2 domain at the C-terminus which contained six conserved cysteines. There was a Linker named Pro/Glu/Ser/Thr-rich (PEST) region between catalytic domain and chitin-binding domain 2. In anyone of these four insect chitinase amino acid sequences, a hydrophobic signal peptide was predicted to precede the N-terminal region of the mature protein.Several putative N-linked glycosylation and O-glycosylation sites that might be necessary for the secretion of the protein and maintenance of their stability were found within the deduced amino acid sequences of the isolated insect chitinases. Two putative N-glycosylation sites were found in the amino acid sequence of S. exigua1 M. sepatata and O. furnacalis while three putative W-glycosylation sites were found in the amino acid sequence of C. suppressalis, respectively. Moreover, the linker domains of these four insect chitinase amino acid sequences were extensively modified by O-glycosylation. There were 23, 21, 29, 29 O-glycosylation sites in the chitinase amino acid sequences from S. exigua, M. sepatata, O. furnacalis and C. suppressalis by NetOGlyc V 2.0 software, respectively.A number of insect chitinases with high sequence homology were gained by BLAST search. The deduced amino acid sequence of chitinase from S. exigua shared 81% identical amino acid residues in the mature part with Manduca sexta, 87 % with Helicoverpa armigera, 83% with Hyphantria cunea and 96% with Spodoptera litura. The comparison of these four deduced amino acid sequences with other lepidopteran insect chitinases also showed high degree of similarity (over 75%). The comparison suggested that the cDNAs encoded chitinase proteins of S. exigua, Mythimna sepatata, Ostrinia fumacalis and Chilo suppressalis The features indicated that these chitinases belonged to family 18 glycosyl hydrolase. These novel chitinase cDNAs, designated as Sechi, Mechi, Ofchi and Cschi, were submitted in GenBank under with accession No. AY658731, No. AY508698, No. AY726548 and No. AY705930.The recombinant protein from the cDNAs of S. exigua and M. sepatata were obtained using two E. co//-based expression systems and one Pachia pastoris yeast expression system. The chitinolytic activities of the recombinant proteins expressed in E. coli and yeast were analyzed by a colorimetric assay method and the result indicated that the recombinant proteins showed apparant levels of chitinolytic activities using CM-Chitin-RBV as the assay substrate.Transcript analysis of S. exigua chitinase cDNA during various developmental stages and in different tissues was determined by semi-quantitative RT-PCR. A specific product was obtained at all stages, but the relative intensity of the band varied at each stage, indicating different mRNA levels. During the larval-larval, larval-pupal transformation, a clear expression of 5. exigua chitinase mRNA could be observed. However, a detectable level of chitinase transcript was also present at the stages when the larvae were not moulting. The tissue specific expression of chitinase transcript was studied in the integument, midgut and the fat bodies by semi-quantitative RT-PCR in the first and second day of sixth instar larvae. The relative gene expression levels from the first day of the sixth instar larva in fat bodies, midgut and integument were not different while in the second day of the sixth instar larva the relative gene expression levels varied significantly. The relative mRNA expression levels in the gut were 2.3 and 1.3 fold to that from fat bodies and integument.