Molecular Cloning And Characterization Of Chitin Metabolism Related Enzymes From The Black Cutworm, Agrotis Ipsilon (Rottemberg)

Several insect chitinase 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 chitin metabolism related enzymes'cDNA sequences from the Agrotis ipsilon(Rottemberg). A detailed understanding of these chitin metabolism related enzymes'molecular characteristics was necessary for the development of insect chitin metabolism related enzymes -based insecticides.One full-length sequence and one fragment of chitinase cDNA 5'fragment were isolated from the prepupae of A. ipsilon. The complete chitinase cDNA sequence, 2823 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 62.5 kDa. The fragment of chitinase cDNA sequence, 1082 base pairs in length, and it encoded a polypeptide of 360 amino acid residues. Two conserved domains were revealed in the full-length of the deduced amino acid sequence. 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. 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 2 N-glycosylation and 20 O-glycosylation sites were found in the amino acid sequence of A. ipsilon by PROSCAN and NetOGlyc V 2.0 software , respectively.Two beta-N-acetylglucosaminidase 3'cDNA sequences were isolated from the prepupae of A. ipsilon. One cDNA sequence, 2267 base pairs in length, contained an open reading frame of 1509 base pairs and it encoded a polypeptide of 503 amino acid residues with a predicted molecular weight of 57.6 kDa. The other cDNA sequence, 2278 base pairs in length, contained an open reading frame of 1509 base pairs and it encoded a polypeptide of 503 amino acid residues with a predicted molecular weight of 57.5 kDa.Two conserved domains were revealed in anyone of the deduced amino acid sequences of beta-N-acetylglucosaminidase. One was the Glycosyl hydrolases catalytic domain at the N-terminus, and the other was Glycosyl hydrolases domain 2.Two putative 3 N-glycosylation and 1 O-glycosylation sites were found in the amino acid sequence of A. ipsilon by PROSCAN and NetOGlyc V 2.0 software , respectively.One fragment of chitin synthase cDNA fragment were isolated from the feeding larvae of A. ipsilon. The fragment is 975 base pairs in length,includes 792 base pairs reading frame,encoded a polypeptide of 264 amino acid residues with a predicted molecular weight of 30.0 kDa. A number of insect chitinases with high sequence homology were gained by BLAST search. The deduced amino acid sequence of chitinase from A.ipsilon shared 80% identical amino acid residues in the mature part with Manduca sexta, 85% with Helicoverpa armigera, 83% with Hyphantria cunea and 89% with Spodoptera litura. The comparison of this deduced amino acid sequence with other lepidopteran insect chitinases also showed high degree of similarity (over 75%).A number of insect beta-N-acetylglucosaminidase with high sequence homology were gained by BLAST search. The deduced amino acid sequence of beta-N-acetylglucosaminidase from A.ipsilon shared 73% identical amino acid residues in the mature part with M. sexta, 37% with Spodoptera frugiperda, and 74.4% with Ostrinia furnacalis.A number of insect chitin synthase sequences with high sequence homology were gained by BLAST search. The deduced amino acid sequence of b chitin synthase from A.ipsilon shared 79.2% identical amino acid residues in the mature part with M. sexta, 92% with Mamestra brassicae, 89.4% with Spodoptera exigua.These novel chitin metabolism related enzymes cDNA sequences were submitted in GenBank under with accession No. EU035316 and EU622802 for chitinase cDNA sequences, EU622803 and EU622804 for beta-N-acetylglucosaminidase and EU601174 for chitin synthase cDNA sequences.Transcript analysis of A. ipsilon chitin metabolism related enzymes during various developmental stages was determined by RT-PCR. During the larval-larval, larval-pupal transformation, a clear expression of A. ipsilon chitinase and beta-N-acetylglucosaminidase mRNA could be observed. However, the level of chitin synthase transcript was present at the stages when the larvae were feeding.