Molecular Cloning And Characterization Of Chitin Degrading Enzymes From Three Lepidopteran Insects

Chitin,a homopolymer ofβ-1,4-linked N-acetylglucosamine,is one of the most abundant, easily obtained,and renewable natural polymers,second only to cellulose.It is commonly found in the exoskeletons or cuticles of many invertebrates and in the cell walls of most fungi and algae. Chitin degrading enzymes can destroy peritrophic membrane and integument because they can hydrolyze chitin.Chitin degrading enzymes,which consist of chitinase,β-N-acetylg lucosaminidase and chitin deacetylase,can damage the tissue structure of insect.The activity of these enzymes can be controlled to influence the growth of insects,and the genetically engineered bacteria and transgenic plants could be constructed by using those gene to against insect pests.In this study,the mRNA was isolated from the prepupal stage.The Agrotis c-nigrumβ-N-acetylglucosaminidase cDNA sequence,Mamestra brassicae chitinase cDNA sequence, Agrotis ipsilon chitinase cDNA sequence and 3' fragment of Agrotis ipsilon chitin deacetylase cDNA sequence were cloned by RT-PCR and Rapid amplification of cDNA ends(RACE).The A.c-nigrumβ-N-acetylglucosaminidase cDNA,2488 base pairs in length,contained an open reading frame of 1785 base pairs coding a polypeptide of 594 amino acid residues with a predicted molecular weight of 67.8 kDa and pI 5.38.The cDNA sequence has been deposited in GenBank with accession No.FJ848784.Two highly conserved regions were found in the deduced amino acid sequence of A. c-nigrumβ-N-acetylglucosaminidase by Prosite software.They were found in all number of family GH20.One was the Glycosyl hydrolases family 20 at the N-terminus,and the other was Glycosyl hydrolases family 20 catalytic domain at 220 of C-terminus.Three putative N-glycosylation sites in the amino acid sequence of the deduced amino acid sequence.But O-glycosylation sites were not foundThe M.brassicae chitinase cDNA,2826 base pairs in length,contained an open reading frame of 1689 base pairs coding for a polypeptide of 562 amino acid residues with a predicted molecular weight of 62.6 kDa and pI 5.30.The deduced amino acid sequence of M.brassicae chitinase was predicted using Prosite software to have two putative N-glycosylation sites and twenty two putative O-glycosylation sites.The cDNA sequence has been deposited in GenBank with accession No.FJ436415.The A.ipsilon chitinase cDNA,2806 base pairs in length,contained an open reading frame of 1680 base pairs coding for a polypeptide of 559 amino acid residues with a predicted molecular weight of 62.5 kDa and pI 5.12.The deduced amino acid sequence of A.ipsilon chitinase was predicted using Prosite software to have two putative N-glycosylation sites and twenty putative O-glycosylation sites.The cDNA sequence has been deposited in GenBank with accession No. FJ899540.Deduced amino acid sequences of the M.brassicae chitinase and A.ipsilon chitinase cDNA are multi-structural domain proteins.Two conserved domains were revealed in the 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. The chitin-binding type 2 of M.brassicae chitinase is at 504-562 and A.ipsilon chitinase is at 499-557.The Chitinases family 18 active site was at 139-147 of M.brassicae chitinase cDNA,and at 138-146 of A.ipsilon chitinase cDNA.The 3'fragment of A.ipsilon chitin deacetylase cDNA sequence,1514 base pairs in length, contained an open reading frame of 1152 base pairs coding for a polypeptide of 383 amino acid residues.The deduced amino acid sequence of A.ipsilon chitinase was predicted using BLAST search to have a Polysaccharide deacetylase 1 in 40 of N-terminus.The predicted protein shared extensive similarities with those from other insects.It can be affirmed a new fragment of chitin deacetylase cDNA sequence.The cDNA sequence has been deposited in GenBank with accession No.FJ899541.Transcript analysis of A.c-nigrumβ-N-acetylglucosaminidase during various developmental stages were determined by RT-PCR.During the larval-larval,larval-pupal transformation,a clear expression of A.c-nigrumβ-N-acetylglucosaminidase mRNA could be observed.