Molecular Cloning, Recombinant Expression Of β-N-acetylglucosaminidase And Hormone Receptor 3 CDNA From Three Noctuid Insects

Insect molting is a complicated physiological process. Many enzymes, hormones and receptors involve in this process.β-N-acetylglucosaminidase, which is the critical degrading enzyme of chitin in cuticle and midgut peritrophic membrane, and molt hormone receptor 3 are studied basically.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. P-N-acetylglucosaminidase is a critical enzyme in the degradation of chitin in insects. It plays an important role in a variety of physiological activities of insects.In this study, the mRNA was isolated from the insect prepupal stage. The cDNA sequence of Agrotis ipsilonβ-N-acetylglucosaminidase was cloned by RT-PCR and rapid amplification of cDNA ends(RACE).The P-N-acetylglucosaminidase gene of Agrotis ipsilon was cloned into the expression vector pTYB11 and expressed in E.coli(BL21)host cell.The Agrotis ipsilonβ-N-acetylglucosaminidase cDNA,2579 base pairs in length, contained an open reading frame of 1788 base pairs, coding for a polypeptide of 595 amino acid residues with a predicted molecular weight of 68.3 kDa and pI 5.48.The cDNA sequence has been deposited in Genbank with accession No. GU985280.Two highly conserved regions were found in the deduced amino acid sequence of Agrotis ipsilonβ-N-acetylglucosaminidase by Prosite software. They were found in all numbers 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 and one O-glycosylation site were found in the amino acid sequence.The P-N-acetylglucosaminidase gene of Agrotis ipsilon was cloned into the expression vector pTYB11 and expressed in E.coli(BL21) host cell. After the expression strain was induced by IPTG, the fusion protein was expressed and detected in the cells.Insect molt hormone receptor 3 (HR3) is a molt-regulating transcription factor. It plays an important role in regulating the insect molting. Total RNA was isolated from the prepupae of Agrotis c-nigrum Linnaeus and Mythimna separata Walker, respectively. The cDNA sequences were cloned by RT-PCR and Rapid amplification of cDNA ends (RACE).The cDNA sequence of A.c-nigrum HR3,1729 base pairs in length, contained an open reading frame of 1533 base pairs coding for a polypeptide of 510 amino acid residues with a predicted molecular weight of 57.5kDa. The cDNA sequence of M. separate HR3,1743 base pairs in length, contained an open reading frame of 1536 base pairs coding for a polypeptide of 511 amino acid residues with a predicted molecular weight of 57.9kDa. The predicted protein of both insects had the characteristics of nuclear receptor superfamily, and shared extensive similarities with those from other insects, especially the lepidopteran insects.The cDNA sequences have been deposited in GenBank with accession numbers GU188853 for A.c-nigrum HR3 and GU188854 for M. separata HR3.