Molecular Cloning And Expression Of Two Different Chitin Deacetylase CDNA Sequences From Mamestra Brassicae

Insect molting is a complicated physiological process, many enzymes, hormones and receptors involve in this process. Chitin deacetylase, which is the critical degrading enzyme of chitin in cuticle and midgut peritrophic membrane, is preliminary studied.Chitin is one of the most abundant, easily obtained, and renewable natural polymers, second only to cellulose. It is commonly found in many invertebrates and in the cell walls of most fungi and algae. chitin deacetylase is a critical enzyme in the degradation of chitin in insects. Chitin deacetylase is a kind of chitin-degrading enzyme which can turn chitin into chitosan.Total RNA was isolated from the fifth instar larvae of Mamestra brassicae. Two CDA cDNA sequences were cloned by RT-PCR and rapid amplification of cDNA ends. One cDNA sequence was 2036 base pairs in length and contained an open reading frame of 1617 base pairs coding for a polypeptide of 539 amino acid residues with a predicted molecular weight of 61.5 KDa. This cDNA sequence was designated as cDNA 1. The other cDNA sequence was 1312 base pairs in length and contained an open reading frame of 1173 base pairs coding for a polypeptide of 390 amino acid residues with a predicted molecular weight of 44.1 KDa. This cDNA sequence was designated as cDNA 2. The deduced amino acid sequence of cDNA 1 contained a polysaccharide deacetylase domain, a chitin-binding peritrophin-A domain and a low-density lipoprotein receptor class A domain. This sequence was a member of chitin deacetylase group I. The deduced amino acid sequence of cDNA 2 only contained a polysaccharide deacetylase domain. This sequence was a member of Chitin deacetylase group V. These two CDAs belonged to two different CDA groups. Transcript analysis on two CDA sequences during various developmental stages and in different tissues was determined by RT-PCR. The results showed that cDNA 1 expressed in salivary glands, midgut, fat body, but cDNA 2 only expressed in midgut. cDNA1 was cloned into the expression vector pET21b and expressed in E. coli(BL21) host cell. Induced by IPTG, the protein was expressed and detected in the cells. The cDNA sequences have been deposited in Genbank with accession numbers HQ680620 for cDNA 1 and HQ680621 for cDNA 2.