Cloning,expression And RNAi Of Chitin Deacetylase(CDA2a/2b)in Monochamus Alternatus(coleoptera,cerambycidae)

Monochamus alternatus Hope?Coleoptera,Cerambycidae?is an important pest to Chinese pine forests.It is the main vector of pine wood nematode,the causative agent of pine wilt disease.C hitin deacetylase?CDA?are members of an extracellular chitin-modifying enzyme family,which deacetylate chitin to chitosan,a polymer of?-1,4-linked D-glucosamine residues,then hydrolyzed by chitinase and?-N-acetyl-D-hexosaminidase.C DA is a key enzyme in insect metabolism and plays an important role in a variety of physiological activities of insects.In this study,3^{t h} instar larvae of M.alternatus was extracted for isolating total RNA.Sequence of ORF encoding Chitin deacetylase2a/2b from M.alternatus,respectively named MaCDA2a and MaCDA2b.The O RF of MaCDA2a contains 1611bp,which encodes 536amino acids.Its predicted molecular weight is 60.9kD,and isoelectric point around 5.00.The ORF of MaC DA2b contains 1590bp,which encodes 529 amino acids.Its predicted molecular weight is 60.0kD,and isoelectric point around 5.08.The hydrophilic proteins,without transmembrane area and glycosylation sites,has a low-density lipoprotein receptor class A domain?LDLa?a chitin-binding peritrophin-A domain?C hBD?and a polysaccharide deacetylase-like domain?CDA?.The length range of signal peptides was from 1-19 amino acids?MANKRIWLCCFLLLGIAYA?.Using amino acid of CDA2 from seven insects to analyzed,t he results concluded that:?1?MaCDA2a/2b and CDA2 from other insects share an deeper homology?82.91%?;?2?Polysaccharide deacetylase-like domain?CDA?was found in possession of five motifs:SFNG?FSLTHK?RAPYL?FVYD?GLHF;?3?The phylogenetic tree analysis found that MaCDA2a/2b belongs to group I of CDA protein.The expression pattern of tissue was analyzed by RT-qPCR showed that MaC DA2a/2b was commonly present in all tissues of larvae,especially high in body wall related to molting.In the development stages,MaCDA2a/2b had obviously different expression patterns,which was related to its different functions.Using ds RNA to research the function of MaCDA2a/2b,the results showed:the expression of gene was inhibited by the ds RNA of two genes effectively,making the mortality of M.alternatus reach 96%,extending the ages of four instars.After injecting dsRNA-MaCDA2a,the larvae die for molting unsuccessfully,and the phenot ype of death was being soft and black.After injecting ds RNA-MaCDA2b,the phenotype of death was different from ds RNA-MaCDA2a.The dead larvae was being soft and grey.In a word,the study showed that MaCDA2a/2b played an important role in the way of chitin degradation.The analyses facilitate further understanding of chitin metabolism in the coleopterous insects,and lay the foundation for finding the potential targets for new biopesticides.