Molecular Cloning And Function Analysis Of Spodoptera Litura Chitin Deacetylase Genes

Chitin deacetylase is an enzyme for one of the pathways of chitin degradation in insects,and it can degrade chitin to chitosan.Spodoptera litura(Lepidoptera,Noctuidae)is considered as one of the most destructive insect pests over the world because of its high reproductive rate and heavy losses of agricultural production.It is imperative to investigate the physiological and biochemical mechanisms of S.litura and explore the effective preventions and control methods.In this study,S.litura was chosen as the target to find out new ideas and methods of controlling this pest by studying the function of chitin deacetylase.The main results are as follows:Six chitin deacetylase genes were screened and cloned from the data of the genus S.litura,named SlCDA1,SlCDA2,SlCDA 3,SlCDA 4,SlCDA 5,SlCDA 6,respectively.Our results showed that open reading frames contained 1620 bp,1446 bp,7224 bp,1131 bp,1167 bp,1125 bp nucleotides,and encoded 540,482,2408,377,389,375 amino acids residues,respectively.The phylogenetic analysis showed that SlCDA1,SlCDA2 and SlCDA3 belonged to the first group(Group Ⅰ),third group(Group Ⅲ)and fourth group(Group Ⅳ),respectively,and SlCDA4,SlCDA5,and SlCDA6 belonged to the fifth group(Group Ⅴ).Furthermore,the homology of the amino acid sequence showed that the chitin deacetylase genes of S.litura had the highest homology to the genes of Helicoverpa armigera,Bombyx mori.The expression profiles of SlCDAs were determined by quantitative real-time PCR(qRT-PCR).The result showed that SlCDA1 and SlCDA2 were highly expressed in epidermis,the express level increased gradually after molting and the highest expression was observed in pupal stage on the first day.Moreover,no specific in the transcript level of SlCDA3 was detected in the epidermis,fat body and midgut in our testing stage.The expression levels of SlCDA4 and SlCDA6 genes were also high on the midgut of the of 2nd 6rd instar larvae.In contrast,the low expression of SlCDA5 was observed in these three tissues.Based on the results,we concluded that SlCDA1 and SlCDA2 genes might be related to the physiological processes of epidermis molting of S.litura,the results also indicated that both SlCDA4 and SlCDA6 might be associated with functions of midgut,whereas SlCDA3 and SlCDA5 might not be much associated with the molting of S.litura.According to the results of expression profiles,two epidermis-specific genes SlCDA1 and SlCDA2,RNA interferenced on S.litura and detected the efficiency.The result showed that both the expression levels of the SlCDA1 and SlCDA2 both were down-regulated injecting after 24 hours but no phenotype or lethality was observed of the tested S.litura.The recombinants of SlCDA1 and SlCDA2 were expessed by baculovirus expression system.The molecular weights of recombinants SlCDA1 and SlCDA2 were approximately 62 kDa and 55 kDa,respectively.We found that both SlCDA1 and SlCDA2 had chitin binding activity.