Molecular Cloning And Functional Analysis Of Spodoptera Litura ?-N-Acetylglucosaminidase Genes

Spodoptera litura(Lepidoptera,Noctuidae)is a destructive agricultural pest on crops among the world and it has a wide range of host plants.In addition,many kinds of chemical pesticides have been used for a long time to control it,resulting in the reducing sensitivity to varieties of pesticides.So it is essential to find effective control methods.We studied the function of ?-N-acetylglucosaminidase genes SlNAG1,Sl NAG2,SlNAG3 and SlFDL,which play very impotant roles in the process of growth and development,and provide theoretical basis for the research and development of novel pesticide based on targeted chitin metabolism.The main results are as follows:1.According to the data of NCBI database and S.litura transcripts,and combining with technique of rapid amplification of cDNA ends(RACE),four ?-N-acetylglucosaminidase genes of S.litura were cloned.Our results showed that the SlNAG1,SlNAG2,SlNAG3 and SlFDL open reading frame(ORF)contained 1770 bp,1854 bp,2067 bp and 2583 bp nucleotides,encoding 590,618,689 and 861 amino acids,respectively.2.The sequence alignment results indicated that the four SlNAGs genes had two hydrolytic regions,which belong to glycosyl hydrolase 20 family.Furthermore,The phylogenetic tree indicated that the ?-N-acetylglucosaminidases of S.litura were collected in different subfamilies,SlNAG1 belonged to subfamily ?,SlNAG2 belonged to subfamily ?,SlNAG3 and SlFDL belonged to subfamily ?,but there was no ?-N-acetylglucosaminidase belonged to subfamily ?.3.The qRT-PCR results showed that the expression level of SlNAG1 was highest in the epidermis of the first day of the pre-pupa.SlNAG2 was mainly expressed in the epidermis and fat body,and peaked in the epidermis of the fourth day of the pupal stage.The expression of SlNAG3 and SlFDL were relatively low,the expression level of SlNAG3 in the epidermis increased in the larvae at each age,and the expresson of SlFDL was mainly expressed in fat body.After injection of 20 E,both SlNAG1 and SlNAG2 showed up-regulation,so it was confirmed that SlNAG1 and SlNAG2 were associated with molting of S.litura.4.RNA interference was performed on four genes by injection method,the results showed that the expression level could be interfered,but no abnormal phenotype was observed.5.The recombinant proteins of SlNAG1 and SlNAG2 were obtained by eukaryotic expression system.The molecular weights of recombinant SlNAG1 and SlNAG2 were 67 kDa and 70 kDa by SDS-PAGE and Western Blotting,respectively.The recombinant protein of SlNAG1 was active with pNP-?-GlcNAc.