Cloning And Characterization Of ?-N-acetylglucosaminidase And Chitinase From The Oriental Armyworm,Mythimna Separata Walker

Chitin is an important component of insects,and the normal function of the chitin metabolic system is crucial for insect molting processes.As the key enzyme in chitin degradation,?-N-acetylglucosaminidase and chitinase are of great significance to insect growing development.M.separata was the main pest of grain crops which occurred in many areas in China,caused high density hazards and threaten to food security of China seriously.Because there is no chitin in higher plants and animals,it is a key link in the study of pest biological control technology to make?-N-Acetylglucosaminidase and chitinase genes as new target genes for biotechnology control of M.separata.The cDNA sequences of the new?-N-acetylglucosaminidase gene MsNAG and the chitinase gene MsCHT7 were cloned and obtained from M.separata,and the expression patterns of the two genes at different developmental stages and in different tissues were studied.The effect of 20-hydroxyecdysone ecdysone on the expression of the two genes were researched.The function of MsNAG gene was investigated by using RNAi technique,which provided a new idea for biological control technology of M.separata.The cDNA sequence of MsNAG was cloned from M.separata by RT-PCR and RACE techniques.The full length cDNA sequence was 2619 bp in length,and contained an open reading frame of 1785 bp,which encodes a polypeptide containing 594 amino acids with an isoelectric point of 5.19 and a molecular weight of 57.8 kDa.Through amino acid sequence analysis,it was found that the gene belongs to the 20 glycosyl hydrolases family,and which was a chitinase NAG I.The gene we cloned was designated as MsNAG and registered on GenBank,the accession number we obtained was No.KY348777.The identities of the amino acid sequence were more than 80%between MsNAG and those of Lepidoptera Noctuidae insects such as M.brassicae,A.ipsilon,T.ni and X.c-nigrum.The cDNA sequence of the chitinase gene MsCHT7 was identified by high-throughput sequencing technology from M.separata.The full-length cDNA of MsCHT7 was 3360 bp with an open reading frame of 2970 bp.It encoded a polypeptide containing 989 amino acids with an isoelectric point of 6.24and a molecular weight of 111.6 kDa.By amino acid sequence analysis,the gene was found to belong to the 18 glycosyl hydrolases family,which has two Glyco₁8 catalytic domains and one chitin binding domain ChtBD₂,and belongs to chitinase Group?CHT7,was designated as MsCHT7.The cDNA squence was registered on GenBank,the accession number we obtained was MG551526.The identities of amino acid sequence were 96%?92%?80%and 78%between MsCHT7 and those of S.exigua,C.punctiferalis,T.castaneum and N.lugens.The expression of MsNAG at different developmental stages and in different tissues was studied using realtime PCR.The result showed that MsNAG was expressed at different developmental stages of M.separata.The expression levels of the gene showed periodic changes before and after each molting period,and the expression levels before molting were higher than those after molting.The expression levels before and after molting raised with the increase of the larval instars.MsNAG expressed a highest level at the prepupal stage,and the level decreased on the first day of pupal stage.In addition,the expression levels did not increase on the last day of pupal stage and adult stage.MsNAG expressed in different tissues,with the highest expression level in the salivary gland,followed by the integument,and the lowest expression level in the hindgut.The expression of MsCHT7 at different developmental stages and in different tissues were researched.The MsCHT7 gene was also expressed at various developmental stages of M.separata,and showed a periodic expression pattern before and after the molting.The expression levels raised with the increase of the larval instars.MsCHT7 expressed a highest level at the prepupal stage.However,on the first day of pupal stage,the expression level decreased,but increased again on the last day of pupal stage,and the expression level at adult was also high.MsCHT7 was expressed in 7 tissues including the foregut,midgut,hindgut,salivary gland,malpighian tubule,integument and fat body.MsCHT7expressed a highest level in the integument and a lowest level in the midgut.20E induced the expression of MsNAG and MsCHT7 genes.Different concentrations of 20E ecdysone were used to treat 5^thh instar larvae of M.separata,and the effect of 20E on the expression of MsNAG and MsCHT7 genes were studied.The result showed that the expression levels of MsNAG and MsCHT7 genes gradually raised with the increase of 20E concentrations at 2.5?g/?L,5?g/?L and 10?g/?L,but decreased at 20?g/?L.The result showed that 20E successfully induced the expression of MsNAG and MsCHT7 genes,and 20E performed best induction effect at the concentration of 10?g/?L.MsNAG might be related to insect molting.RNAi was used to perform functional study on MsNAG gene.At 24 and 48 h after dsMsNAG injection,the expression levels of MsNAG gene in insect bodies were repressed by 76.5%and 80.6%,respectively.The expression levels of MsNAG in integuments were repressed by 67.9%and 81.1%,respectively.After RNA interference,some insects could not complete normal molting process and even died.The result proved that the expression of MsNAG was inhibited by RNAi successfully,and MsNAG might be related to physiological activities such as insect molting.