Genetic Characteristics,Functional Identification And Regulation Analysis Of Glycogen Synthase And Glycogen Phosphorylase In Nilaparvata Lugens

Glycogen is an animal's storage polysaccharide,a large branched biological macromolecule consisting of multiple glucose,with a molecular weight of 106-108 kD.It's a form of storage of glucose.Nilaparvata lugens is one of the main monophagous pests in rice.In this paper,we synthesized the Glycogen synthase(GS)and the Glycogen phosphorylase(GP)sequence of glycogen metabolic pathway obtained by the transcriptional group sequencing(RNA-Seq).Bioinformatics analysis of N.lugens GS and GP protein sequence,and uses the RNA interference technology(RNA interference,RNAi)to study its potential role and function in trehalose metabolism,chitin metabolism and insulin regulation pathway,in order to provide a basis for controlling the molecular mechanism of insect targets by regulating GS and GP genes.First of all,according to the known protein sequences of N.lugens GS and GP bioinformatics analysis,found that GS and GP open reading frame are 2199 bp and 2532 bp,encoding 733 and 844 amino acids,respectively.The predicted molecular weight of 84.1 kD and 97.3 kD respectively;the prediction of protein isoelectric 6.20 and 6.10 points respectively.DsRNA(double-stranded RNA)was synthesized in vitro,it was injected into the body of nymphs of five instar N.lugens.Taking the experimental materials after 48 h and 72 h after injection.The samples of RNA injected with dsGFP as the control group were detected by fluorescence real-time quantitative PCR(qRT-PCR).The results showed that compared with the injection of dsGFP,the mRNA level of GS and GP genes of the N.lugens was significantly decreased,which indicated that the effect of RNAi was obvious.After that,RNA samples from dsGS,dsGP and dsGFP were selected to be sequenced in the transcriptional group.RNA-Seq sequencing results showed that: compared with the sample injected with dsGFP RNA,after injection of dsGS RNA,the up regulation gene was 920,and the down regulated gene was 9412,while after injection of dsGS RNA,the expression of up regulation gene was 1055,and the down regulated gene was 6257.The relative expression levels of GS and GP genes in different developmental stages of N.lugens were detected by qRT-PCR.It was found that GS gene had relatively high expression level in five nstar nymphs in 12 h,48 h and 72 h of adult N.lugens,and the relative expression amount of other developmental stages was relatively low.The relative expression of GP gene was higher in the stage of five nstar 12 h to 48 h,and the relative expression of other stages was lower.After injecting RNA of dsGP or dsGP,the N.lugens has the phenomenon of chitin synthesis disorder or molting disorder,with high deformity rate and mortality.The rates of deformity were 21.27% and 27.50%,respectively,and the mortality rate was 23.40% and 25.40%,respectively.QRT-PCR was used to detect the metabolic pathways of trehalose and glycogen,and the changes in the expression of genes related to chitin.The results found the relative expression of PGM1 gene significantly increased after 48 h injection of dsGS,the relative expression of GS,GP,TPS3,TRE1-1,GFAT,G6PI1 gene decreased significantly,after injection of 72 h the relative expression of TRE1-1,TRE1-2,G6 Pase,TPS3,GS,HK and GFAT gene decreased significantly.After 48 h injection of dsGP,the relative expression of PGM1,G6PI1,CHS1,CHS1 a,CHS1b,GNPNA and InR2 increased significantly,while the relative expression of GS and GP decreased significantly.After 72 h injection,the relative expression of TRE1-1,TRE1-2,TRE2,TPS1,TPS2,UAP,PGM1,G6PI1,G6PI2 and GNPNA increased significantly.There was no significant change in trehalose content in the body of N.Lugens after RNAi GS gene in 48 h,and the content of glycogen and glucose had little increase but no significant change.There were no significant changes in trehalose,glycogen and glucose after 48 h low expression of the N.lugens GP gene.The expression of insulin signaling pathway related genes was detected by qRTPCR.It was found that after 48 h injection of dsGS,the relative expression of Ilp4 gene in N.lugens decreased significantly.After 72 h injection,the relative expression of Ilp2 and Ilp3 genes increased significantly,and Ilp4 gene decreased significantly.After 48 h injection of dsGP,the relative expression of InR2 gene increased significantly,while the relative expression of InR1,Ilp1 and Ilp2 genes decreased significantly.Ilp4 gene was significantly down regulated after injection of 72 h.After the low expression of GS and GP genes of the N.lugens 48 h and 72 h,the content of triglyceride was slightly increased,but there was no significant change.These results indicate that GS and GP genes have functions of regulating the chitin metabolism pathway by regulating trehalose metabolism pathway,resulting in the difficulty of chitin synthesis and molting disorder of N.lugens.