Study On The Expression Of ApGFAT By Using Antheraea Pernyi Nucleopolyhedrovirus Expression Vector System

Glutamine: fructose-6-phosphate amidotransferase(GFAT)is the rate-limiting enzyme in the hexosamine biosynthetic pathway,which is mainly located in the cytoplasm and is conserved among different species.The hexosamine biosynthetic pathway as an energy sensor and can integrate glucose,amino acid,lipid and nucleotide metabolism and other metabolic pathways.UDP-GlcNAc,the end product of hexosamine synthesis pathway,is involved in the synthesis of glycoprotein,proteoglycan and glycolipid,which is of great significance in cell biology.In insects,the hexosamine biosynthetic pathway is an integral part of chitin metabolism.Antheraea pernyi is an important silk insect unique to China,its growth and development are inseparable from energy metabolism and bone remodeling.The outer and inner layers of the Antheraea pernyi body wall are composed of chitin and protein,and the mass fraction of chitin in the Antheraea pernyi pupa is about 2.8%.However,hexosamine synthetic pathway has not been found in Antheraea pernyi,and GFAT,as the rate-limiting enzyme in this pathway,is of great significance to its research.Phosphorylation modification is one of the important ways to regulate GFAT activity.GFAT phosphorylation has been reported in different species such as humans,mice,and drosophilid.Due to the unstable activity of this protein,it is difficult to expression and purification in eukaryotic expression systems.This study aimed to express and purify the active Antheraea pernyi glutamine: fructose-6-phosphate amidotransferase(ApGFAT)by using the Antheraea pernyi nucleopolyhedrovirus expression vector system.First,the cDNA sequence of ApGFAT was obtained from the Antheraea pernyi transcriptome database.The expression of ApGFAT mRNA in different tissues was detected by real-time quantitative PCR.The results showed that the expression of this gene in different tissues was significantly different,and the expression in the testis was much higher than that in other tissues.Bioinformatics analysis revealed that the cDNA sequence of ApGFAT is composed of 2025 bases,encodes 674 amino acids.The molecular weight of ApGFAT is about 75.5 kDa,without a signal peptide structure.Through hydrophobic clustering analysis and three-dimensional structure superposition comparison,it was found that GFAT is highly conserved among different species,and the structure of ApGFAT is similar to human GFAT protein.It consists of two domains: glutamine domain and sugar isomerase domain.Then,the total RNA of Antheraea pernyi pupa was extracted and the cDNA library was synthesized by reverse transcription and we cloned the cDNA sequence of ApGFAT of Antheraea pernyi successfully.The fragment was inserted into the ApNPV transfer vector pApBacDual-ApGFAT,and then the transformed E.coli DH10 Bac was transformed to obtain recombinant bacmid.The bacmid DNA was extracted and then transfected into Tn-High 5cells.Screen out the recombinant virus with the gene of interest.The pupae were injected with the virus,and hemolymph was collected 7-10 days after the onset.Immunoblotting analysis using His antibody showed that the recombinant protein ApGFAT was expressed in pupae.The recombinant protein ApGFAT was purified by His affinity column,and its activity was0.978 ?mol /(min · mg).In this study,the cDNA sequence of ApGFAT was cloned and expressed in pupae,which laid the foundation for the follow-up research on the hexosamine pathway and chitin synthesis of Antheraea pernyi.