Cloning And Expression Of β-N-acetylglucosaminidase (OfOGA)and β-N-acetylglucosaminyltransferase (OfOGT) Genes From Ostrinia Furnacalis

O-GlcNAcsylation is a dynamic and reversible post-translational modification of proteins, participates in the important physiological functions. The process of unique O-linked N-acetylglucosamine (O-GlcNAc) on Ser/Thr residues of targeted proteins is regulated by two key enzymes:O-GlcNAc transferase (OGT) for the addition of GlcNAc; and O-GlcNAcase (OGA) for the hydrolyzation of the mono suger unit.In this research, the β-N-acetylglucosaminidase (OfOGA) and β-N-acetylglucosaminyltransferase (OfOGT) genes from the agricultural pest, Asian corn borer Ostrinia furnacalis, were cloned and recombinant expressin, which may facilitate the understanding of their roles in insect development and growth. This research mainly includes:1. The clone of OfOGA gene of the Asian corn borer Ostrinia furnacalis (Guenee).Based on sequences alignment of the reported β-N-acetylglucosaminidase (OGA) of insects, highly conserved sequences were selected and primers were designed. The OfOGA gene from the fifth instar larva of the Asian corn borer Ostrinia furnacalis (Guenee) was obtained by RT-PCR and RACE. Full length OfOGA gene is3541bp, containing241bp5’-UTR,3165bp coding region and132bp3’-UTR, which encodes1055amino acids. The OfOGA gene has been submitted to GenBank and its accession number is JQ666007.2. The recombinant expression, purification, and characterization of OfOGA of the Asian corn borer Ostrinia furnacalis (Guenee).The OfOGA gene was cloned into the expression vector pET28a with six-histidine tag, then was transformed into E.coli host strain, BL21(DE3)plysS. With IPTG induction, the restructured OfOGA expressed accordingly.The Ni-NTA agarose affinity column was used to purify OfOGA. The theory molecular weight of OfOGA is118kDa, but SDS-PAGE shows its molecular weight is about130kDa; Its predicted isoelectric point pi is5.12. The optimal pH of OfOGA is5.5and optimal temperature is55℃, when select the pNP-GlcNAc as the substrate.3. The clone and recombinant expression of OfOGT of the Asian corn borer Ostrinia furnacalis (Guenee). Based on sequences alignment of the reported μ-N-acetylglucosaminyltransferase (OGT) of insects, highly conserved sequences were selected and primers were designed. The OfOGT gene from the fifth instar larva of the Asian corn borer Ostrinia furnacalis (Guenee) was obtained by RT-PCR and RACE. Coding region of OfOGT gene is3177bp, which encodes1058amino acids. ProtParam predicts its weight is about118kDa, its isoelectric point pI is6.31. The OfOGT gene was cloned into the expression vector pET28a with six-histidine tag, then was transformed into E.coli host strain, BL21(DE3)plysS. With IPTG induction, the restructured OfOGT expressed accordingly. SDS-PAGE shows its molecular weight is about118kDa...