梯棱羊肚菌谷氨酸脱氢酶基因克隆及原核表达OA

The glutamate dehydrogenase gene from Morchella importuna(MiGDH)was cloned using RT-PCR and subjected to bioinformatics analyses.A prokaryotic expression vector for MiGDH,pEB-MiGDH,was constructed and then induced for heterologous expression in Escherichia coli BL21(DE3).The results showed that the cDNA sequence of MiGDH contains an open reading frame of 1 377 bp,encoding a protein of 458 amino acids.The full-length genomic DNA of MiGDH is 1 529 bp,comprising three exons and two introns.The deduced MiGDH is a stable hydrophilic protein,and has a relative molecular mass of 50 167.51 and a theoretical isoelectric point(pI)of 5.91.It lacks a signal peptide and transmembrane domains,indicating that it is localized in the cytoplasm.The N-terminus of MiGDH contains an ELFV_dehydrogenase domain,which is identified as an NADP(H)-specific glutamate dehydrogenase.The secondary structure of MiGDH primarily consists of α-helix and random coils,with a tertiary structure forming a homologous hexamer.MiGDH was successfully expressed in the prokaryotic expression system.The heterologous expression of MiGDH provided a reference for further investigations into the biological functions of MiGDH in M.importuna.