Abstract: 【Objective】In this paper, Genes of GDH were cloned from mulberry and characteristics of their structures, tissue expressions，regulation factors were studied and GDH proteins were obtained in order to reveal the role of glutamate dehydrogenase (GDH) in nitrogen metabolism. The results could provide data for studying other perennial woody plants on GDH.【Method】Primers were designed according to the homologous sequences of GDH downloaded from the Morus genome database. Using Guisangyou 62 (M. atropurpurea Roxb.) cDNA as template, MaGDHs were cloned by RT-PCR. The similarity of GDH sequences was analyzed by BLASTN and converse structure domains were analyzed by CDD on line of NCBI. The composition of the amino acid, theoretical molecular mass and pI were deduced by Expasy software online. A phylogenetic tree was constructed by MEGA 5.0 software performed with the neighbor-joining (NJ) method. Expressions of MaGDHs under the condition of different concentrations of sucrose, NH4+ and 6-BA solutions were measured by qRT-PCR. The data were analyzed by StepOne Software V2.1 using method of 2^-△△Ct. MaGDHs recombinant plasmids were constructed with vectors of pET-28a(+), pET-32a(+), pcold-TF and transformed to E. coli to produce MaGDHs recombinant protein.【Result】Both two obtained genes MaGDHs were 1236 bp in length with an open reading frame encoding a polypeptide of 411 amino acids, named MaGDH1 and MaGDH2. The predicted molecular mass of MaGDH1 was 44.1 kD and a predicted isoelectric point was 5.84. The predicted molecular mass of MaGDH2 was 44.2 kD and a predicted isoelectric point was 6.68. Both two sequences have 9 exons and 8 introns including a mitochondrial transfer peptide and two features domains: a Glu/aKG binding domain and an NAD(P) binding domain. The MaGDHs protein showed identity of 99% with Morus notabilis, about 90% with dicotyledons plants and 85% with monocotyledons plants. The proteins of MaGDHs were successfully expressed in E.coli BL21 (DE3) in the form of inclusion bodies. After purification and renaturation, active enzymes were obtained and the activity of recombinant protein pET-28a(+)-MaGDH1 was the highest with 10.07 nmol?min^-1?mL^-1. MaGDH1 and MaGDH2 showed organ specificity but distinct transcript intensity. The expression level of flower was the highest and the expression level of young leaf was taken the second place. They hardly expressed in fruit. Transcript abundance of MaGDHs were up-regulated by sucrose in this study, MaGDHs were also expressed without NH₄⁺ in media and excess NH4+ promoted the transcript of MaGDHs. 6-benayl aminopurine first inhibited and then promoted the expressions of MaGDHs and MaGDH1 was promoted after 24 h and MaGDH2 was promoted after 48 h.【Conclusion】Two genes of GDH were obtained from mulberry named MaGDH1 and MaGDH2 respectively encoding β and α subunit. The proteins of MaGDHs were successfully expressed in E. coli BL21 (DE3) in the form of inclusion bodies. The activities of recombinant proteins were different with different vectors. The expression of MaGDHs showed organ specificity and expressed higher in vigorous organs than others. They were up-regulated by excess NH₄⁺. MaGDH1 were promoted by 6-BA earlier than MaGDH2.

Key words: mulberry, glutamate dehydrogenase (GDH), tissue expression, prokaryotic expression, regulation