Abstract

Glutamate dehydrogenase (GDH; EC1.4.1.2) catalyses the reversible amination of 2-oxoglutarate for the synthesis of glutamate using ammonium as a substrate. Herein, ahomology-based reverse transcription polymerase chain reaction (RT-PCR) strategy was employed to search for cDNAs encoding GDH from Camellia sinensis cv Longjing 43, an excellent variety of tea tree most suitable for processing into green tea in China. The full-length cDNA encoding GDH a-subunit, designated as CsGDH2(GenBank accession No. EU715396) was amplified from the tea plant. The cDNA ofCsGDH2 was 1667 bp with a 1236 bp open reading frame which encodes a 411 amino acid polypeptide. The bioinformatics analysis showed that CsGDH2 was highly homologous to plant GDH2 which encodes GDH a-subunit of plant. Phylogenetic treereconstructed for GDH protein family confirmed the result because CsGDH2 was distinctly clustered into the GDH2 clade of plant. Then, to improve the expression of CsGDH2 in soluble form in Escherichia coli, its full-length cDNA was cloned and expressed with several different N-terminal fusion tags in E. coli, such as B1 immunoglobulin binding domain of protein G (GB1), glutathione-S-transferase (GST), maltose-binding protein (MBP) and N-utilization substance protein (NusA). The results reveal that without any solublizing partners, the fusion CsGDH2 was predominantly found in insoluble bodies and no soluble protein was detected by either sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or western blot, whereas GB1, MBP and NusA tags may enhance the soluble expression of CsGDH2 to different extents. The MBP tag was found to be the best fusion partner for the soluble expression of CsGDH2. Isolation and cloning of important functional genes have significances on elucidating the molecular mechanism of high quality, yield and resistance for tea plant, as well as the genetic manipulating. This work would provide the possibility for further insights into the physiological role of GDH in nitrogen metabolism in plants.

Key words: Glutamate dehydrogenase, Camellia sinensis, RT-PCR, cDNA, bioinformatic analysis, soluble expression.

Abbreviation

CsGDH2, A gdh gene from Camellia sinensis encoding GDH a-subunit of plant; RT-PCR, reverse transcription polymerase chain reaction; RACE, rapid amplification of cDNA ends; ORF, open reading frame; cDNA, complementary DNA; IPTG, isopropyl β-D-1-thiogalactopyranoside; BLAST, the basic local alignment search tool; GB1, B1 immunoglobulin binding domain of protein G; GST, glutathione-S-transferase; MBP, maltose-binding protein; NusA, N-utilization substance protein;HGT, horizontal gene transfer; LGT, lateral gene transfer.