Cloning and characterization of a cytosolic glutamine synthetase from Camellia sinensis (L.) O. Kuntze that is upregulated by ABA, SA, and H2O2.
A cDNA encoding glutamine synthetase, one of the enzymes of the GS/GOGAT pathway, was cloned from Camellia sinensis (CsGS). The isolated cDNA consists of 1,071 nucleotides encoding a polypeptide of 356 amino acids with an estimated isoelectric point of 6.13. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of 39.2 kDa. The purified protein was confirmed by blotting with anti-His antibodies. Catalytic parameters of the protein were determined using glutamate and ATP as substrates. The observed Km was 9 mM and Vmax was 93 U/mg protein with glutamate as substrate, while with ATP Km and Vmax values were 6 mM and 70 U/mg protein, respectively. Purified enzyme showed pH optima at 8. Cations were found to be showing enhancing effect on the activity of GS enzyme and Mg2+ ion exhibited maximum enhancing effect among the various ions used in this study. This enzyme activity increased by 25% in presence of DTT and decreased by 18% when incubated with PMSF. Transcript analysis in tea bud, youngest leaf, showed that CsGS gene expression is stimulated in response to abscisic acid (ABA), salicylic acid (SA), and hydrogen peroxide (H2O2), while gibberellic acid (GA3) has no influence on its expression levels.
Rana
Nisha K
NK
Biotechnology Division, Institute of Himalayan Bioresource Technology (CSIR), Palampur, 176061, Himachal Pradesh, India.
Mohanpuria
Prashant
P
Yadav
Sudesh Kumar
SK
D000040:Abscisic Acid / Q000494:pharmacology*; D000595:Amino Acid Sequence; D028241:Camellia sinensis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D030562:Databases, Protein; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D020156:Salicylic Acid / Q000494:pharmacology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015854:Up-Regulation / Q000187:drug effects*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D000040:Abscisic Acid; D006861:Hydrogen Peroxide; D005974:Glutamate-Ammonia Ligase; D020156:Salicylic Acid