ABSTRACT
As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min (-1) mg (-1) which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG.
Subject(s)
Oryza/enzymology , Oryza/physiology , Pyruvaldehyde/pharmacology , Stress, Physiological/drug effects , Triose-Phosphate Isomerase/biosynthesis , Amino Acid Sequence , Chromosomes, Plant/metabolism , Enzyme Induction/drug effects , Gene Expression Regulation, Plant/drug effects , Genes, Plant/genetics , Genetic Complementation Test , Kinetics , Molecular Sequence Data , Mutation/genetics , Oryza/drug effects , Oryza/genetics , Protein Transport/drug effects , Protein Transport/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Stress, Physiological/genetics , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/geneticsABSTRACT
For studying genetic diversity in natural populations of Terminalia, a medicinal plant, our attempts to isolate high quality DNA using several previously reported protocols and even modifications were unsuccessful. We therefore combined CTAB based isolation, and column based purification step, to isolate DNA from Terminalia arjuna. The DNA isolated using this standardized protocol was high in quality and suitable for restriction digestion and generation of random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP).
