Arora, SandeepNegi, Bhawana2018-03-052018-03-052017-04http://krishikosh.egranth.ac.in/handle/1/5810041522Oxidative stress, a convergent point of various abiotic and biotic stresses, is a major threat for food security all over the world. Oxidative stress generated due to accumulation of excessive reactive oxygen species (ROS), leads to the oxidation of vital cellular biomolecules and to cell death in extreme circumstances. Plants have evolved an antioxidant defense system to detoxify ROS and maintain redox homeostasis. Ascorbic acid, a small water soluble antioxidant molecule, plays a crucial role in detoxification of excess ROS. A high reduced:oxidized ascorbate ratio in plants is required for adaptation of plants to environmental stresses. Reduced ascorbate pool is maintained in cells through efficient reduction of oxidized forms of ascorbate.The present study delineates the role of monodehydroascorbate reductase, one of the enzymes responsible for maintaining cellular reduced ascorbate pool, under environmental stresses. The expression of monodehydroascorbate reductase (EC 1.6.5.4), as quantified through real-time PCR, was found to increase under salinity, water deficit as well as ultra violet radiation stress. The qPCR expression profiling of Ecmdar under stress indicated that mdar is an early responsive gene. Monodehydroascorbate reductase gene (mdar) was then cloned from a drought tolerant variety of Eleusine coracana (PR202). For structural validation, the cloned Ecmdar gene was subjected to in-silico analysis and for functional validation the gene was over-expressed in Brassica juncea and Arabidopsis thaliana. Structural analysis indicated that the cloned Ecmdar gene comprises of a 1437bp CDS, encoding a 478 amino acid long polypeptide. The amino-acid sequence analysis suggested that the cloned EcMDAR is a membrane bound peroxisomal isoform. The active site analysis showed presence of conserved Tyr348residue having the catalytic activity in electron transfer mechanism. String based analysis of EcMDAR predicted that mdar is coexpressed with ascorbate peroxidase and PLANT-CARE analysis of Osmdar showed presence of light responsive elements in the promoter region. Functional analysis of the transgenic lines over-expressing Ecmdar, indicated thatthe transgenic plants have better stress tolerance potential, measured in terms of standard biochemical markers, than the wild type plants. The present results clearly indicate that Ecmdar can be used as a promising candidate for developing stress tolerant transgenic plants.ennullThesis