Overexpression and downregulation of dof1 transcription factor for elucidating its role in carbon and nitrogen metabolism in Eleusine coracana and Arabidopsis thaliana
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Date
2018-08
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Dof1 is a transcription factor belonging to one of the well characterized plant specific transcription factor family Dof, presented on chromosome1. Dof1 was first recognized in maize and by its over expression in Arabidopsis thaliana it was revealed that dof1 is involved in the regulation of genes which play important role in carbon/nitrogen metabolism like phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase (PK), citrate synthase (CS), and isocitrate dehydrogenase (ICDH), GS (Glutamate Synthetase) and GOGAT (Glutamine oxoglutarate aminotransferase). In our laboratory, full-length gene sequence of Dof1 was cloned and sequenced from finger millet genotype. Upon comparing the EcDof1 protein sequence with Dof1 of other cereals revealed high sequence similarity with rice Dof1. Real time expression analysis of Dof1 has been carried out in three genotypes of finger millet differing in grain protein content revealed that expression of Dof1 is higher for high protein genotype as compare to low protein genotype. Hence, Dof1 appears to be the key regulator in the expression of genes involved in carbon and nitrogen metabolism. Molecular cloning and characterization of dof1, from a high protein genotype of Eleusine coracana (GE-3885), was done to elucidate the role of Dof1 transcription factor. Dof1 was cloned in expression vector for its overexpression and downregulation in Eleusine coracana and Arabidopsis thaliana. Physiological and biochemical analysis of dof1 gene in transgenic lines was done to validate its role in carbon and nitrogen metabolism. Prior to this, the high protein genotype of Eleusine coracana was studied to see the expression of dof1 along with different genes involved in carbon and nitrogen assimilation under different treatments of carbon and nitrogen. And also, to transform finger millet with dof1 transcription factor (cis-trangenic), complete plant regeneration protocol was standardized via direct organogenesis and somatic embryogenesis using mature seeds as explants. Functional analysis of the transgenic plants clearly reveals that Dof1 transcription factor controls the expression of genes involved in carbon assimilation or organic acid metabolism including PEPC and PK. No significant increase in expression of GS, GOGAT and NiR was observed in Dof1transgenic plants but there was an increase in both chlorophyll and protein content in them. These results indicated that, Dof1 is indirectly involved in assimilation of nitrogen by enhancing the amount of carbon metabolites/intermediates which are needed for assimilation. Hence, Dof1 transcription factor could be targeted for transforming various plants to generate nitrogen use efficient crops.
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