Functional validation of down regulation of Dof1 transcription factor using RNA interference in Arabidopsis thaliana
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Date
2017-08
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Nitrogen (N) is one of the most important mineral elements which directly affect on crop yield. Due to this reason, in the last 40 years, the amount of synthetic nitrogen (N) applied to crops has risen dramatically, from 12 Tg/year to 104 Tg/year, resulting in increase in yield but with considerable impacts on the global environment because plants consume much less than half of the fertilizers applied, means more than half of the fertilizer are leach into surrounding lake, ponds, river and cause environmental problem. To solve this problem, various studies of different regulatory genes were carried out in recent years. One of the regulatory gene is Dof1 (DNA Binding with one Finger domain 1). Dof1 gene was first recognized in Maize and by its over expression in Arabidopsis thaliana it was concluded that, it regulates the enzymes which participate in Carbon/Nitrogen assimilation like nitrate reductase, phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase (PK), citrate synthase (CS), and isocitrate dehydrogenase (ICDH), GS(Glutamate Synthetase) and GOGAT (Glutamine oxoglutarate aminotransferase) enzyme at transcription level. Most of the studies for defining its role have been carried out by over expression of this (Dof1) gene in different plants, but still its proper role is yet to be validated and for that another approach can be used i.e. its down regulation study which could be done by gene silencing. For this, pRNAi golden gate vector present within agrobacterium cell suspension was transformed in Arabidopsis plant through floral dip method. Selection of transgenic line was done in kanamycin selection medium. After that different morphological, biochemical and molecular assays were done to validate the functions of dof TF in NUE. It was clearly seen that upto 20-32 % decrease has been observed in case of different biochemical analysis of enzyme activities which are directly controlled by this Dof1 transcription factor. Total chlorophyll content has also reduced which clearly indicated that photosynthetic activity is hampered. 25-40% down regulation of gene has been observed in real time PCR analysis of Dof 1 transcription factor. This transformation technique clearly indicated the functional validation of dof1 transcription factor which directly and indirectly control the enzyme of carbon and nitrogen metabolism.
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