To Improve Dof1 Expression Levels To Improve Tobacco Nitrogen Utilization

Nitrogen is one of three major nutrition elements and also one of the rate-limiting factor in plant growth. Although many measures for increasing nitrogen use effiency is taken, the effects of improving plants nitrogen utilization are not obvious. Dofl transcription factor is unique to plants^[1]. It is an activator for multiple gene expressions associated with the organic acid metabolism, including phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase (PK) gene expression, and then improving the nitrogen-utilization of plants^[2]. In study, Dofl transcription factor was cloned from Arabidopisis and overexpressed in the tobacco for improving tobacco nitrogen utilization by enhancing PEPC and PK activities. The main results were as follows:PCR-primers were designed according to the sequence reports in Genbank and the Dofl transcription factor was obtained from Arabidopisis genome DNA by Polymerase Chain Reaction (PCR). The constitutive plant expression vector pK2 35S-Dofl and light inducible plant expression vector pPZP221-rbcS-Dofl were constructed and these plant expression vectors were transformed to the wild type tobacco via Agrobacterium-mediated method and got the transgenic tobacco. We confirmed that Dofl transgenic tobacco and light inducible Dofl transgenic tobacco were obtained through antibiotics selection and PCR. RT-PCR showed that Dofl transgenic tobacco and light inducible Dofl transgenic tobacco were expressed in tobacco. The mensuration of PK and PEPC activities showed that the Dofl gene had improved these enzymes' transcriptional activities and had physiological and biochemical impact on the tobacco. The potted experiment of light inducible Dofl transgenic tobacco on perlite medium under low (1 m Mol), moderate (5 mMol) and high (10 mMol) nitrogen nutrition condition showed the heights of transgenic tobacco were 1.3, 1.7 and 1.1 folds taller than the control; the fresh weights of transgenic tobacco were 1.3, 1.1 and 1.05 folds heavier than the control; the dry weights of transgenic tobacco were 1.3, 1.2 and 1.06 folds heavier than the control. The PK and PEPC activities of the transgenic tobacco grown on the low nitrogen perlite medium were 1.1².1 and 1.5—2.4 higher than the control, respectively. The tissue culture experiment of T2 light inducible Dofl transgenic tobacco on low (1 mMol), moderate (5 mMol) and high (10mMol) nitrogen mediums showed that the heights of transgenic tobacco were 1.63, 1.38 and 1.17 folds higher than the control; the fresh weights of transgenic tobacco were 1.94, 1.31and 1.28 folds heavier than the control; the dry weights of transgenic tobacco were 1.62, 1.32 and 1.32 folds heavier than the control. The contents of total N and chlorophyll in T2 transgenic tobacco were 1.41 and 2.18 folds higher than the control, the wild type tobacco, respectively under the low nitrogen condition.All these results indicated the Dofl transcription factor cold activate the PK and PEPC activities, enhance contents of total N and chlorophyll and improve the growth and nitrogen utilization of plant, especially under the low nitrogen-condition.