The Identification Of Dof Transcription Factors Responsed To Low Nitrogen Stress And The Development Of Low-nitrogen Tolerant Transgenic Rice

Rice is one of the most important crops and the main food for half the people of the world. Nitrogen is very important during the growth and development of the plant. Howerver, the low nitrogen-utilization efficiency and a vast amount of nitrogen fertilizers poured into the fields severely pollute the environment and increase the cost. Now, many reports have showed that transformation is an efficient way to improve nitrogen-utilization efficiency of rice.The objective of this research is to find the putative similar functional genes to ZmDof1 by the way of Blast in both Arabidopsis and Oryza sativa, and identify of the genes which responsed to the low nitrogen stress in rice Dof transcription factor family. As the rules, three genes (At2g37590, OsDof-6, OsDof-13) were selected and over-expressed in rice. The main results are as follows:1. At2g37590 is the most similar Dof gene to ZmDof1 in Arabidopsis by Blast and two expression vectors with two different selection gene hpt and bar were constructed respectively, which named p13at and p33at and introduced into an elite indica restorer Minghui 86 by Agrogacterium-mediated transformation method, 19 and 2 positive transgenic plants were got respectively.2. Two interesting genes, OsDof-6 (compareing the Dof transcription factor family of Oryza sativa with the gene ZmDof1) and OsDof-13 (identifying throught the expression of the Dof transcription factor family of rice responsed to the low nitrogen stress), are from rice, and over-expression vectors of them were constructed and introduced into Hejiang 19 by Agrogacterium-mediated transformation method.3. Sub-cellular localization analysis showed that At2g37590,OsDof-6 and OsDof-13 were located in nucleus.4. Yeast one-hybrid assay showed that At2g37590 can bind to the promoters of PEPC and IDH, while OsDof-6 and OsDof-13 can not.5. Southern blotting assay indicated that OsDof-13 was successfully integrated into the genome of the recipient rice with 1-4 gene copies.6. Expression level and copy number of At2g37590 T₀ regenerated plants were analyzed by Northern and Southern blotting respectively. At2g37590-overexpressing T₁ transgenic plants display better growth. The dry weight and the nitrogen content of per plant were increased under the normal nitrogen condition. Under the low nitrogen condition, only the nitrogen content of gram were increased significantly, at least by 7.56% up.