Identification And Function Research On Genes That Are Related To Nitrogen Metabolism Of Rice

Nitrogen is one of the most important nutrient elements that influence plants growth. Over the past two or three decades, the nitrogen fertilizer has been applied excessively, resulting in significant negative impact on the environment. In such a context, improvement of utilization of the nitrogen fertilizer enables to improve agricultural productivity while benefiting the ecological system. The molecular mechanism of nitrogen absorption and transportation of plants has been made clear by using the molecular biology and genetic engineering techniques. In order to improve nitrogen use efficiency, the current research should focus on screening efficient plants varieties and identifying relevant new genes. This study aims to screen new genes associated with nitrogen metabolism in rice using the genome-wide cDNA microarray technology. Major results are described as follows:1. Nipponbare and Zhenshan97as the test material were grown hydroponically to the five-leaf stage, when the nitrogen stress treatment was conducted. Samples were collect at different time points. Seedlings cultured in the normal nutrient solution were used as the control. Using genome-wide cDNA chip,15differentially expressed genes that reacted similarly to changes of the nitrogen environment were selected from the two materials.2. The Real-time PCR was used to further screen eight genes based on changes in expression of15genes under different nitrogen treatments.3. Based on information of these genes, the coding sequence and the upstream promoter sequence of each gene were determined through the bio-informational analysis. The coding sequences and the promoter sequences of eight genes under test were amplified by PCR. The coding sequences were used to construct under-and over-expression vector. The promoter sequences were cloned to DX2181b vector and fused with the GUS reporter gene.32vectors were constructed in total.4. Through the Agrobacterium mediated transformation method, we transferred the constitutive plasmid into the japonica variety ZH-11. Each vector was introduced to30-70transgenic rice seedlings. The seedlings were indentified to discover positive plants and gene expression level was determined for further research on functionalities of the genes.5. Analysis on the Gus Report gene expression and GUS activity detected two specific promoters induced by the changing nitrogen environment.6. Through sequence analysis, these two specific promoters were truncated into small fragments. After the truncated promoter fragments were connected to Gus, they were introduced to rice. Real-time PCR validation was carried out to determine expression of the various truncated GUS reporter gene fragments so that the corresponding cis-regulatory elements were discovered.