Functional Analysis Of Over-Expression For Nitrate Transport Accessory Protein Gene OsNAR2.1in Rice

With the growing population and the decreasing land area, increasing crop productivity is becoming more and more important. As one of the major staple food crops, rice (Oryza sativa L.) plays a key role in coping with this problem. Crop productivity relies heavily on nitrogen fertilization. Excess application of nitrogen fertilizer not only increases costs of agriculture production, but also it is detrimental to the environment. Thus, improving nitrogen use efficiency (NUE) is essential for the development of sustainable agriculture.There have been evidences indicating that rice and other wetland plants may take up significant amounts of nitrate formed by nitrification of ammonium in the rhizosphere, though ammonium is the predominant form in anaerobic-flooded soil. In the rice genome, the genes (4NRT2and2NAR2) encoding HATS components have been identified. OsNAR2.1mainly expresses in root and plays a very important role in enabling the plant to cope with variable nitrate supply. Both high-and low-affinity nitrate transports were greatly impaired after OsNAR2.1knockdown.In this work, two independent lines (Ov-199and Ov-200) were selected from T3transgenic plants after transforming OsNAR2.1over-expression vector into rice plant (Nipponbare). Then Ov-199and Ov-200were used to study the effect of OsNAR2.1over-expression on rice photosynthesis, growth, nitrogen uptake and translocation. The results were as follows:1. Acquisition of OsNAR2.1over-expression plants. Firstly, to constuct OsNAR2.1gene into PTCk303vector with Ubi promoter and then transform the construct into Nipponbare cell callus. To positive plants were selected by RT-PCR. Further identification was carried in T1, T2and T3plants through RT-PCR and Southern blot. The T3two lines were named Ov-200and Ov-199respectively and their T3generations were used for further physiological and field experiments.2. Analyses of the the effect of OsNAR2.1over-expression on photosynthesis by hydroponic and soil culture experiments. The hydroponic experiment results showed:under normal nitrogen supply,the xylem bleeding sap pH of the transgenic rice plants were significantly higher than wild type (WT). Furthermore, photosynthetic rate, stomatal conductance, transpiration rate and chlorophyll content of Ov-200were significantly higher than WT. Under low nitrogen supply, there were no significant difference in the xylem bleeding sap pH and chlorophyll content, however, the intercellular CO2concentration, stomatal conductance and transpiration rate of Ov-200and Ov-199were significantly higher than WT, the photosynthetic rate was also increased compared with WT. In the soil culture experiments, both the chlorophyll content and total nitrogen content were increased significantly in transgenic plants.3. Analyses of the the effect of OsNAR2.1over-expression on growth and agronomic characters in the field treated with67.5and135kg N/ha nitrogen fertilizer respectively. Through the field observation, the growth period of over-expression lines were delayed in the field. When WT was in the period of fructification, Ov-200and Ov-199were in the period of grain filling and ear sprouting respectively. The over-expression plants height increased but the tiller number showed no significant difference under135kg N/ha nitrogen fertilizer treatment. In contrast, at67.5kg N/ha supply, both height and the tiller number increased in Ov-200but no significant change in till number was observed in Ov-199from WT.s. At mature stage, the flag leaf areas and the shoot biomass of Ov-200and Ov-199were significantly increased and the stems became thicker in the over-expression lines. In addition, significant changes on the grain were also shown in over-expression lines such as the ear shape, the seed size, panicle length and panicle weight. The grain yield of per plant increased significantly in Ov-200under67.5or135kg N/ha nitrogen fertilizer supply (increased about13%-19%), but Ov-199only increased significantly at67.5kg N/ha nitrogen fertizer supply(increased about16%).4. Analyses of the the effect of OsNAR2.1over-expression on nitrogen uptake and translocation. The biomass, the nitrogen concentration and the nitrogen content of different parts of over-expression plants and WT were tested at flowing and harvest stage under67.5and135kg N/ha nitrogen fertilizer. The results showed:there were more biomass and total nitrogen concentration of shoot in Ov-200and Ov-199under67.5and135kg N/ha nitrogen fertilizer supply. At flowering stage, the total nitrogen was mainly accumulated in stem and leaves. At haversted stage, the total nitrogen was mainly translocated into ears. However the nitrogen transfer ratio from vegetative organs to reproductive organs in over-expression plants was less than that in WT, the reason may be that the growth period was later than WT. This study also found that both the nitrogen uptake efficiency and nitrogen fertilizer use efficiency in over-expression plants increased under67.5and135kg N/ha nitrogen fertilizer supply.