Function Analyses Of High-Affinity Nitrate Transporter Gene Osnrt2.3a/b In Rice

Rice(Oryza. sativa L.) is one of the most important staple food crops that provides food for more than half of the world’s population, accounting for1/3of the global cereal crop acreage. Nitrogen is one of the major factors limiting crop growth and yield among the necessary nutrient elements for plant. Both ammonium and nitrate are the major sources of soil N for plants, however, ammonium is the predominant form of N in a paddy soils. Some physiological experiments have shown that lowland rice was exceptionally efficient at acquiring NO3-formed by nitrification in the rhizosphere, and nitrate was main source of N in soil at later growth and development stages of rice under the intermittent irrigation. It has been predicted that25%～40%of the total N taken up by rice roots grown under wetland conditions might be in the form of nitrate and the rate of nitrate uptake can be comparable with that of ammonium. To meet the different NO3-concentration in the soil, higher plants have low-affinity nitrate transport system (LATS) and high-affinity nitrate transport system (HATS) to uptake nitrate. In higher plants, the molecular basis of root nitrate uptake has been the matter of intensive studies during the last decade. So far, two gene families have been identified:the NRT1and NRT2families involved in the low-affinity nitrate transport system (LATS) and high-affinity nitrate transport system (HATS), respectively. Many genes involved in LATs and HATs have been cloned and studied in multiple plant species, especially in model plant Arabidopsis.Five genes involved in nitrate transport which belonged to NRT2gene family have been cloned in rice, however, the functional properties study of these genes was unclear. In this thesis, we focus on the high-affinity nitrate transport gene OsNRT2.3a/b in rice. In order to understand the in planta function of OsNRT2.3a/b, the over-expression and RNA interference transgenic mutant plants were obtained. RT-PCR, qRT-PCR, TAIL-PCR, Southern blot and Western blot methods were employed to identify the transgenic plants. The nitrate uptake and translocation of transgenic plants were analyzed by15N labeled NH4+and NO3". The yields of transgenic plants and the potential genes directly or indirectly regulated by OsNRT2.3a/b were also analyzed in the thsis. The main results were as follows:1. Sequence alignment between genomic and cDNA sequences showed that one gene OsNRT2.3was mRNA spliced into two genes, OsNRT2.3a and OsNRT2.3b. There was only90bp difference between OsNRT2.3a and OsNRT2.3b. The results of rice chip database showed that OsNRT2.3a/b were mainly expressed in roots and little in shoots under field condition. Also, the expression level was increaseed gradually as the plant growth, up to the most at the stage of vegetative growth to reproductive growth. The protoplast subcellular localization showed that OsNRT2.3a and OsNRT2.3b were both localized on plasma membrane.2. Over-expression of OsNRT2.3a/b and knock-down of OsNRT2.3a transgenic plants were successfully obtained through transgenic manipulation. RT-PCR, TAIL-PCR, Southern blot and Western blot methods were employed to identify the stably inherited transgenic lines.3. There was no significant difference in phenotype when over-expression OsNRT2.3a alone; but the OsNRT2.3b over-expression plants were obviously higher and bigger than WT. Expression patterns of OsNRT2s and OsNAR2.1were analyzed in over-expression transgenic plants. OsNRT2.3a over expression did not affect the expression of OsNRT2.1, OsNRT2.2and OsNAR2.1. The expression of OsNRT2.1, OsNRT2.3a and OsNAR2.1were up-regulated in OsNRT2.3b over expression plants, however, the expression of OsNRT2.4was not affected. Over expression of OsNRT2.3a alone could not improve the growth of rice plants and enhance the nitrate uptake in rice. The biomass of OsNRT2.3b over expression plants was increased a lot compared with WT, and the whole plant nitrogen content of OsNRT2.3b over expression plants was significantly higher than WT, but the total nitrogen concentration was lower than WT under low nitrogen condition.4. The field experiment with different nitrogen level showed that OsNRT2.3b over expression plants grow better than WT in all nitrogen level, the yields and nitrogen use efficiency were higher than WT both. However, delaying Heading stage of OsNRT2.3b over expression plants resulted in longer growth period than WT. Further study of the growth period difference between OsNRT2.3b over expression plants and WT showed that over expression of OsNRT2.3b in rice not only increased accumulation and transfer amounts of dry matter but also increased the ratio of dry matter transfer to grain and contribution ratio of transferred dry matter to grain. It also revealed that over expression of OsNRT2.3b in rice not only increased accumulation and transfer amounts of nitrogen but also increased the ratio of nitrogen transfer to grain and contribution ratio of transferred nitrogen to grain.5. The analyses of OsNRT2.3a RNAi mutants showed that knockdown of OsNRT2.3a did not significantly affect nitrate uptake by rice roots with low nitrate supply. However, knockdown of OsNRT2.3a in rice suppressed nitrate transport from root to shoot under low nitrate condition, resulted in nitrate accumulation in rice roots and down regulation of OsNRT2.1/2.2, which decreased nitrate uptake by rice roots.6. The results of the whole-genome expression of OsNRT2.3a over expression plants and OsNRT2.3a knockdown mutants showed that Carbon and Nitrogen cycle was suppressed in osnrt2.3a knockdown mutants due to nitrogen deficiency in shoots, furthermore, knockdown of OsNRT2.3a affected the plants photosynthesis and resulted in decreasing accumulation of dry matter and growth retardation. The results of the whole-genome expression of OsNRT2.3b over expression plants showed that suppressing photorespiration appropriately and improving antioxidant activity in OsNRT2.3b over expression plants may resulted in the delayed aging and increased biomass and yields.Taken together, high-affinity nitrate transporter gene OsNRT2.3a was a key factor in nitrate transport from root to shoot. Over expression of OsNRT2.3b can improve nitrate transportation significantly, and then improve nitrogen use efficiency and yields in rice.