Relationship Between Expression Of DsNRT2.1 And Characteristics Of NO₃～- Uptake In Dunaliella Salina And Transgenetic Alfalfa

Nitrogen is a necessary nutrient element for plant, which is converted to animo acid and nucleotide for the need of plants.Nitrate as an important inorganic nitrogen,its loss cause deficiency of nitrogen in the soil,Unfortunately,due to the mixture and fluctuation of the oxidative and reductive environment in submerged soil, the utilization rate of the nitrogen fertilizer in irrigated fields is reduced to low, especially the plant that live in saline-alkali soid has the lower recovery rate of nitrogen fertilizer. All of these reasons inhibit the growth of plants and reduce the production of crops. Alfalfa is an excellent grazing that has high nutrition and high yield, it also can improve the quality of soil. So it is important to improve the stress-resistance of alfalfa for stockbreeding and entiroment. Dunaliella.Salina, an enkaryotic unicellular green algae, is able to proliferate under high saline conditions The reseach demonstrates that the ability of salt-tolerance of Dunaliella.Salina is correlated to its special mechanism of NO3-uptake and assimilation. The purpose of this reseach is understanding the mechanisms of how Dunaliella.Salina takes up and assimilates nitrate under high saline conditions at the genetic level and transform DsNRT2.1 into alfalfa in order to improve its ability of NO3- uptake in saline-alkali soid, utimately select transgenic alfalfa which has strong salt-resistance and high NO3- uptake rate.The experiments and results of my thesis are including:1.The study of NO3- uptake of Dunaliella.Salina which was cultivated in different concentrations of salt confirms the NO3- uptake rule of Dunaliella.Salina,and the study of the relationship between NO3- uptake and NH4+ also demonstrates that NH4+ has a repressive effect on NO3- influx of Dunaliella.Salina.2.The study of the relationship between expression of DsNRT2.1 and NO3-influx has shown that DsNRT2.1 has a higher affinity at low NO3- concentration. It confirms the hypothesis predicted by bioinformatics analysis that DsNRT2.1 is a putative high affinity nitrate transpoter of Dunaliella. Salina . Meanwhile we can further confirm that NO3- influx is inhibited by NH4+ at expression level.3.we constructed pCAMBIA2301G-DsNRT2.1 vector, then the pCAMBIA2301G-DsNRT2.1 was introduced into Agrobacterhun tumefaciens using the freeze-thaw method. After infected by A.tumefaciens, the leaf of alfalfa were selected on UM inducement medium containing 50mg/L kanamycin.After four weeks, there are some embryoids on the callus of leave, then the embryoids develop to regeneration buds which grow to regenerated seedlings.4.The regenerated seedlings were identified by PCR and Southern blot analysis. PCR analysis showed that approximate 20% tested plants are transgenic plants, the results of Southern blot showed DsNRT2.1 had been inserted into the genome of alfalfa. Based on these results, we can carry on the continued studies of identification salt-tolerance transgenic alfalfa .