Identify TILLING Mutant Of Rice OsNRT2.3 Gene And Phenotypic Analysis

The overuse of nitrogen fertilizer resulted into environment pollution.Therefore,improving nitrogen use efficiency becomes an important target in sustainable development of China’s agricultural system.Previous studies have shown that rice can absorb ammonium as well as nitrate from the nitrification of ammonium by bacteria in rhizosphere.The nitrate transport protein is divided into high affinity nitrate transporters and low affinity nitrate transporters.OsNRT2.3 belongs to high affinity nitrate transport genes,and the OsNRT2.3 gene could be transcribed into two mRNA products as OsNRT2.3a and OsNRT2.3b.OsNRT2.3b plays an independent function in transfering of NO3-,and studies have shown that its expression can greatly improve the utilization rate of nitrogen and grain yield(Tang,2012).In this thesis,through the sequencing of TILLING material of OsNRT2.3 gene,I directed several mutation lines in the promoter position.I tested the expression of the mutants,and compared the impact of OsNRT2.3 point mutant on rice growth with wild type(WT)and the over-expression lines of OsNRT2.3b.The results are as follows:1.I obtained 25 mutants and selected the promoter mutation lines named as T6,T8,T11,T12,T13,T20,T21 and other mutation lines in Open Reading Fragment(ORF)as T14,T16 by TILLING technology.T22 line had no point mutations.Through promoter motif analysis by software,I found that there was a TATA BOX at 83 bp upstream from ATG of OsNRT2.3,which was point mutated in T8,T11 and T12 lines.Through analyzing the structures of crossing the membrane,I also found that the mutation in T16 line resulted into insertion mutation of amino acids of OsNRT2.3b as only have half of the transmembrane regions was expressed out.2.I chose T8,T11,T12 lines with 83 bp point mutation as the research materials in my thesis and T20 as blank control to compare the effect of this mutant on rice.I measured plant height in different periods and checked the expression of OsNRT2.3b,nitrogen utilization in hydroponic and field experiments.Comparing with WT,plant height,tiller number and biomass of T8,T11,T12 increased in the hydroponic experiment,however T20 showed no growth difference from WT in seedling stage.Furthermore OsNRT2.3b expression of T8,T11,T12 lines increased in tillering stage compared with WT and T20 line.The field experiment showed that the plant growth and the OsNRT2.3b expression in T8,T11,T12 leaves was higher than WT only before the blooming stage,which confirmed the hydroponic result that 83 bp mutation increased OsNRT2.3b expression in tillering stage and improved the rice growth in this stage.3.In order to test the hypothesis that 83 bp motif in may involved in the expression regulation during the growth stage,I investigated the growth and nitrogen usage in OsNRT2.3b over expression lines(369 and 366 lines)in the tillering stage in hydroponic conditions and field experiment.The results showed that both biomass and grain yield were significantly increased by OsNRT2.3b higher expression in 369 and 366 lines,compared with WT.Total nitrogen content of transgenic lines was also increased significantly.By comparing OsNRT2.3 TILLING materials(T8,T11 and T12 lines)with OsNRT2.3b over-expression lines(369 and 366 lines),I found that T8,T11 and T12 lines only showed higher expression of OsNRT2.3b in tillering stage and in jointing stage than WT while 366 and 369 lines showing higher expression pattern during the whole growth stages since 35S promoter drived OsNRT2.3b expression in over expression lines.Furthermore the super-growth trade was kept through whole growth period in 35S drived over expression lines.However the super-growth in tillering stage and in jointing stage of 83 bp mutants was slowly disappeared as the mature growth stage,as the OsNRT2.3b expression in T8,T11 and T12 lines did not show significantly higher than WT at grain filling stage.In a conclusion,OsNRT2.3b high expression could improve rice growth and 83bp motif upstream from ATG of OsNRT2.3 was involved in OsNRT2.3b expression regulation before the blooming stage.