The Mechanism Reasearch Of Nitrate Abosorption And Assimilation On Foliar Fertilizers Of ’Pyrus Bretschneideri’

In this paper,the effects of different concentrations of potassium nitrate on growth and physiological characteristics in leaves of‘Pyrus bretschneideri’under foliar fertilizers were studied;The 86 PbrNRTl members are identified using bioinformatics tools based on genome wide data of‘Pyrus bretschneideri’,the content of analysis including phylogeny,gene structure,synteny and expression patterns of PbrNRT1 and so on;Through cloning the genes that related absorption and assimilation of pear and constructing the recombinant expression vector,the cell localization of genes were observed through laser confocal microscope using protoplast transformation method;The temperature and humidity conditions that satisfied leaf nitrogen were selected through setting different temperature and humidity gradient in the artificial climate box.Based on this result,through sampling pear leaf that in different period of time and different areas after fertilization,the absorption and assimilation related genes expression characteristics of space and time were studied using real-time quantitative PCR.The results are as follows:1.The effects of different concentrations of potassium nitrate(10 mM,N1;30 mM,N2;50 mM,N3)on growth and physiological characteristics in leaves of‘Pyrus bretschneideri’ seedlings under foliar fertilizers were studied.The results indicated that the optimal conditions for the growth of‘Pyrus bretschneideri’was N2 treatment,with higher plant height,stem diameter,shoot FM and leaf area.And also,the higher chlorophyll content and nitrate reductase(NR)activity of leaves were also shown in N2 treatment.In addition,the soluble sugar,proline(Pro)content and malondialdehyde(MDA)concentration of leaves of N2 treatment were relatively lower,while the activity of superoxide dismutase(SOD)was relatively higher.Overall,when the concentration was 30 mM,the degree of stress of seedlings was minimum,and the growth and physiological characteristics were optimal.2.86 PbrNRT1 members of gene family were successfully identified.The phylogenetic relationship and gene structure analysis revealed that NRT1 proteins were grouped into four subfamiles and their structure are complex;Synteny analyses revealed during family evolution happened two duplication events,segmental duplication and tandem duplication and there are 38 pairs genes have relationship of collinearity,these made gene family and function expansion;Expression profile showed that in the different stages of fruit and the process of pollen germination,60 genes expressed in different stages of pear fruit and 22 genes expressed in the process of pollen germination.The results lay a basis for gene cloning and function identification in‘Pyrus bretschneideri’.3.In order to study the absorption and assimilation related genes functions,the open reading frame(ORF)with restriction sites of PbrNRT1.84(Pbr023734.1),PbrNRT1.1(Pbr023555.1),PbrNRT1.2(Pbr019204.1),PbrNIA1(Pbr008178.1),PbrNIA2(Pbr008534.1)and PbrNiR(Pbr040385.1)were respectively obtained by PCR,and then recombinant expression vector were constructed respectively by using the GFP protein.Fluorescence microscopy was used to observe the distribution of the expression vectors in protoplast to determine the subcellular localization of the genes.The results indicated that PbrNRTl.84,PbrNRT1.1 and PbrNRT1.2 had membrane localization,PbrNIAl was located in membrane and nucleus while PbrNIA2 was located in membrane,PbrNiR had chloroplast localization.4.This study selected 14 temperature and humidity conditions that suit foliar fertilization of‘Pyrus bretschneideri’seedlings,and the condition of 30℃,80%humidity is optimum.After foliar fertilization with 5 mM KNO3 in ’Arabidopsis thaliana’,the result showed that NRT1.1 gene was induced by nitrate.The absorption and assimilation related genes expression characteristics of space and time were studied after precise foliar fertilization with 30 mM KNO3 in ’Pyrus bretschneideri’,the result showed that PbrNRT1.84,PbrNRT1.1 and PbrNRT1.2 genes expression patterns were basically similar,genes expression in the place of applying fertilizer initially increased and then decreased with the extension of time,with the highest expression in 2 to 3 hours,genes response in symmetric place were retardation compared with the place of applying fertilizer;PbrNIA1 and PbrNIA2 genes transcription in the place of applying fertilizer began to rise after 6 hours,with active nitrogen assimilation between 24 to 72 hours;The gene response speed of PbrNiR was retardation compared with PbrNIAl and PbrNIA2 genes,but it still maintain a high transcription level in 72 hours,it indicated that fertilizer efficiency of nitrate after foliar fertilizer can sustain at least 72 hours.