Expression And Function Analysis Of A Nitrate Transpoter Gene PtrNPF5.2 Associated With Arbuscular Mycorrhizal Symbiosis In Poncirus Trifolata

Citrus and its related species have few root hairs,they mainly rely on symbiosis with arbuscular mycorrhizal fungi to absorb soil nutrients,of which nitrogen is one of the essential nutrients for citrus growth and development.In order to improve crop yield by applying large amount of nitrogen fertilizer is easy to cause nitrogen nutrition waste,studying the nitrogen absorption mechanism of citrus mycorrhizal pathway can provide a basis for improving the nitrogen absorption efficiency and nitrogen utilization efficiency of citrus roots.Based on mycorrhizal transcriptome analysis showed that a nitrate transporter gene PtrNPF5.2 in Poncirus trifoliata was induced by arbuscular mycorrhizal fungi,suggesting that PtrNPF5.2 may be involved in nitrate transport during citrus mycorrhizal symbiosis.Therefore,the temporal and spatial expression pattern of PtrNPF5.2 was analyzed in this study through transient transformation of medicago hairy root and tobacco,RNAi and Tnt1 insertion mutants of the homologous gene Mt NPF5.3 in medicago and CRISPR analysis of the homologous gene SLNPF5.2 in tomato were analyzed to explore the function of these genes on mycorrhizal symbiosis.The main results are as follows:1.Quantitative expression analysis showed that PtrNPF5.2 gene was significantly up-regulated after inoculation with arbuscular mycorrhizal fungi,and gradually increased with the inoculation time,reaching the peak 30 th day after inoculation.The promoter-GUS signal of PtrNPF5.2 gene was mainly expressed in arbuscule-containing root cells,but not in root cells without arbuscules,suggesting that PtrNPF5.2 gene may be involved in the regulation of mycorrhizal symbiosis.2.Protein structure prediction result showed that PtrNPF5.2 was a membrane protein with 12 transmembrane domains.Transient expression experiments showed that PtrNPF5.2-GFP fusion protein was located in tobacco cell membrane,which was consistent with the characteristics of nitrate transporter.3.This study explored the function of Mt NPF5.3,a homologous gene of PtrNPF5.2in medicago,though RNA interference and Tnt1 insertion mutant analysis.The results showed that compared with wild-type or empty-transgenic plants,the infection rate and the development of arbuscules of Mt NPF5.3 interference or Tnt1 insertion lines were not affected,it may be due to the functional redundancy of other homologous genes in medicago.4.Similarly,the two CRISPR editing strains of SLNPF5.2,a homologous gene of PtrNPF5.2 in tomato had no difference in infection rate and arbuscular development compared with the wild type.Quantitative analysis of tomato root genes showed that,compared with the wild type,SLNRT1.1 and SLAIT genes related to nitrate uptake were down regulated in the CRISPR homozygous strain of SLNPF5.2,while SLNi R and SLNIA genes related to nitrate assimilation were up-regulated,the nitrate uptake of CRISPR homozygous plants of SLNPF5.2 was decreased and the nitrate assimilation was increased,resulting in the nitrate content of CRISPR editing plants significantly lower than that of wild type.These results suggest that SLNPF5.2 may affect nitrate uptake and assimilation in mycorrhizal pathway,thereby reducing nitrate content.