Expression And Function Analysis Of Ammonium Transporter Gene LbAMT3-1 Induced By Arbuscular Mycorrhizal Fungi In Lycium Barbarum

Lycium barbarum L.,a deciduous shrub of the genus Lycium in the Solanaceae family,is a traditional medicinal and edible plant in China.L.barbarum is widely planted in arid and semi-arid areas in China.The planting area is rainless,and the soil is barren.There is a common phenomenon of excessive use of nitrogen（N）fertilizer to ensure yield in production,which leads to problems such as soil hardening,salinization and alkalization.To realize the green organic production and improve the yield and quality of L.barbarum,the application of AM fungi as biological fertilizer in L.barbarum planting has broad prospects.Studying the N absorption mechanism of the mycorrhizal pathway in L.barbarum can provide a basis for improving the N absorption efficiency and N use efficiency of L.barbarum.Then,an ammonium transporter gene LbAMT3-1 specifically induced by AM fungi was identified through the L.barbarum transcriptome data,suggesting that it may play a role in N transport in the mycorrhizal pathway.Through the quantitative real-time PCR analysis,promoter-GUS assay,yeast mutant complementation test and LbAMT3-1 overexpression experiment,this study explored the biological function and expression mode of LbAMT3-1 and its effect on mycorrhizal symbiosis and nutrient absorption.The main results are as follows:（1）A well symbiotic relationship was established between AM fungi and L.barbarum.The colonization intensity was more than 50%and the arbuscular abundance was more than30%.The colonization intensity of AM fungi increased with the increase of N concentration,and the colonization intensity and arbuscular abundance under NO₃^-supply were significantly higher than those under NH₄⁺supply.（2）AM fungal colonization can improve the biomass,N and phosphorus content,net photosynthetic rate,root activity and other growth indicators of L.barbarum under 7.5 m M,15 m M and 30 m M N supply,but not 1.5 m M N supply.（3）An ammonium transporter gene LbAMT3-1 specifically induced by AM fungi was identified in the mycorrhizal roots of L.barbarum.The CDS region of the gene is 1473 bp,encoding 490 amino acids,molecular weight 53572.42 Da and theoretical isoelectric point5.75.LbAMT3-1 existed in the form of trimer assembly,and there were central pores surrounded by 11 transmembrane helices in each subunit and the important functional site in LbAMT3-1 was conserved.Phylogenetic analysis showed that LbAMT3-1 was closely related to Sl AMT3-1 in tomato.The promoter sequence analysis results showed that LbAMT3-1promoter contained multiple cis regulatory elements related to mycorrhizal or root nodule,as well as cis acting elements specific to the promoter of phosphate transporter gene in response to mycorrhizal induction.（4）LbAMT3-1 functionally complemented the yeast mutant efficiently,which proved that the coding product of LbAMT3-1 gene could transport NH₄⁺.Histochemical GUS assays showed that GUS staining was detected in mycorrhizal roots and GUS activity driven by the LbAMT3-1 promoter was exclusively confined to cells containing arbuscules,which proving that LbAMT3-1was specifically induced by AM fungi only in arbuscule cells.（5）LbAMT3-1 overexpression increased colonization intensity and arbuscular abundance under normal and limited N conditions.The overexpression LbAMT3-1 plants showed similar growth performance with the wild-type plants under the condition of non-inoculation.After inoculation with AM fungi,the overexpression plants showed significantly enhanced nutrient acquisition ability and growth advantages compared with the wild-type plants,indicating that the LbAMT3-1 did not improve the nutrient acquisition ability of tobacco roots directly,but strengthened plant nutrient absorption and improved plant growth through mycorrhizal pathway.