Cloning And Functional Characterization Of Key K~+/Na~+ Balance Genes In Lycium Ruthenicum Murr.

Soil salinization has a serious impact on the normal growth of plants.Maintaining the balance of potassium and sodium in plants is essential for improving the salt tolerance of plants.In recent years,some researches have been made on the key genes of potassium and sodium balance in plants.Studies have shown that the absorption and transport of K+in plants mainly depends on the two transport systems of potassium channels and potassium transporters.In this study,based on the transcriptome data of under salt stress in the previous group,two genes LrSKOR and LrKUP8 responding to salt stress were obtained by homologous gene cloning.The coding region of LrSKOR is 2448 bp in length and is a hydrophilic external rectifier potassium channel gene;the coding region of LrKUP8 is 2316 bp in length and is a low affinity potassium transporter.In this study,the functions of two salt-tolerant genes were analyzed by expression analysis,vector construction,subcellular localization,and homologous transformation.The main results are as follows:1.Tissue expression showed that under the long-term medium concentration of salt stress,the relative expression levels of the two genes in different organs were new leaves,old leaves,old stems and new stems from high to low;Under high salt stress for a short time,the relative expression levels of the two genes in leaves showed a decrease and then increased.2.Subcellular localization results showed that both LrSKOR and LrKUP8 were localized on the plasma membrane,which laid the foundation for further revealing the functions of the two genes of interest.3.Callus harvesting and transformation of ruthenicum:The leaves of ruthenicum were used as explants to sterilize and induce callus.Through transformation of callus,transgenic callus of pBI121-GFP empty vector,pBI121-LrSKOR-GFP and pBI121-LrKUP8-GFP were successfully obtained.4.Salt treatment and analysis of transgenic callus showed that the transgenic callus of transformed two target genes maintained a higher potassium to sodium ratio by inhibiting Na+inflow and K+effusion,thus increasing the salt tolerance of Lycium ruthenicum.5.Exploration of callus redifferentiation system of Lycium ruthenicum.:A suitable medium for callus differentiation of SY3 genotype was obtained:MS+KT 1.0 mg/L+6-BA 0.4 mg/L+NAA 0.1 mg/L.6.Leaf transformation of Lycium ruthenicum:the genetic transformation of two target genes was carried out with the leaves of Some resistant seedlings were obtained.And two Gemd2 genotypes transformed with LrKUP8 has rooted.In this study,two genes LrSKOR and LrKUP8 related to the regulation of salt-alkali stress were cloned from Lycium ruthenicum for the first time,and their expressions under different salt stress were analyzed.After construction of recombinant vectors,it was found that both genes were located in the plasma membrane of the cell,and for the first time,the callus of the Lycium ruthenicum was transgenic,and the transgenic callus of the Lycium ruthenicum was obtained.The salt stress treatment and analysis of the transgenic callus were carried out.Both genes were found to maintain a higher potassium to sodium ratio by inhibiting Na+ influx and K efflux.The application of Agrobacterium-mediated leaf disc transformation in Lycium ruthenicum was explored,and some resistant seedlings were obtained by directly transforming the leaves of Lycium ruthenicum.This research provides important basic materials for future plant molecular breeding and new varieties of stress-resistant varieties.Relevant research has important theoretical and practical significance.