Identification And Functional Verification Of NHX And SOS1 Genes In Banana

Banana（Musa spp.）is one of the most popular fruits in the world.Salinization of land has seriously affected the development of the banana industry.Na⁺/H⁺antiporters such as NHX and SOS1 play an important role in the salt tolerance mechanism of plants.The identification and functional analysis of NHX and SOS1 genes in banana have not been reported.This study used Brazilian banana（M.acuminata L.AAA group）and Fenjiao banana（M.ABB group,Pisang Awak）seedlings（10-12 cm,consistent growth conditions）as the experiment materials.The genes and protein sequences of NHX and SOS1 were identified and obtained from banana A genome database and B genome database respectively;the gene structure,cis-acting element,transmembrane structure and protein function of MaNHXs,MbNHXs,MaSOS1 and MbSOS1 were analyzed by bioinformatics;qRT-PCR was used to detect the expression of NHX and SOS1 genes in banana seedlings under 200 mmol L^-1 NaCl treatment.MaNHX5,MaSOS1 and MbSOS1 genes were cloned and be performed subcellular localization experiments and site-directed mutagenesis.Functional verification of transgenic yeast were carried out to explore the functional differences of Na⁺/H⁺reverse transporters in different genomes.The results are as follows:（1）11 MaNHX gene family genes were identified from banana A genome database.The analysis of physical and chemical properties found that most of the MaNHXs protein had an isoelectric point（5.86-8.88）greater than 8,and the protein molecular mass was 57-62 k Da.Subcellular localization analysis showed that MaNHXs were all located in vacuoles.The phylogenetic trees constructed with NHX proteins of Arabidopsis thaliana and rice were divided into two cluster groups.Most banana NHX genes were closely related to Arabidopsis thaliana and rice.Most of the exons（14-22）of MaNHXs gene are 14,and MaNHX10 contains 22 exons.In this study,9 MbNHXs gene family genes were identified from banana B genome database.Physical and chemical properties found that most of the MbNHXs protein has an isoelectric point（6.01-8.69）greater than 8,and the protein molecular mass is 50-171 k Da.Subcellular localization analysis showed that MbNHXs was located in vacuoles and cell membranes.The phylogenetic tree constructed with banana A genome,Arabidopsis thaliana and rice NHX protein was divided into two cluster groups.There are 11-19 exons of MbNHXs gene and 19 exons of MbNHX1.（2）Two banana plasma membrane Na⁺/H⁺reverse transporter genes,named MaSOS1（Ma08＿t18780.1）and MbSOS1（Mb08＿t18680.1）,were obtained from banana A genome database and B genome database respectively.Bioinformatics analysis showed that both proteins had PF00999 conserved domain;MaSOS1 and MbSOS1 contained 5 kinds of plant hormone response elements（Me JA,GA,SA,IAA,ABA）and 4 kinds of plant hormone response elements（GA,IAA,ABA,Me JA）respectively;MaSOS1 and MbSOS1 proteins both contained 12 transmembrane structures;MaSOS1 protein encoded by 1143 aa amino acid,MbSOS1 protein encoded by 964 aa amino acid.In the amino acid regions of530～630 aa and 670～710 aa,MbSOS1 protein had about 100 aa and 40 aa amino acid deletions,and the results of protein homology alignment showed that MaSOS1 and MbSOS1 proteins were closely related to Elaeis guineensis,Phoenix dactylifera,Ananas comosus,Phragmites australis,Nelumbo nucifera and Sorghum bicolor homologous proteins.（3）qRT-PCR results showed that MaNHXs were expressed in banana roots,pseudostems and leaves under 200 mmol·L^-1NaCl salt stress.After 3 hours of treatment,most of the MaNHXs expression in roots was up-regulated,while the expression of MaNHXs was down-regulated after 12 hours of treatment,but recovered after 24 hours of treatment.In pseudostems,the expression of most MaNHXs was up-regulated at 3 h,6 h and 24 h,respectively.The expression of MaNHX3 was up-regulated 3.42 times at 24 h.The expression of MaNHXs in the leaves was significantly up-regulated at 12 h and the expression of MaNHX10 was up-regulated by 4.05 times.The expression of MaSOS1 gene was induced by NaCl in roots,pseudostems and leaves,while the expression of MbSOS1gene was mainly induced by NaCl in pseudostems.（4）MaNHX5 subcellular localization experiment showed that GFP signal and tonoplast Marker were co-located on the tonoplast,indicating that MaNHX5 protein was mainly expressed on the tonoplast.MaSOS1 subcellular localization experiments show that MaSOS1 protein is mainly expressed on the cell membrane.（5）After the mutation of serine 276 of MaNHX5 gene to aspartic acid,yeast complementary experiment showed that MaNHX5’-AXT3 is more salt-tolerant than MaNHX5-AXT3.MaSOS1 gene and MbSOS1 gene could significantly improve the salt tolerance and high temperature tolerance of yeast AXT3.Compared with the AXT3 yeast that only transferred the p416 vector,the results showed that the growth of p416-AXT3was significantly inhibited under 100 mmol·L^-1 NaCl treatment,MaSOS1-AXT3 could grow normally under 300 mmol·L^-1 NaCl treatment,and MbSOS1-AXT3 could grow normally under 500 mmol·L^-1 NaCl treatment.The yeast p416-AXT3 could not grow at40℃,while MaSOS1-AXT3 grew well at 40℃.2 mmol·L^-1 Ca Cl₂ is beneficial to the growth of MaSOS1-AXT3 and MbSOS1-AXT3,and 400 mmol·L^-1 and 500 mmol·L^-1 KCl can inhibit the growth of MbSOS1-AXT3.