Expression Characteristics Of Na⁺/H⁺ Antiporter Genes In Mulberry（Morus Multicaulis） And Salt-tolerance Function Of Mul-NHX5 Gene

Mulberry is not only the only feed tree species of silkworm,but also the important material basis of sericulture production,and also has high edible,medicinal and feeding values.In addition,as an ecological tree species,mulberry also plays an important role in saline-alkali land development,soil and water conservation,forestry carbon sequestration etc.Salt stress is an important abiotic stress factor affecting plant growth and development,and soil salinization has become an important factor affecting agricultural and forestry development and ecological environment construction in China.Therefore,it is of great significance to analyze and clear the molecular mechanism of mulberry salt-tolerance,clone salt tolerance genes,and cultivate new salt tolerance varieties,for the restoration and reconstruction of vegetation in saline-alkali land,and the development and utilization of saline-alkali land resources.In this study,the genome-wide identification and analysis of the Na⁺/H⁺antiporter（NHX）genes of mulberry was carried out,and the potential transcriptional regulation factor of NHX genes was predicted,and its regulatory effect on the expression of NHX genes was explored.The salt tolerance functions of the transcription factor and the NHX genes were studied,and their transgenic hairy root mulberry plants with strong salt tolerance were obtained.The information provided here not only lay a foundation for in-depth research on the function and expression regulation mechanism of NHX genes,and also provide new salt-tolerant germplasm for mulberry breeding.Moreover,it will be great significance to better play the ecological value of mulberry and promote the development of mulberry industry.The main results are as follows:（1）Genome-wide identification and analysis of mulberry NHX gene family.Seven NHX genes（Named Mul-NHX1～7）were identified in the genome of Husang 32.According to their subcellular localizations,Mul-NHX genes can be divided into three subfamilies:vacuolar membrane,plasma membrane and endosomal membrane.It was found that amino acid sequences among the members of different subfamilies are less conserved,but members of the same subfamily have the same conserved motifs,and all the members of the three subfamilies have Na⁺/H⁺exchange domains.Protein interaction network analysis revealed that Mul-NHX proteins interact with CIPK/CBL kinase network,and are regulated by abscisic acid（ABA）signaling pathway and MAPK kinase cascade pathway.（2）The expression pattern analysis of Mul-NHX genes.q RT-PCR analysis showed that the expression of all the Mul-NHX genes were induced by Na Cl stress,but had different induced expression patterns in the aerial and underground parts of mulberry plants,and different Mul-NHX members also had different expression patterns.In addition,the expressions of Mul-NHX genes were also regulated by ABA,SA,and Me JA.The induced expression patterns of different genes in different tissues of mulberry were also varied.The promoters（p Mul-NHXs）of Mul-NHX genes were cloned,and it was found that their nucleotide sequences contained various plant hormone response elements such as ABA,SA and Me JA,as well as multiple binding sites of the transcription factors,such as MYC,MYB and ARE.Combining transient tobacco infection and GUS histochemical staining analysis,it was found that p Mul-NHXs have ABA,Me JA and SA inducible expression activities.（3）Regulation of Mul-MYC2 on Mul-NHX5 gene expression and analysis of their salt tolerance functions.The MYC2 transcription factor gene（Mul-MYC2）of mulberry was cloned.Combining transient infection of tobacco leaves and GUS histochemical staining analysis,it was found that Mul-MYC2 could enhance the expression activity of p Mul-NHX5 and the expression of Mul-NHX5 gene.Mul-NHX5 gene was successfully transformed into Arabidopsis thaliana,and it was found that overexpression of Mul-NHX5 gene in Arabidopsis thaliana can enhance the salt tolerance of the transgenic seeds during germination,improve the activities of SOD,CAT and POD in transgenic plants under salt stress,reduce the damage caused by superoxide radicals,and enhance the salt tolerance of transgenic plants.Using hairy root transformation system,the transgenic Mul-NHX5 or Mul-MYC2 hairy root mulberry plants were successfully obtained,respectively.Salt stress test showed that overexpression of Mul-NHX5 or Mul-MYC2 gene in hairy roots of transgenic mulberry plants confers the transgenic plants more tolerance to salt stress.