Abstract: 【Objective】Based on RNA-Seq profiling of the homeodomain leucine zipper protein (HD-Zip) transcription factor family in soybean (Glycine max) during salt stress, screening and cloning of a salt-induced gene (GmHAT5) from soybean were conducted. GmHAT5 was transformed into Lotus japonicus, the legume model system, to further understand the mechanism of salt tolerance. 【Method】The ORF of GmHAT5, the protein molecular weight, isoelectric point, sequence structure and protein localization were analyzed by some bioinformatics programs. Meanwhile, the homologous protein alignments with 10 other species, and relative expression levels of GmHAT5 in different organs and under saline stress were also analyzed. The overexpression vector of GmHAT5 was constructed and transformed into Agrobacterium rhizogenes strain LBA1334 to obtain the “composite” Lotus japonicus plants and the salt resistant phenotype was analyzed under salt stress. The overexpression vector of GmHAT5 was also transformed into Agrobacterium tumefaciens strain EHA105 to obtain the stable transgenic Lotus japonicus plants and then the phenotype and related physiological indexes were analyzed under salt stress. 【Result】Bioinformatics software analysis showed that the GmHAT5 contained an ORF with 1 038 bp, encoded 345 amino acid. The theoretical molecular weight and isoelectric point of GmHAT5 protein were 39.17 kD and 4.63, respectively. GmHAT5, located in the nucleus as other HD-Zip family proteins, was a typical nuclear protein. Sequence analysis showed that GmHAT5, which belonged to the first class of plant HD-Zip protein, contained a homeobox domain and a leucine zipper domain. The homologous protein alignments showed that GmHAT5 had a high similarity with GsHAT5. The hairy root transformation result showed that, after being treated with 200 mmol·L^-1 NaCl for 7 d, the “composite” plants grew well, while the empty vector control plants exhibited discoloration and stunted growth. Transgenic hairy roots in vitro culture was treated with a different concentration of NaCl for 14 d. Compared with the overexpressing transgenic hairy roots, the control group shriveled up and its growth was inhibited significantly. Stable transformation result showed that, after being treated with different concentration of NaCl for 14 d, the transgenic plants grew well compared to the two control groups. There were significantly changes in malondialdehyde content and relative membrane permeability caused by saline stress in transgenic plants compared to the two control groups (P<0.05). Moreover, transgenic plants had higher levels of chlorophyll content and root activity compared with the two control groups under saline stress conditions (P<0.05). Compared with the control plants, transgenic plants had lower levels of Na⁺ content in leaves and roots, while K⁺ and Ca²⁺ contents in leaves and roots increased significantly in transgenic plants.【Conclusion】 A HD-ZipⅠclass homeodomain leucine zipper protein gene (GmHAT5) was cloned from soybean, the expression level was significantly increased under salt stress. Over-expression of GmHAT5 gene could enhance resistance to saline in Lotus japonicus. Hairy root transformation method could be used as a quick effective means for screening of salt tolerance candidate gene. These imply that GmHAT5 may play an important role in salt stress regulation in soybean.

Key words: Glycine max, homeodomain leucine zipper protein (HD-Zip), transcription factor, Lotus japonicus, saline tolerance