Salt stress can severely damage plant cells, inhibit plant growth and development, and consequently lead to a substantial reduction in yield. Kunitz trypsin inhibitor (KTI), a representative type of serine protease inhibitors, is primarily involved in regulating physiological processes in plants, such as growth and development, pest and disease resistance, and responses to abiotic stress. Exploring and analyzing the molecular mechanisms underlying the regulation of salt stress by the alfalfa KTI would contribute to the provision of novel candidate genes for molecular breeding of salt-tolerant alfalfa.

In this study, a salt stress induced KTI was cloned from Medicago sativa ‘Zhongmu No. 4’, which was named MsKTI3. Through bioinformatics methods, the structural characteristics of the MsKTI3 and its encoded protein were deeply analyzed. Sequence alignment and evolutionary analysis were carried out with homologous genes of other species. The real-time fluorescence quantitative PCR (RT-qPCR) method was used to analyze the expression patterns of the MsKTI3 in different tissues and under different stress conditions. With the aid of the tobacco transient expression system, the subcellular localization of the MsKTI3 protein was analyzed. An MsKTI3 overexpression vector was constructed, and the MsKTI3 overexpressing lines of Arabidopsis thaliana were successfully obtained by the Agrobacterium tumefaciens-mediated method. Meanwhile, the Medicago sativa plants with MsKTI3 overexpression in roots were obtained by the Agrobacterium rhizogenes-mediated method. Phenotypic analysis and physiological index determination of the related lines were carried out under salt stress conditions.

Bioinformatics analysis indicated that the coding sequence (CDS) of the MsKTI3 gene was 627 bp, encoding 208 amino acids. The relative molecular weight was 23 220.81 Da, and the theoretical isoelectric point was 8.57. Phylogenetic tree analysis revealed that the amino acid sequence of MsKTI3 shared a high homology with that of MtKTI3 of Medicago truncatula, reaching 97%. RT-qPCR was employed to analyze the expression pattern of the MsKTI3. The results demonstrated that the expression level of the MsKTI3 was the highest in roots. Moreover, during the initial stage of NaCl (200 mmol·L^-1) and ABA (150 μmol·L^-1) stress, the expression level generally exhibited an up-regulation trend. Subcellular localization results showed that the MsKTI3 protein was located in the plasma membrane. Twelve overexpressing Arabidopsis lines were generated via the Agrobacterium-mediated method. Under salt-stress conditions, the germination rate of the Arabidopsis lines overexpressing the MsKTI3 was higher than that of the wild type, and the damage degree of the overexpressing seedlings was lower than that of the wild type. The relative electrolyte leakage (IEL) and malondialdehyde (MDA) content of the overexpressing plants were significantly lower than those of the wild type (P<0.05), while the chlorophyll (Chl) content and catalase (CAT) activity were significantly higher than those of the wild type (P<0.05). MsKTI3 was transferred into the roots of alfalfa using Agrobacterium rhizogenes. Phenotypic analysis indicated that overexpression of MsKTI3 in roots enhanced the salt tolerance of alfalfa. Additionally, the CAT activity of the overexpressing plant roots was higher than that of the control plants.

The MsKTI3 gene played a positive regulatory role in responding to salt stress, and overexpression of MsKTI3 could improve the salt tolerance of plant.