Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (21): 4497-4511.doi: 10.3864/j.issn.0578-1752.2025.21.018

• EXPLORATION OF SALT-ALKALI AND DROUGHT RESISTANT GENES FOR ALFALFA BREEDING • Previous Articles     Next Articles

Cloning and Salt Tolerance Function Analysis of MsKTI3 Gene in Alfalfa

LÜ HuanHuan1,2(), LI RuYue1,2, LIU QingSong3, XU Lei1, XU YanRan1, YU HaoJie1, GUO ChangHong2, LONG RuiCai1()   

  1. 1 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
    2 College of Life Science and Technology, Harbin Normal University/Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province, Harbin 150025
    3 Cangzhou Academy of Agriculture and Forestry Sciences, Cangzhou 061001, Hebei
  • Received:2025-02-11 Accepted:2025-05-30 Online:2025-11-01 Published:2025-11-06
  • Contact: LONG RuiCai

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Abstract:

【Objective】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. 【Method】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. 【Result】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. 【Conclusion】The MsKTI3 gene played a positive regulatory role in responding to salt stress, and overexpression of MsKTI3 could improve the salt tolerance of plant.

Key words: alfalfa, trypsin inhibitor, overexpression, salt stress, physiological indexes

Table 1

The primer list"

引物名称Primer name 引物序列Primer sequence(5'-3') 用途Application
MsKTI3-F ATGCTAGCATTTCTTCTTCTCTTT 基因克隆Gene cloning
MsKTI3-R TCAAACCTTCTGAAACTTACTTTAAA 基因克隆Gene cloning
Y-MsKTI3-F CAGGCCTGGCGCGCCACTAGT ATGCTAGCAT TTCTTCTTCTCTTT 载体构建Vector construction
Y-MsKTI3-R TTGCTCCATCCCGGGACTAGTTCAAACCTTCTGAAACTTACTTTAAA 载体构建Vector construction
q-MsKTI3-F CGTCGGAGAATCTTGCCCTC 实时荧光定量RT-qPCR
q-MsKTI3-R GTGGAGACACGAATAACGCC 实时荧光定量RT-qPCR
Atactin-F GAAATCACAGCACTTGCACC 实时荧光定量RT-qPCR
Atactin-R AAGCCTTTGATCTTGAGAGC 实时荧光定量RT-qPCR
E-MsKTI3-F GGACTCTAGAGGATCCCCGGGATGCTAGCATTTCTTCTTCTCTTT 亚细胞定位Subcellular localization
E-MsKTI3-R GCTCACCATGGTACCCCCGGGTCAAACCTTCTGAAACTTACTTTAAA 亚细胞定位Subcellular localization
Msactin-F CAAAAGATGGCAGATGCTGAGGAT RT-qPCR
Msactin-R CATGCACCAGTATGACGAGGTCG RT-qPCR

Table 2

Bioinformatics tools"

生物信息学工具
Bioinformatics tool		 网址
Website		 用途
Application
ExPASy http://web.expasy.org/protparam/ 蛋白理化性质分析
Physicochemical property analysis of protein
TMHMM Server 2. 0 http://www.cbs.dtu.dk/services/TMHMM 蛋白质跨膜结构域预测
Transmembrane domain prediction of protein
DNAMAN https://npsa-prabi. ibcp. fr/cgi-bin/npsa_automat. pl?page=npsa_sopma.html 多序列比对 Multiple sequence alignment
MEGA 6 https://www. megasoftware.net/ 进化树构建 Construction of evolutionary tree
SMART https://smart.embl.de/ 蛋白质保守结构域分析
Conserved domain analysis of protein
Plant CARE https://bioinformatics. psb. ugent. be/webtools/plantcare/html/ 顺式作用元件分析 Cis-acting element analysis
PRABI https://npsa-prabi. ibcp. fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html 蛋白质二级结构分析
Secondary structure analysis of protein
Cell-PLoc 2.0 http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 亚细胞定位预测
Subcellular localization prediction
Blast https://blast. ncbi. nlm. nih. gov/Blast. cgi 同源序列比对分析
Homologous sequence alignment analysis

Fig. 1

Cloning and bioinformatics analysis of MsKTI3 A：The bands for full-length amplification of the MsKTI3 cDNA; B: Hydrophilicity analysis of MsKTI3 protein; C: Prediction of the conserved domains of MsKTI3 protein, pink region: low complexity domain; Blue region: STI (Soybean trypsin inhibitor) conserved domain); D: Some cis-acting elements are contained in the upstream promoter sequence of the MsKTI3 gene"

Fig. 2

Construction of the phylogenetic tree of alfalfa MsKTI3 protein and its orthologs in other species Numbers at node indicates bootstrap value derived from 1000 replicates. The numbers on the scale represent the genetic distances between different species"

Fig. 3

Subcellular localization analysis of MsKTI3 in tobacco (Nicotiana benthamiana) leaves A-C: Results of the empty vector (GFP); D-F: Results of the fusion vector (MsKTI3 - GFP). Bar=50 µm"

Fig. 4

The expression analysis of MsKTI3 in alfalfa tissues, under abiotic stress conditions, and in Arabidopsis thaliana transgenic lines A: Expression levels of MsKTI3 gene in various tissues of alfalfa; B, C: Expression levels of MsKTI3 in roots and leaves under 150 μmol·L-1 ABA stress; D, E: Expression levels of MsKTI3 in roots and leaves under 200 mmol·L-1 NaCl stress; F: The expression level of MsKTI3 gene in Arabidopsis thaliana transgenic lines and WT lines. Different lowercase letters represent significant differences at the P<0.05 level"

Fig. 5

Comparison of the germination and growth of wild-type (WT) and MsKTI3 overexpressing lines of Arabidopsis thaliana A-D: The growth status of wild-type (WT) Arabidopsis thaliana and Arabidopsis thaliana overexpressing the MsKTI3 under different sodium chloride concentrations. E, F: Germination rates of wild-type (WT) Arabidopsis thaliana and Arabidopsis thaliana overexpressing the MsKTI3 on NaCl media with concentrations of 0 (A), 100 (B), 150 (C), and 200 mmol·L-1 (D)"

Fig. 6

Phenotypes of vertically cultured wild-type (WT)and MsKTI3 overexpressing Arabidopsis thaliana under different NaCl stresses A-C: Phenotypes of vertically cultured wild-type (WT) and MsKTI3 overexpressing Arabidopsis thaliana seedlings with different salt concentrations (0、150、200 mmol·L-1 NaCl). Bar=1 cm；D-F：Phenotypes of plant fresh weight (D), root length (E) and the number of lateral roots (F) of wild-type and MsKTI3 overexpressing Arabidopsis thaliana seedlings under different salt concentrations on vertical culture plates"

Fig. 7

Phenotypes comparison of transgenic and WT Arabidopsis thaliana in soil before and after salt stress A, B: Phenotypes of WT and overexpressing Arabidopsis thaliana before NaCl stress; C, D: Phenotypes of WT and overexpressing plants after 17 days of NaCl stress with 0 (C) and 250 mmol·L-1 (D); E, F: Plant height phenotypes of WT and overexpressing plants after 17 days of NaCl stress with 0 (E) and 250 mmol·L-1 (F) (E: Before cutting off the flowers and stems; F: Before cutting off the flowers and stems). Bar=1 cm"

Fig. 8

Determination of physiological indexes of Arabidopsis thaliana under salt stress A: plant height; B: the number of rosette leaves; C: Relative electrical conductivity; D: MDA content; E: Chlorophyll content; F: CAT activity detection"

Fig. 9

Detection of oxidative damage in Arabidopsis thaliana leaves under different salt stresses A: DAB staining, NBT staining; B: The darker the color, the higher the degree of oxidative damage"

Fig. 10

Phenotypic analysis of salt tolerance in plants overexpressing the MsKTI3 gene in alfalfa hairy roots A: Phenotype of normally growing plants. B: Phenotype of plants after 200 mmol·L-1 NaCl treatment for 4 days, scale bar = 1 cm. C: Electrophoretogram of genomic PCR products. WT: non-transgenic plants, —: negative control, +: positive control, K: MsKTI3 gene overexpression in hairy root of alfalfa; D: Expression analysis of MsKTI3 in roots of WT and transgenic alfalfa seedlings; E: Root length before and after treatment; F: Detection of CAT (catalase) activity. Lowercase letters indicate significant differences (P<0.05)"

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