Objective To explore the function of the MfWRKY40 gene in Medicago falcata L. and to investigate the functional mechanism of the WRKY40 homologous gene in plants.

Methods The full length of MfWRKY40 gene was successfully cloned from alfalfa using PCR technology, and bioinformatics analysis, subcellular localization, and expression pattern analysis were performed.

Results The MfWRKY40 gene related to alfalfa was discovered, with an ORF containing 954 bases and encoding 317 amino acids. This gene does not contain transmembrane regions or signal sequences. Peptides are a protein structure with hydrophilicity. Since the secondary structure of the protein consists mainly of helices and random convolutions, some basic results have been obtained from predictive analyses of the protein's tertiary structure. These results have a high degree of confidence and have confirmed that the protein has a total of 44 phosphorylation sites, and of these sites, the serine phosphorylation sites are the most numerous. Through subcellular localization analysis, the MfWRKY40 protein is located in the nucleus. Based on the results of phylogenetic tree analyses, it can be seen that the MfWRKY40 protein is evolutionarily closely related to the alfalfa protein. Through RT qPCR detection, it was confirmed that the MfWRKY40 gene is most significantly expressed in the leaves, followed by the roots, and has lower expression in the flowers and stems. Therefore, the MfWRKY40 gene mainly plays a role in the leaves and roots.

Conclusion The cloning of the CDS sequence of the MfWRKY40 gene has been successfully achieved and the amino acid sequence of the gene has been characterised bioinformatically, subcellular localization, and expression pattern characteristics of the amino acid sequence encoded by this gene were analyzed, providing theoretical support for further exploring the function of the MfWRKY40 gene in alfalfa.