Genome-wide identification and characterization of Calcium-Dependent Protein Kinase (CDPK) gene family in autotetraploid cultivated alfalfa (Medicago sativa subsp. sativa) and expression analysis under abiotic stresses.

Background: Calcium-dependent protein kinases (CDPKs), play multiple roles in plant development, growth and response to bio- or abiotic stresses. Calmodulin-like domains typically contain four EF-hand motifs for Ca²⁺ binding. The CDPK gene family can be divided into four subgroups in Arabidopsis, and it has been identified in many plants, such as rice, tomato, but has not been investigated in alfalfa (Medicago sativa subsp. sativa) yet.

Results: In our study, 38 non-redundant MsCDPK genes were identified from the "XinJiangDaYe" alfalfa genome. They can be divided into four subgroups which is the same as in Arabidopsis and Medicago truncatula, and there were 15, 12,10 and 1 in CDPK I, II, III and IV, respectively. RNA-seq analysis revealed tissue-specificity of 38 MsCDPK genes. After researching the transcriptome data, we found these 38 MsCDPK members responsive to drought, salt, and cold stress treatments. Further analysis showed that the expression of almost all the MsCDPKs is regulated by abiotic stresses. In addition, we chose MsCDPK03, MsCDPK26, MsCDPK31 and MsCDPK36 for RT-qPCR validation which was from CDPK I-IV subgroups respectively. The result showed that the expression of these four genes was significantly induced by drought, salt and cold treatments. The subcellular location experiment showed that these four proteins were all located in nucleus.

Conclusion: In our study, we identified 38 distinct MsCDPK genes within the alfalfa genome, which were classified into four groups. We conducted a comprehensive analysis of various gene features, including physicochemical properties, phylogenetic relationships, exon-intron structures, conserved motifs, chromosomal locations, gene duplication events, cis-regulatory elements, 3D structures, and tissue-specific expression patterns, as well as responses to drought, salt, and cold stresses. These results also provide a solid foundation for further investigations into the functions of MsCDPKs aimed at improving drought tolerance in autotetraploid cultivated alfalfa through genetic engineering.