Genome-wide characterization of the CIPK gene family in mung bean and functional validation of VrCIPK5 in drought stress response

Abstract

Calcineurin B-like protein-interacting protein kinases (CIPKs) act as core regulators in plant calcium (Ca2+) signaling pathways and mediate abiotic stress adaptation. Mung bean (Vigna radiata L.) is an economically important legume that is widely grown in arid and semi-arid regions; however, drought stress significantly reduces its yield and quality. Despite this, genome-wide identification and functional analysis of the CIPK gene family have not been reported in mung bean, limiting our understanding of drought-resistance mechanisms and stress-tolerant variety breeding. In this study, 23 VrCIPK genes were identified from the mung bean genome, which were unevenly distributed across 6 chromosomes, with the remaining genes located on scaffolds. Phylogenetic analysis classified the VrCIPK family into five groups (A–E), characterized by high structural conservation of the N-terminal kinase and the C-terminal NAF/FISL regulatory domains. Gene Ontology annotation further indicated their conserved involvement in protein phosphorylation and calcium signal transduction. Collinearity analysis revealed that CIPK genes in mung bean and other species have relatively conserved evolutionary patterns. Segmental duplication contributed to VrCIPK gene family expansion, and most duplicated gene pairs underwent purifying selection during evolution. Gene structure and motif analyses showed that VrCIPK genes within the same group shared conserved structural features. Cis-acting element profiling revealed abundant hormone- and stress-responsive elements in VrCIPK promoters, indicating diverse transcriptional regulatory potential. Transcriptomics analysis and quantitative real-time PCR demonstrated that VrCIPK5 was significantly induced under drought stress. The heterologous overexpression of VrCIPK5 in tobacco conferred enhanced drought tolerance by promoting proline accumulation, increasing antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities, accelerating stomatal closure, and upregulating downstream stress-responsive genes (NtSOD, NtCAT, NtP5CS1, NtLEA5, and NtRD29A), thus alleviating reactive oxygen species overaccumulation and membrane lipid peroxidation. Collectively, these findings fill the research gap in the genome-wide identification and functional analysis of the CIPK gene family in mung bean and provide key genetic resources and theoretical support for breeding drought-resistant mung bean varieties.