Genome-wide identification and expression analysis of the cips gene family in lettuce

CBL-interacting protein kinases serve as pivotal regulators within the plant Ca signalling pathway, playing a crucial role in responding to abiotic stresses such as salinity and drought. However, systematic studies on the CIPK gene family in lettuce remain relatively scarce. This research comprehensively identified, structurally characterised, evolutionarily analysed, and assessed the stress responses of the CIPK gene family based on the lettuce reference genome. A total of 32 LsCIPK genes were identified, distributed across nine chromosomes with marked unevenness. Phylogenetic analysis grouped them into five evolutionary branches, exhibiting good clustering consistency with Arabidopsis homologues. All LsCIPK proteins contain the characteristic protein kinase domain and NAF domain, with gene structures exhibiting either "single-exon" or "multi-exon" characteristics. Promoter cis-element analysis revealed that most LsCIPK genes are rich in cis-elements associated with light response, hormone response, and abiotic stress. Colinearity analysis indicated that the expansion of this family was primarily driven by segmental duplication. The Ka/Ks ratios for all eight duplicate gene pairs were less than 1, suggesting that the family as a whole is under purifying selection. Transcriptome data from salt stress and drought stress treatments revealed significant expression changes in multiple LsCIPK genes under stress conditions. Notably, LsCIPK7, LsCIPK26, and LsCIPK32 exhibited strong induction in both stress types. Further qRT-PCR results corroborated the transcriptional trends. Subcellular localisation analysis of LsCIPK32 using a tobacco transient expression system revealed that this protein is primarily distributed in the cytoplasm and plasma membrane. In summary, this study systematically elucidates the composition, structural characteristics, evolutionary patterns, and non-biotic stress response mechanisms of the lettuce CIPK gene family. It provides crucial genetic resources and theoretical foundations for further functional elucidation and the development of stress-tolerant lettuce cultivars.