Molecular feature and gene spatio-temporal expression profile of Apis mellifera CKI1 protein

In this study, the expression of Apis mellifera Cyclin-dependent kinase inhibitor 1 (CKI1) gene AmCKI1 was validated, and the physicochemical properties and molecular features of the AmCKI1 protein were investigated, followed by determination of the expression profile of AmCKI1 in various tissues and developmental stages of worker bees, aiming to provide reference and foundation for further functional research. PCR and Sanger sequencing were performed to verify the expression of AmCKI1. Relative software were employed to predict the physicochemical properties and molecular features of AmCKI1, followed by phylogenetic analysis. RT-qPCR was utilized to detect the relative expression level of AmCKI1 in seven different tissues of the workers (antennae, brain, hypopharyngeal gland, midgut, venom gland, cuticle and fat body), across various developmental stages (egg, larva, pre-pupa, pupa and adults at 1, 2, 6, 12, 15, and 17 days). The results demonstrated that AmCKI1 was truly expressed in worker bees. The molecular formula of AmCKI1 was C₁₁₀₄H₁₇₉₃N₃₃₃O₃₃₁S₇, and the molecular weight was approximately 25.26 kDa, and the lipophilicity coefficient, isoelectric point and the average hydrophilicity index were 71.88, 10.18 and -0.674, respectively. AmCKI1 were potentially localized in the nucleus, mitochondria and cytoplasm simultaneously. It included 32 phosphorylation sites but lacked typical transmembrane domain and signal peptide. One CDI structural domain and three identical conserved motifs were included in CKI1 proteins from A. mellifera and six other bee species. The highest homology was found in CKI1 between A. mellifera and Apis florea. AmCKI1 was expressed in the brain, hypopharyngeal gland, midgut, antennae, venom gland, cuticle and fat body of workers, without significant difference in expression levels (P > 0.05). AmCKI1 was differentially expressed in egg, 3-day-old larva, 7-day-old and 8-day-old pre-pupae and 12-day-old pupa, the expression levels in egg and pre-pupae were significantly higher than those in 3-day-old larva and 12-day-old pupa (P < 0.05). AmCKI1 was also differentially expressed in 1-, 2-, 6-, 12-, 15- and 17-day-old adults, while the expression level was the highest in 17-day-old adult and significantly higher than that in 1-, 2-, 6-, 12- and 15-day-old adults. AmCKI1 was a putative intracellular and hydrophilic protein. CKI1 was highly conserved among A. mellifera, A. florea, Bombus terrestris, Bombus impatiens, Apis laboriosa, Apis ceranae, and Apis dorsata. The homology of CKI1 between A. mellifera and A. florea was the highest. AmCKI1 was specifically and highly expressed in worker egg and 17-day-old adult.