Abstract: 【Aim】 This study aims to offer reference and foundation for continuous and further study on RNA m⁶A methyltransferase 14 (METTL14) gene of Apis mellifera AmMETTL14 through cloning AmMETTL14, analyzing the physicochemical properties and molecular characteristics of AmMETTL14, detecting the expression pattern of AmMETTL14 in different developmental stages and various tissues of workers, and preparing the polyclonal antibody to AmMETTL14. 【Methods】 The CDS of AmMETTL14 in A. mellifera was amplified by PCR followed by Sanger sequencing validation. Bioinformatic analysis and construction of phylogenetic tree of AmMETTL14 were conducted using relevant bioinformatics software. Inducible expression of AmMETTL14 fusion protein was performed through prokaryotic expression system, followed by immunization of New Zealand white rabbits to prepare the polyclonal antibody, and the titer and specificity of the antibody were evaluated by ELISA and Western blot, respectively. RT-qPCR was used to detect the relative expression level of AmMETTL14 in the egg, larva, prepupa, pupa and adult worker, and seven tissues including antenna, venom gland, brain, midgut, hypopharyngeal gland, fat body and cuticle of the newly emerged adult worker.【Results】 The CDS of AmMETTL14 of A. mellifera was successfully cloned. The molecular formula of AmMETTL14 is C₁₉₅₇H₃₁₁₃N_567O589S₁₅, the molecular weight is about 44.49 kD, the liposoluble coefficient is 79.97, the theoretical isoelectric point is 8.58, and the average hydrophilic coefficient is -0.59. AmMETTL14 contains no typical transmembrane domain and signal peptide, and is simultaneously located to the nucleus, mitochondria and cytoplasm. METTL14s in A. mellifera, A. cerana cerana, A. laboriosa, A. cerana, A. dorsata, A. florae, Bombus terrestris, Vespa mandarinia, B. impatien and Drosophila melanogaster contain the structural domain MT-A70 and three same conserved motifs. AmMETTL14 and METTL14 of A. laboriosa had the highest amino acid sequence identity (65.60%) and the closest relationship. The prepared polyclonal antibody to AmMETTL14 had high titer (more than 512 K) and strong specificity. The expression levels of AmMETTL14 in the 7- and 8-day-old prepupae and eggs were similar and significantly higher than that in the 3-day-old larva, that in the 12-day-old pupa was significantly higher than those in the egg, larva and prepupa, and those in the 6-, 12- and 15-day-old adult workers were significantly higher than that in the 1-day-old adult worker. Additionally, the expression levels of AmMETTL14 in the brain and hypopharyngeal gland were similar to that in the antenna, and significantly higher than those in the venom gland, midgut, fat body and cuticle of the newly emerged adult worker. 【Conclusion】 This study result indicates that AmMETTL14 may be a hydrophilic, non-transmembrane and intracellular protein, AmMETTL14 plays potential regulatory role in larval growth and development, and the prepared polyclonal antibody to AmMETTL14 has high titer, sensitivity and specificity, laying a foundation for further exploring the function of AmMETTL14 and the involved epigenetic regulation mechanisms.

Key words: Apis mellifera, N⁶-methyladenosine, methyltransferase 14, spatiotemporal expression pattern, MT-A70, antibody