关键词: 东方蜜蜂, DNA甲基转移酶3, 生物信息学, 分子特征, 系统进化, 发育, 宿主应答

Abstract: 【Aim】 To analyze the molecular features, phylogeny and gene expression profiles of DNA methyltransferase 3 (DNMT3) from Apis cerana (AcDNMT3), so as to provide a reference and foundation for further functional analysis of AcDNMT3. 【Methods】 Bioinformatics tools were used to predict and analyze the physicochemical properties, molecular features, structural domains, conserved motifs and phylogeny of AcDNMT3 in Ap. cerana. RT-qPCR was used to measure the relative expression levels of AcDNMT3 in different developmental stages (egg, 3-day-old larva, 1-day-old prepupa, 2-day-old prepupa, 4-day-old pupa, and 1-, 2-,6-,12-,15- and 18-day-old adults) of Ap. cerana workers, and various adult tissues (antennae, hypopharyngeal gland, brain, cuticle, midgut, fat body and venom gland) of Ap. cerana workers, as well as in the guts of the 4-6-day-old larval workers of Ap. cerana after inoculating the 3-day-old larval workers with Ascosphaera apis and the midguts of the 2-5-day-old adults after inoculating the 1-day-old adult workers with Nosema ceranae. 【Results】 AcDNMT3 contains 758 amino acids with the approximate molecular weight of 88.24 kD, the molecular formula of C₃₉₄₅H₆₁₈₂N₁₀₇₆O₁₁₃₄S₄₄, the theoretical isoelectric point (pI) of 8.32, and the average hydrophilic index of -0.454. AcDNMT3 was predicted to interact with ten proteins, including adenosylhomocysteinase (A0A2A3ECJ8). DNMT3 proteins from Ap. cerana, Ap. mellifera, Ap. dorsata, Ap. laboriosa, Bombus terrestris, Frieseomelitta varia, Osmia lignaria, Colletes gigas, Eciton burchellii, Nylanderia fulva, Linepithema humile, Solenopsis invicta, Monomorium pharaonis and Vollenhovia emeryi contained four common structural domains (PWWP_DNMT3, ADDz_Dnmt3, Dcm domain and PWWP domain) and five common conserved motifs (Motif1, Motif2, Motif3, Motif4 and Motif5). AcDNMT3 was clustered with Ap. mellifera DNMT3 on the phylogenetic tree. AcDNMT3 was differentially expressed in the eggs, larvae, prepupae and pupae of Ap. cerana workers, with the highest expression level in the 2-day-old prepupae which was significantly higher than those in eggs, the 3-day-old larvae, 1-day-old prepupae and 4-day-old pupae. AcDNMT3 was differentially expressed in adult workers of Ap. cerana at the 1-, 2-, 6-, 12-, 15- and 18-day-old, with the highest expression level in the 1-day-old adults which was significantly higher than those in the 2-, 6-, 12-, 15- and 18-day-old adults. Differential expression of AcDNMT3 was observed across various adult worker tissues including antennae, venom gland, brain, midgut, fat body, cuticle and hypopharyngeal gland of Ap. cerana, with the highest expression level in the antennae and the lowest expression level in the midgut. After infecting the 3--day-old larval workers of Ap. cerana with As. apis, the expression level of AcDNMT3 in the gut of the 4-day-old larval workers was significantly down-regulated as compared with that of the control. After inoculation of the 1-day-old adult workers of Ap. cerana with N. ceranae, the expression levels of AcDNMT3 in the midguts of the 2-, 4- and 5-day-old adult workers were significantly up-regulated and that in the 3-day-old adult workers was extremely significantly down-regulated as compared with those of the control. 【Conclusion】 AcDNMT3 is a putative hydrophilic, intracellular, non-transmembrane protein that is highly conserved across Ap. cerana and the above other insect species. AcDNMT3 shows the highest homology with DNMT3 of Ap. mellifera.AcDNMT3 may play a role in the development of Ap. cerana workers, in the larval responses to As. apis infection, and in the adult responses to N. ceranae infection.

Key words: Apis cerana, DNA methyltransferase 3, bioinformatics, molecular features, phylogeny, development, host response