Cloning, molecular characterization, and expression pattern of N6-adenine-specific DNA methyltransferase gene in Nosema ceranae

Abstract:

[Objective] Nosema ceranae exclusively infects adult honey bees and causes nosemosis, resulting in great losses for the beekeeping industry. Little is known about the N. ceranae N6-adenine-specific methyltransferase gene NcN6AMT. This study cloned the coding sequence (CDS) region of NcN6AMT, investigated the physicochemical properties and molecular characteristics of NcN6AMT, and then determined the relative expression level of NcN6AMT during the infection process of N. ceranae in Apis mellifera ligustica and Apis cerana cerana workers. This study aim to enrich the information about NcN6AMT and lay a foundation for exploring the function and epigenetic regulation mechanism of NcN6AMT during the infection process of N. ceranae. [Methods] Protparam and ProtScale were used to analyze the isoelectric point and hydrophilia of NcN6AMT. The signal peptide, phosphorylation site, transmembrane domain, secondary structure, and tertiary structure of NcN6AMT were predicted by SignalP 5.0, NetPhos 3.1, TMHMM-2.0, SOPMA, and SWISS-MODEL, respectively. WoLF PSORT II was employed to predict the subcellular localization of NcN6AMT. TBtools was employed to predict the domains in the amino acid sequences of N6AMTs in Homo sapiens, Mus musculus, Nilaparvata lugens, Encephalitozoon cuniculi, Encephalitozoon intestinalis ATCC 50506, Encephalitozoon Romaleae SJ-2008, Spraguea lophii 42_110, Nosema bombycis CQ1, Nitzschia inconspicua, and N. ceranae. MEME and MEGA 11.0 were employed to predict the conserved motifs and build the phylogenetic tree of N6AMTs. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to determine the relative expression level of NcN6AMT during the N. ceranae infection of A. m. ligustica and A. c. cerana workers. [Results] The target fragment with a size of about 500 bp was amplified via PCR, and the cloning and sequencing results showed that it was consistent with the predicted sequence in GenBank. The deduced NcN6AMT protein was composed of 166 residues and had the molecular weight of 18.7 kDa, the formula of C₈₄₅H₁₃₇₄N₂₁₄O₂₄₉S₆, the theoretical isoelectric point of 5.88, the lipid solubility coefficient of 119.76, the instability coefficient of 37.47, the average hydrophilic coefficient of 0.025, and 15 phosphorylation sites, with no typical transmembrane domain or signal peptide. NcN6AMT was located in cytoplasm, mitochondria, nucleus, and vacuole membrane. It had one structural domain MTS, which also existed in the N6AMTs of other eight species such as N. bombycis CQ1 and E. cuniculi. Five same conserved motifs were predicted in the N6AMTs of N. ceranae, E. cuniculi,E. intestinalis ATCC 50506, and E. romaleae SJ-2008. The sequence identity of N6AMTs in N. ceranae, N. bombycis CQ1, E. cuniculi, E. intestinalis ATCC 50506, and E. romaleae SJ-2008 was 70.92%. The N6AMTs in N. ceranae and N. bombycis CQ1 were grouped into one clade. The expression of NcN6AMT was first up-regulated and then down-regulated within 1-4 days post inoculation (dpi) of N. ceranae in A. m. ligustica and A. c. cerana workers. [Conclusion] The CDS region of NcN6AMT was successfully cloned, and the physiochemical properties and molecular characteristics of NcN6AMT protein were clarified. The N6AMT proteins in N. ceranae and N. bombycis were highly conserved. During the first proliferation cycle (1-4 dpi) of N. ceranae in A. m. ligustica and A. c. cerana workers, the expression of NcN6AMT exhibited a bell-shaped pattern.