Expression profiles of nce-miR-34537 and the target gene PIP5KI during the infection process of Nosema ceranae

Abstract:

[Objective] To validate expression and sequence of the previously identified nce-miR-34537, predict the target gene of nce-miR-34537 and the molecular characteristics of the gene, determine the expression profiles of nce-miR-34537 and its target gene during the Nosema ceranae infection in Apis mellifera ligustica workers, so as to provide a foundation for revealing the role of nce-miR-34537 in N. ceranae infection. [Methods] Stem-loop-RT-PCR and Sanger sequencing were performed to verify the expression and sequence of nce-miR-34537. The molecular properties and conserved motifs of the target gene of nce-miR-34537, PIP5KI (encoding type I phosphatidylinositol 4-phosphate 5-kinase), were predicted via bioinformatics tools. The phylogenetic tree was constructed based on amino acid sequences. The expression profiles of nce-miR-34537 and its target genes were determined via RT-qPCR. [Results] The existence and expression of nce-miR-34537 were confirmed in N. ceranae spores. nce-miR-34537 can target 151 genes including PIP5KI. The deduced PIP5KI protein was predicted to have the molecular formula of C₈₈₂H_{1 364}N₂₂₆O₂₅₁S₇ and the molecular weight of 19.37 kDa and contain 160 residues, 17 phosphorylation sites, and no classical signal peptide. PIP5KI can simultaneously locate in the nucleus, mitochondrion, cytoplasm, and secretory vesicles. N. ceranae PIP5KI (AAJ76_2700017870) contained four conserved motifs, while the other PIP5KI (EEQ82436.1) contained five motifs. PIP5KI proteins in N. ceranae, N. apis, and Spraguea lophii were grouped into one clade in the phylogenetic tree, and N. ceranae PIP5KI (EEQ82436) and N. apis PIP5KI (EQB60549.1) had the closest evolutionary distance. Compared with that 1 dpi (day post inoculation), the expression level of nce-miR-34537 was up-regulated at 2 dpi (P>0.05) and down-regulated at 4, 6, and 8 dpi (P<0.05), and that of PIP5KI was up-regulated at 2 dpi (P<0.05) and down-regulated at 4, 6, and 8 dpi (P<0.05).[Conclusion] nce-miR-34537 is expressed in N. ceranae spores. N. ceranae PIP5KI (EEQ82436) and N. apis PIP5KI (EQB60549.1) have the closest genetic relationship. The expression of both nce-miR-34537 and the target gene PIP5KI is first up-regulated and then down-regulated during N. ceranae infection in A. m. ligustica workers. The results indicate that nce-miR-34537 may participate in the infection process via positive regulation of PIP5KI expression.