东方蜜蜂微孢子虫侵染过程中nce-miR-34537和靶基因PIP5KI的表达谱

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

摘要:

【目的】本研究旨在对前期鉴定到的nce-miR-34537进行表达和序列验证，预测nce-miR-34537的靶基因并明确其分子特性，进而检测nce-miR-34537及其靶基因在东方蜜蜂微孢子虫(Nosema ceranae)侵染意大利蜜蜂(Apis mellifera ligustica)工蜂过程的表达谱，为进一步探究nce-miR-34537调控东方蜜蜂微孢子虫侵染的功能和作用机制提供基础。【方法】通过Stem-loop-RT-PCR和Sanger测序验证nce-miR-34537的表达和序列。通过生物信息学软件预测nce-miR-34537的靶基因PIP5KI (I型磷脂酰肌醇4-磷酸-5-激酶基因)的理化性质等分子特性和保守基序，并构建基于氨基酸序列的系统进化树。采用RT-qPCR检测nce-miR-34537及其靶基因的表达谱。【结果】nce-miR-34537在东方蜜蜂微孢子虫孢子中真实存在和表达。nce-miR-34537共靶向PIP5KI等151个基因。PIP5KI蛋白的分子式为C₈₈₂H_{1 364}N₂₂₆O₂₅₁S₇，分子量约为19.37 kDa，含160个氨基酸和17个磷酸化位点，但不含信号肽，可同时定位于细胞核、线粒体、细胞质和分泌囊泡。东方蜜蜂微孢子虫的1个PIP5KI (AAJ76_2700017870)含4个保守基序，另1个PIP5KI (EEQ82436.1)含5个保守基序。东方蜜蜂微孢子虫、蜜蜂微孢子虫(N. apis)和鮟鱇思普雷格孢虫(Spraguea lophii)的PIP5KI在进化树中聚为一支，且东方蜜蜂微孢子虫的PIP5KI (EEQ82436)与蜜蜂微孢子虫的PIP5KI (EQB60549.1)的进化距离最近。相较于接种后1 dpi (day post inoculation)，nce-miR-34537在2 dpi上调表达(P>0.05)，而在4 dpi、6 dpi和8 dpi的表达量均显著下调(P<0.05)；PIP5KI在2 dpi表达量显著上调(P<0.05)，在4 dpi、6 dpi和8 dpi的表达量均显著下调(P<0.05)。【结论】nce-miR-34537在东方蜜蜂微孢子虫孢子中真实存在和表达，东方蜜蜂微孢子虫的PIP5KI (EEQ82436)与蜜蜂微孢子虫的PIP5KI (EQB60549.1)之间亲缘关系最近，nce-miR-34537及其靶基因PIP5KI在东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中均表现出上升-下降的表达规律，nce-miR-34537通过正调控PIP5KI表达潜在参与调节侵染过程。

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.