东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中nce-miR-12220及其靶基因的表达谱

Expression profiles of nce-miR-12220 and its target genes during the Nosema ceranae infection process of Apis mellifera ligustica workers

东方蜜蜂微孢子虫Nosema ceranae侵染成年蜜蜂导致蜜蜂微孢子虫病。本研究旨在验证东方蜜蜂微孢子虫nce-miR-12220的存在和表达,并检测nce-miR-12220及其靶基因在病原侵染意大利蜜蜂Apis mellifera ligustica(简称意蜂)工蜂过程的表达谱。Stem-loop RT-PCR和Sanger测序结果显示nce-miR-12220真实存在和表达。靶向预测结果显示nce-miR-12220共靶向KRAB-A和γtubulin等15个基因。上述靶基因可注释到19个GO条目和3条KEGG通路。RT-qPCR结果显示,相较于接种后1 d(1 day post infection,1 dpi),nce-miR-12220在2 dpi上调表达,而在3–12 dpi阶段总体表现出显著下调表达的趋势。类似地,与1 dpi相比,靶基因KRAB-A在2 dpi上调表达,而在3–12 dpi阶段总体呈下调表达的趋势。另外,与1dpi相比,靶基因γtubulin在2–12dpi阶段总体表现出显著下调表达的趋势。上述结果表明nce-miR-12220与KRAB-A和γtubulin之间存在潜在的靶向结合和正向调控关系;东方蜜蜂微孢子虫通过下调表达nce-miR-12220抑制KRAB-A的表达进而促进增殖;意蜂工蜂可能通过抑制东方蜜蜂微孢子虫的γtubulin表达抵御病原侵染。研究结果明确了nce-miR-12220及其靶基因KRAB-A和γtubulin在东方蜜蜂微孢子虫侵染意蜂工蜂过程中的动态表达规律,为深入探究nce-miR-12220在病原侵染中的功能及调控机制提供了理论和实验依据。

Nosema ceranae infects adult bee and results in nosemosis.This study aims at verifying the existence and expression of nce-miR-12220 and detecting the expression profiles of nce-miR-12220 and its target genes during the N.ceranae infection of Apis mellifera ligustica workers.Stem-loop RT-PCR and Sanger sequencing confirmed the true existence and expression of nce-miR-12220.Targeting prediction showed that nce-miR-12220 can target a total of 15 genes.These targets could be annotated to 19 GO terms and 3 KEGG pathways,respectively.RT-qPCR test indicated that compared with nce-miR-12220 at 1 day post infection(1 dpi),nce-miR-12220was up-regulated at 2 dpi and significantly down-regulated during the stage of 3–12 dpi.Similarly,in comparison with KRAB-A at 1 dpi,the target gene KRAB-A was up-regulated at 2 dpi and down-regulated during the stage of 3–12 dpi.The target geneγtubulin presented an overall significantly down-regulated trend during the stage of 2–12 dpi as compared with that at 1 dpi.These results suggest that there is potential target binding and positive regulation relationship between nce-miR-12220 and KRAB-A andγtubulin;N.ceranae likely suppressed the expression of KRAB-A by down-regulating nce-miR-12220,further promoting its own proliferation;A.m.ligustica workers probably resist N.ceranae invasion through inhibiting theγtubulin expression.The findings not only reveal the dynamic expression rule of nce-miR-12220 and target genes KRAB-A andγtubulin during the N.ceranae infection of A.m.ligustica workers,but also provide theoretical and experimental bases for further investigating the function and regulatory mechanism of nce-miR-12220 in the infection process.