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

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γ tubulin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1 dpi相比,靶基因γ tubulin在2-12 dpi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γ tubulin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γ tubulin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γ tubulin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。

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

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-12220 was 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 gene γ tubulin 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 and γ tubulin; 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 the γ tubulin expression. The findings not only reveal the dynamic expression rule of nce-miR-12220 and target genes KRAB-A and γ tubulin 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.