东方蜜蜂微孢子虫侵染过程中nce-miR-15325及其靶基因NCCS和DTIII分析
Analysis of the nce-miR-15325 and its target genes NCCS and DTIII during the process of infection by Nosema ceranae
钱加珺，荆 欣，张佳欣，范小雪，张文德，吉 挺，蔺哲广，陈大福，郭 睿
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DOI：10.7679/j.issn.2095-1353.2023.075
作者单位：福建农林大学动物科学学院（蜂学学院），福州 350002；福建省蜂疗研究所，福州 350002
中文关键词：蜜蜂；意大利蜜蜂；东方蜜蜂微孢子虫；nce-miR-15325；靶基因；相对表达量
英文关键词：honey bee; Apis mellifera ligustica; Nosema ceranae; nce-miR-15325; target gene; relative expression
中文摘要：
【目的】 东方蜜蜂微孢子虫Nosema ceranae侵染蜜蜂导致微孢子虫病，但侵染机制未明。本研究拟在前期工作基础上，验证nce-miR-15325的相对表达量及序列信息，并进一步检测nce-miR-15325及其靶基因在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程的相对表达量，为进一步探究nce-miR-15325调控侵染的功能和机制提供依据。【方法】 利用Stem-loop RT-PCR和Sanger测序对nce-miR-15325进行相对表达量及序列信息验证。利用相关生物信息学软件对nce-miR-15325的靶基因进行信息预测及分析。通过RT-qPCR检测nce-miR-15325及其靶基因的相对表达量。【结果】 鉴定到在东方蜜蜂微孢子虫孢子中nce-miR-15325真实存在并表达。预测到nce-miR-15325的11个靶基因，其中分别有3、4、7和4个靶基因可注释到KEGG、GO、Nr和Swissprot数据库。与接种后1 d相比，nce-miR-15325在2 dpi（day post inoculation, dpi）的表达量变化不显著，而在4、6和8 dpi时，相对表达量均显著下调（P<0.05），总体表达趋势呈下调。相较于1 dpi，靶基因NCSS在2、4、6和8 dpi的表达量均显著下调（P<0.05）；类似的，靶基因DTIII在2、4、6和8 dpi的表达量均显著下调（P<0.05），总体上表现出下降的表达趋势。【结论】 证实了东方蜜蜂微孢子虫孢子中nce-miR-15325存在和表达的真实性，明确了在东方蜜蜂微孢子虫侵染过程的nce-miR-15325及其靶基因的动态表达模式，揭示nce-miR-15325通过正调控NCSS和DTIII表达潜在调节侵染过程。
英文摘要：
[Objectives] The infection of Nosema ceranae caused nosemiosis, but the mechanism of infection was unknown. Based on the previous work, this study was intended to further verify the expression level and sequencing results of nce-miR-15325, and to detect the relative expression levels of nce-miR-15325 and its target genes during the infection of Apis mellifera ligustica by Nosema ceranae. This study provided the basis for further exploring the function and mechanism of nce-miR-15325 in regulating infection [Methods] Stem-loop RT-PCR and Sanger sequencing were employed to confirm the expression of, and to sequence, nce-miR-15325. Related software was used to predict and analyze relevant target genes. The expression profiles of nce-miR-15325 and its target genes were determined using RT-qPCR. [Results] nce-miR-15325 was present and expressed in Nosema ceranae spores. Eleven target genes were predicted, of these, three, four, seven and four, respectively, were annotated to the KEGG, GO, Nr, and Swissprot databases. Compared to its expression at 1 dpi (1 day post-inoculation), the expression of nce-miR-23928 was unchanged at 2 dpi, but was progressively less at 4, 6, and 8 dpi (P<0.05). Compared to its expression at 1 dpi, the expression of the target gene NCCS was also progressively less (P<0.05) at 2, 4, 6 and 8 dpi, as was that of the target gene DTIII. [Conclusion] These results confirm the existence and expression of nce-miR-15325 in Nosema ceranae spores, reveal its expression dynamics and that of its target genes during Nosema ceranae infection, and suggest that nce-miR-15325 potentially regulates the infection process via positive modulation of the expression of NCSS and DTIII.