有翅和无翅豌豆蚜中翅型分化信号通路相关微小RNA及其靶基因的表达差异

【目的】前期研究发现麦长管蚜Sitobion avenae孤雌蚜有翅和无翅个体中存在很多差异表达的微小RNA(microRNA, miRNA)，本研究旨在进一步明确这些miRNA在豌豆蚜Acyrthosiphon pisum中发挥作用的发育阶段，探索miRNA调控孤雌蚜翅两型性分化的机制。【方法】选择在麦长管蚜有翅蚜和无翅蚜中显著差异表达，且靶基因为蜕皮激素、胰岛素信号通路及翅型发育关键基因的5个miRNA(Let-7,miR-92a, miR-92b, miR-92a-1-p5和miR-277)，利用qPCR检测这些miRNA及其靶标基因在豌豆蚜3-4龄若蚜和成虫有翅和无翅个体中的表达谱；同时利用双荧光素酶活性检测法对上述miRNA的靶基因进行验证。【结果】表达谱分析发现，这5个miRNA在豌豆蚜成虫中表达量均高于其在若蚜中的表达量，而其预测的靶基因在4龄若蚜中的表达量均高于其在成虫中的表达量，表明miRNA对其靶基因的调控作用可能集中在成虫阶段。分析豌豆蚜有翅和无翅个体中5个miRNA的表达情况发现，在成虫有翅个体中5个miRNA的表达量均高于无翅个体中的，其中miR-277表达差异最显著，成虫有翅个体中的表达量是无翅个体中表达量的7.5倍;其次为Let-7，表达差异达3倍。而Let-7在3龄有翅若蚜和无翅若蚜中表达差异最显著，有翅个体中的表达量是无翅个体中的37.8倍;其次为miR-277，表达差异达7.6倍。比较5个miRNA与其靶基因在豌豆蚜3-4龄若蚜及成虫有翅和无翅个体中的表达发现，miRNA Let-7和miR-92b的表达趋势分别与其靶基因abrupt和Foxo的基本相反。荧光素酶活性检测结果显示，Let-7的真实靶标为abrupt，共转染Let-7模拟物后与对照相比，荧光素酶活性下降53%，达极显著水平。其他miRNA与靶标基因的互作不显著。【结论】首次发现miRNA对豌豆蚜孤雌蚜翅型分化相关基因的调控可能发生在成虫阶段。Let-7可能通过调控abrupt基因参与孤雌蚜翅型分化。该研究为进一步探索miRNA参与孤雌蚜翅两型性分化的机制奠定了基础。

Differences in the expression of microRNAs related to wing-morph differentiation and theirtarget genes in the winged and wingless pea aphid,Acyrthosiphon pisum (Hemiptera:Aphididae)

【Aim】 Previous studies revealed that many microRNAs (miRNAs) showeddifferential expression in the winged and wingless individuals of Sitobion avenae. Thisstudy aims to further clarify the exact stage, during which these miRNAs play roles, and tostudy the functions of miRNAs in the wing dimorphism of the parthenogenetic pea aphid,Acyrthosiphon pisum. 【Methods】 Five miRNAs (Let-7, miR-92a, miR-92b, miR-92a-1-p5and miR-277) with significantly differential expression levels between winged and winglessindividuals of S. avenae and their target genes related to ecdysone, insulin signalingpathway and wing development were selected, and the expression profiles of the miRNAs andtheir target genes in the 3rd-4th instar nymphs and adults of A. pisum as well as in wingedand wingless individuals were detected by qPCR. The above target genes of miRNAs were alsoverified by dual luciferase activity assay. 【Results】 The expression profiling resultsindicated that the expression levels of the five miRNAs in adults of A. pisum were higherthan those in nymphs, but the expression levels of the predicted target genes in the 4thinstar nymphs were higher than those in adults, suggesting that miRNAs might playregulatory roles on their target genes in the adult stage. The expression levels of fivemiRNAs in winged individuals were higher than those in wingless ones of adults, especiallyfor the miR-277, whose expression level in winged individuals was 7.5 times as high asthat in wingless individuals of adults, then followed by Let-7 with three fold expressiondifference. Moreover, the expression difference of Let-7 between winged and winglessindividuals of the 3rd instar nymphs was the most significant, its expression level in thewinged individuals being 37.8 times as high as that in wingless ones, then followed by miR-277 with 7.6-fold expression difference. The expression patterns of predicted targetgenes abrupt and Foxo in the 3rd-4th instar nymphs and winged and wingless individuals ofadults of A. pisum were opposite to those of miRNA Let-7 and miR-92b, respectively.Luciferase activity assay results showed that the target gene of Let-7 was abrupt, and theluciferase activity was extremely significantly decreased by 53% compared to the controlafter co-transfection with the mimics of Let-7. The interaction of other miRNAs withtheir target genes was not obvious. 【Conclusion】 We first found that the regulation ofmiRNAs on genes related to wing-morph differentiation of parthenogenetic A. pisum mightoccur during the adult stage. Let-7 may participate in the wing-morph differentiation ofparthenogenetic aphids by regulating abrupt gene. This study lays a foundation for furtherexploring the mechanism of miRNAs participating in the wing dimorphism of parthenogeneticaphids.