昆虫学报 ›› 2021, Vol. 64 ›› Issue (4): 419-427.doi: 10.16380/j.kcxb.2021.04.001

• 研究论文 •    下一篇

豌豆蚜翅分化相关miRNA及其预测靶基因对蜕皮激素的应答及miR-92a-1-p5靶基因的验证

马田田, 杨宗霖, 常美玲, 霍春月, 阚云超, 李丹丹*   

  1.  (南阳师范学院, 河南省伏牛山昆虫生物学重点实验室, 河南南阳 473061)
  • 出版日期:2021-04-20 发布日期:2021-04-25

Response of miRNAs related to wing differentiation and their predicted target genes to ecdysone and the confirmation of target gene of miR-92a-1-p5 in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphidae)

MA Tian-Tian, YANG Zong-Lin, CHANG Mei-Ling, HUO Chun-Yue, KAN Yun-Chao, LI Dan-Dan*   

  1.  (Henan Key Laboratory of Insect Biology in Funiu Mountain, Nanyang Normal University, Nanyang, Henan 473061, China)
  • Online:2021-04-20 Published:2021-04-25

摘要:

【目的】蜕皮激素对孤雌蚜翅两型性分化具有重要调控作用。在前期研究中我们发现5个微小RNA(microRNA, miRNA)在豌豆蚜Acyrthosiphon pisum翅两型性分化中也发挥关键作用,但蜕皮激素是否与miRNA互作参与蚜虫翅型分化未知。本研究旨在探索蜕皮激素对5个miRNA及其预测靶基因表达的影响,揭示蜕皮激素与miRNA协同调控蚜虫翅型分化的机理。【方法】选择5个与豌豆蚜翅两型性分化相关的miRNA (Let-7, miR-92a, miR-92b, miR-92a-1-p5和miR-277),使用0.1 mol/L蜕皮激素类似物20-羟基蜕皮酮(20E)分别处理孤雌生殖豌豆蚜2龄若蚜10 min和30 min,48 h后取样;qPCR检测这些miRNA及其预测靶基因在20E处理后的表达量;利用双荧光素酶活性检测法对miR-92a-1-p5的预测靶基因flightin进行验证;利用纳米载体/变性剂在孤雌生殖豌豆蚜4龄若蚜中敲低miR-92a-1-p5表达,验证miR-92a-1-p5与其预测靶基因flightin的互作关系。【结果】0.1 mol/L 20E处理30 min可极显著诱导孤雌生殖豌豆蚜2龄若蚜5个miRNA的表达;处理10 min则miR-92a-1-p5和miR-92b表达量较对照极显著下降,而Let-7和miR-277较对照极显著上升。Let-7与预测靶基因abrupt在0.1 mol/L 20E处理后孤雌生殖豌豆蚜豌2龄若蚜中表达趋势相反,miR-92a和miR-277的预测靶基因winglessUba1在0.1 mol/L 20E处理10 min时表达量极显著下降,与两个对应的miRNA表达趋势相反;miR-92a-1-p5的预测靶基因flightin在0.1 mol/L 20E处理30 min后表达量极显著下降,与miRNA表达趋势相反。双荧光素酶活性检测结果显示,共转染miR-92a-1-p5模拟物和flightin CDS过表达载体pmirGlOflightin]后与转染对照NC mimics相比,荧光素酶活性下降40%,达极显著水平;敲低孤雌生殖豌豆蚜4龄若蚜中miR92a1p5后其表达量下调83%,其预测靶基因flightin表达量上升48%,均达极显著水平,表明flightin是miR-92a-1-p5的真实靶基因。【结论】蜕皮激素能够诱导豌豆蚜体内miRNA的表达;miR-92a-1-p5可能通过调控flightin参与孤雌蚜翅型分化;纳米载体/变性剂可以在蚜虫个体中实现miRNA的有效干涉。该研究为进一步探索蜕皮激素和miRNA互作参与孤雌蚜翅两型性分化机制奠定了基础。

关键词: 豌豆蚜, 翅型分化, 蜕皮激素, miRNA, 靶基因

Abstract:
 Abstract: 【Aim】 Ecdysone plays important roles in wing dimorphism of the parthenogenetic aphid. In the previous study we found that five microRNAs (miRNAs) also play pivotal roles in the wing dimorphism of the pea aphid, Acyrthosiphon pisum, but whether ecdysone and miRNAs have interaction in wing differentiation of aphids is unclear. This study aims to explore the effect of ecdysone on the expression of miRNAs and their predicted target genes, and to reveal the interaction of ecdysone and miRNAs in wing differentiation of aphids. 【Methods】 The five miRNAs (Let-7, miR-92a, miR-92b, miR-92a-1-p5 and miR-277) related to the wing dimorphism of A. pisum were selected. The 2nd instar nymphs of parthenogenetic A. pisum were exposed to 0.1 mol/L ecdysone analog 20-hydroxyecdysone (20E) for 10 min and 30 min, respectively, and sampled at 48 h after treatment. The expression levels of the five miRNAs and their predicted target genes after 20E treatment were detected by qPCR. The predicted target gene of miR-92a-1-p5, flightin, was verified by dual luciferase activity assay. Finally, the expression of miR-92a-1-p5 in the 4th instar nymphs of parthenogenetic A. pisum was knocked down with nanocarrier/detergent to verify the interaction between miR-92a-1-p5 and its predicted target gene flightin. 【Results】 The expression of the five miRNAs in the 2nd instar nymphs of parthenogenetic A. pisum could be extremely significantly induced by the treatment of 0.1 mol/L 20E for 30 min. But when the 2nd instar nymphs were exposed to 0.1 mol/L 20E for 10 min, the expression levels of miR-92a-1-p5 and miR-92b extremely significantly decreased, while those of Let-7 and miR-277 increased extremely significantly compared with the control. The expression trends of Let-7 and its predicted target gene abrupt were opposite in the 2nd instar nymphs of parthenogenetic A. pisum after 20E treatment. The expression levels of wingless and Uba1, which are the predicted target genes of miR-92a and miR-277, respectively, decreased extremely significantly in the 2nd instar nymphs of parthenogenetic A. pisum exposed to 0.1 mol/L 20E for 10 min, showing the opposite trend with those of the corresponding two miRNAs. The expression level of flightin, the predicted target gene of miR92a-1-p5, decreased extremely significantly in the 2nd instar nymphs of parthenogenetic A. pisum exposed to 0.1 mol/L 20E for 30 min, exhibiting an opposite expression trend to that of miR-92a-1-p5. Dual luciferase activity assay results showed that after co-transfection of the mimics of miR-92a-1-p5 and the flightin CDS overexpression vector pmirGlO [flightin] the luciferase activity was extremely significantly decreased by 40% compared to the control transfected with NC mimics. Knocking down the expression of miR-92a-1-p5 in the 4th instar nymphs of parthenogenetic A. pisum extremely significantly decreased its expression by 83%, while extremely significantly enhanced the expression of its predicted target gene flightin by 48%, confirming that flightin is the target gene of miR-92a-1-p5. 【Conclusion】 Ecdysone can induce the expression of miRNAs in A. pisum. miR-92a1-p5 may be involved in wing differentiation of parthenogenetic aphids by regulating flightin gene. Nanocarrier/detergent can achieve effective miRNA interference in aphids. This study lays a foundation for further exploring the interaction of ecdysone and miRNAs in wing differentiation of parthenogenetic aphids.

Key words: Acyrthosiphon pisum, wing differentiation, ecdysone, miRNA, target gene