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)
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)
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DOI：
10.16380/j.kcxb.2021.04.001
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发表时间：
2021-04-20
期刊：
影响因子：
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通讯作者：
Li Dan-Dan
中科院分区：
文献类型：
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作者：
Ma Tian-Tian;Yang Zong-Lin;Li Dan-Dan
[Aim]蜕皮激素在孤雌生殖蚜虫翅的二型性中起重要作用。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】筛选出5个与A.选择豌豆。孤雌生殖A.用0.1mol/L蜕皮激素类似物20-羟基蜕皮激素（20 E）分别处理豌豆10 min和30 min，于处理后48 h取样。采用qPCR检测20 E处理后5种miRNA及其预测靶基因的表达水平。通过双荧光素酶活性测定验证了miR-92 a-1-p5的预测靶基因flightin。最后，研究了miR-92 a-1-p5在孤雌生殖四龄若虫中的表达。目的：探讨miR-92 a-1-p5与其预测的靶基因flightin的相互作用。【结果】5种miRNA在孤雌生殖的拟除虫菊酯若虫2龄若虫中的表达量均为0。0.1mol/L20 E处理豌豆2龄若虫30 min后，miR-92 a-1-p5和miR-92 b的表达量极显著低于对照，而Let-7和miR-277的表达量极显著高于对照。Let-7基因及其预测的目的基因突变体在孤雌生殖的2龄若虫中的表达趋势相反。20 E处理后的豌豆。预测的miR-92 a和miR-277的靶基因wingless和Uba 1在孤雌生殖2龄若虫中的表达量极显著下降。0.1mol/L20 E作用10 min后，豌豆中miRNA的表达变化趋势与相应的两种miRNA相反。miR-92 a-1-p5的预测靶基因flightin在孤雌生殖的2龄若虫中的表达量极显著下降。0.1 mol/L 20 E作用30 min后，miR-92 a-1-p5的表达趋势与miR-92 a-1-p5的表达趋势相反。双荧光素酶活性测定结果显示，在miR-92 a-1-p5的模拟物和flightin CDS过表达载体pmirGlO [flightin]的共转染后，与用NC模拟物转染的对照相比，荧光素酶活性极显著地降低了40%。在孤雌生殖的四龄若虫中敲除miR-92 a-1-p5的表达。豌豆极显著降低了其表达83%，而极显著提高了其预测靶基因flightin的表达48%，证实了flightin是miR-92 a-1-p5的靶基因。[结论]蜕皮激素可以诱导拟南芥miRNA的表达。豌豆miR-92 a-1-p5可能通过调控flightin基因参与孤雌生殖蚜虫翅膀的分化。纳米载体/去污剂可以有效地干扰蚜虫体内的miRNA。本研究为进一步探讨蜕皮激素和miRNA在孤雌生殖蚜虫翅膀分化中的相互作用奠定了基础。
[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.