在发育激素拮抗剂对森蛙胚后发育影响的节律性基因表达研究中,鉴定用于RT-qPCR数据归一化的特定参考基因。

IF 2.4 Q1 ENTOMOLOGY
Frontiers in insect science Pub Date : 2024-06-28 eCollection Date: 2024-01-01 DOI:10.3389/finsc.2024.1362473
Minurani Dalai, Anita Jagota
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引用次数: 0

摘要

褐飞虱是一种鳞翅目全代谢昆虫,具有不同的发育阶段:卵、幼虫、蛹和成虫。鳞翅目昆虫的中枢神经系统(CNS)发生了重大变化,以适应这些具有特定栖息地和功能的不同阶段的生活方式,从贪婪觅食的幼虫阶段到通过休眠蛹阶段飞行繁殖的成虫阶段。这种转变与转录、表观遗传和翻译的复杂性有关。因此,研究不同发育阶段中枢神经系统的节律性基因表达以及拮抗剂对发育激素的影响需要非常稳定的参考基因(RG)。为了便于使用反转录定量聚合酶链式反应(RT-qPCR)研究森蛙的节律性基因表达以及发育激素幼年激素(JH)和 20-羟基蜕皮激素(20 HE)的影响,研究人员分别使用了拮抗剂 Precocene 1 和睾酮。评估了八种候选 RG,即翻译起始因子 3 亚基 4(TI3S4)、翻译起始因子 3 亚基 5(TI3S5)、核糖体蛋白亚基 7(RPs7)、TATA 结合蛋白关联因子(TAF13)、翻译起始因子 4 A(TI4A)、核糖体蛋白(RPL32)、延伸因子 1(EF1)和精氨酸激酶(AK)在 B. mori 中枢神经系统中的作用。mori。所使用的胚后发育(PED)阶段为第五期晚幼虫、早期蛹、中期蛹、晚期蛹和成虫。评估在四个不同的时间点(Zeitgeber 时间(ZT)0、6、12 和 18)进行,以发现 24 小时节律表达的稳定性。进行了 RefFinder、geNORM 和 Ct 值分析。RefFinder 和 geNORM 研究表明稳定性顺序为 TI3S4 > TI3S5 > RPs7,但 Ct 值评估显示稳定性顺序为 TI3S5 > TI3S4 > RPs7。因此,我们证明了 TI3S4、TI3S5 和 RPs7 可用作森蝶(品系:CB-杂交,PM×CSR2)中枢神经系统中不同 PED 阶段的 RG,用于 JH 和 20 种 HE 拮抗剂作用的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Identification of specific reference gene for normalization of RT-qPCR data in rhythmic gene expression studies of the effect of developmental hormone antagonist in postembryonic development in Bombyx mori.

Bombyx mori is a lepidopteran holometabolous insect with distinct developmental stages: egg, larvae, pupae, and adult. The lepidopteran insect undergoes major modifications in the central nervous system (CNS) so as to adapt to the lifestyle of these distinct stages with specific habitats and functions from voraciously feeding larval stages to flying reproductive adults via dormant pupal stages. Such transitions are linked to transcriptional, epigenetic, and translational complexities. Therefore, studying rhythmic gene expression in CNS of various developmental stages and the effects of antagonists on developmental hormones requires a very stable reference gene (RG). To facilitate rhythmic gene expression studies using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in B. mori and the effect of developmental hormone juvenile hormone (JH) and 20-hydroxy ecdysone hormone (20 HE), antagonists Precocene 1 and testosterone, respectively, were used. Eight candidate RGs, namely, Translational initiation factor 3 subunit 4 (TI3S4), Translational initiation factor 3 subunit 5 (TI3S5), Ribosomal protein subunit 7 (RPs7), TATA-binding protein association factor (TAF13), Translational initiation factor 4 A (TI4A), Ribosomal protein (RPL32), Elongation factor 1 (EF1), and Arginine kinase (AK), were assessed in the CNS of B. mori. The postembryonic developmental (PED) stages used were the fifth late larval instar, early pupa, mid pupa, late pupa, and adult. The assessments were done at four different time points, Zeitgeber time (ZT) 0, 6, 12, and 18, to find stability towards 24-h rhythmic expression. RefFinder, geNorm, and Ct value analysis were performed. RefFinder and geNORM studies suggested stability order as TI3S4 > TI3S5 > RPs7, but Ct value evaluation showed stability order as TI3S5 > TI3S4 > RPs7. We therefore demonstrated that TI3S4, TI3S5, and RPs7 can be used as RG in various PED stages in CNS of B. mori (Strain: CB-hybrid, PM×CSR2) towards studies with effects of JH and 20 HE antagonists.

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