Bombyxmori是一种鳞翅目全代谢昆虫,具有不同的发育阶段:卵,幼虫,蛹,和成人。鳞翅目昆虫在中枢神经系统(CNS)中经历了重大变化,以适应具有特定栖息地和功能的这些不同阶段的生活方式,从贪婪地喂养幼虫阶段到通过休眠p飞行的成虫。这种转换与转录相关,表观遗传,和翻译的复杂性。因此,研究中枢神经系统各个发育阶段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)。为了促进使用逆转录定量聚合酶链反应(RT-qPCR)进行的有节奏的基因表达研究,以及发育激素保幼激素(JH)和20-羟基蜕皮激素(20HE)的作用,拮抗剂Precocene1和睾酮,分别,被使用。八个候选RGs,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结合蛋白相关因子(TAF13),平移启动因子4A(TI4A),核糖体蛋白(RPL32),延长系数1(EF1),和精氨酸激酶(AK),在B.mori的中枢神经系统中进行了评估。使用的胚胎后发育(PED)阶段是第5个晚期幼虫龄,早期的蛹,中蛹,晚期蛹,和成人。评估是在四个不同的时间点进行的,Zeitgeber时间(ZT)0、6、12和18,以找到对24小时节律表达的稳定性。RefFinder,geNorm,并进行Ct值分析。RefFinder和geNORM研究建议稳定性顺序为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可以在B.moriCNS的各个PED阶段用作RG(菌株:CB-hybrid,PM×CSR2)对JH和20种HE拮抗剂作用的研究。
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.