Chrysoperla nipponensis (Okamoto), which has the unique diapause phenotype distinguishable from nondiapause adult, is an ideal model organism for studying the mechanism of reproductive diapause. However, there is no reliable and effective reference genes used for the reproductive diapause study of C. nipponensis. Therefore, in this study, we evaluated the expression stability of 10 candidate reference genes (Tub1, Arpc5, EF1a, 128up, RpS5, RpS26e, GAPDH, Arp3, Actin, α-Tub) in adults under diapause and nondiapause induction conditions using four statistical algorithms including GeNorm, NormFinder, Bestkeeper, and ∆CT method. Results showed that Arp3 and Tub1 were the most stable reference genes in all samples and in the adult tissues group. Arp3 and RpS5 were the most stable reference genes in the development degree group. α-Tub and EF1a were unstable reference genes under the conditions of this study. Meanwhile, to verify the reliability of the reference genes, we evaluated the relative expression levels of Vg and VgR in different treatments. Significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was, respectively, observed when using Arp3 as the reference gene but not when using an unstable reference gene. The reference genes identified in this work provided not only the basis for future functional genomics research in diapause of C. nipponensis and will also identify reliable normalization factors for real-time quantitative real-time polymerase chain reaction data for other related insects.

中文翻译：

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在繁殖和滞育下的组织下，日本Chrysoperla nipponensis（Neuroptera：Chrysopidae）实时定量PCR参考基因的选择。

日本Chrysoperla nipponensis（冈本）具有独特的滞育表型，可以区别于非滞育成年人，是研究生殖滞育机制的理想模型生物。然而，用于生殖滞育的研究没有可靠和有效的参考基因C.青虾。因此，在这项研究中，我们评估了10个候选参考基因（Tub1，Arpc5，EF1a，128up，RpS5，RpS26e，GAPDH，Arp3，肌动蛋白，α- Tub的表达稳定性）在包括滞育和非滞育诱导条件下的成年人中，使用GeNorm，NormFinder，Bestkeeper和∆CT方法等四种统计算法。结果表明，Arp3和Tub1是所有样品和成人组织组中最稳定的参考基因。Arp3和RpS5是发育程度组中最稳定的参考基因。在本研究条件下，α- Tub和EF1a是不稳定的参考基因。同时，为了验证参考基因的可靠性，我们评估了Vg和VgR的相对表达水平在不同的治疗方法。使用Arp3作为参考基因时，分别观察到滞育终止和滞育脂肪体组织中两个基因表达水平的显着上调和下调，而使用不稳定的参照基因时则没有观察到。这项工作中确定的参考基因不仅为今后日本滞留念珠菌的功能基因组学研究提供了基础，还将为其他相关昆虫的实时定量实时聚合酶链反应数据确定可靠的归一化因子。