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Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae).
Journal of Insect Science ( IF 2.2 ) Pub Date : 2020-01-01 , DOI: 10.1093/jisesa/iez130
Zheng Wang 1 , Qianqian Meng 1 , Xi Zhu 1, 2 , Shiwei Sun 1 , Aiqin Liu 1, 2 , Shengfeng Gao 1 , Yafeng Gou 1
Affiliation  

Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

中文翻译:

鉴定和评估参考基因,以使Caesalis Diaphania(Lepidoptera,Pyralidae)在发育阶段,性别和组织中的基因表达正常化。

盲es(Walker)是一种重要的无聊昆虫,主要分布于亚热带和热带地区,并袭击了热带木纹作物,例如面包果的淀粉状植物。实时定量聚合酶链反应(qRT-PCR)是一种在转录水平上研究目标基因表达谱的强大方法。然而,在通过qRT-PCR分析靶基因表达之前,内部参考基因的鉴定和选择通常是被忽略的,这是最重要的步骤。到目前为止,还没有研究在某些条件下可靠的内部参照基因。因此,本研究评估了八个候选参考基因在不同发育阶段的表达稳定性,包括ACT,β-TUB,GAPDH,G6PDH,RPS3a,RPL13a,EF1α和EIF4A,使用geNorm,NormFinder和BestKeeper算法处理组织和性别。为了验证推荐的内部参考基因的稳定性,在不同治疗条件下分析了DcaeOBP5的表达水平。结果表明,ACT,RPL13a,β-TUB,RPS3a和EF1α被认为是最稳定的参考基因,可用于进一步研究ca菜不同发育阶段的靶基因表达。ACT和EIF4A被推荐为不同组织的稳定参考基因。此外,根据三种算法,ACT,EF1α和RPS3a被评为不同性别的最佳参考基因。
更新日期:2020-01-01
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