Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae).

Shiwei Sun,Yafeng Gou,Zheng Wang,Xi Zhu,Qianqian Meng,Shengfeng Gao,Aiqin Liu,Joanna Chiu

doi:10.1093/jisesa/iez130

Abstract

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.

Highlights

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
The guidelines should ensure the selection of reference genes to be transcribed are constitutively the same level in all samples, regardless of what developmental stages, tissues or other experimental conditions are used, multiple reference genes were strongly recommended to be employed for normalization (Appukuttan et al 2018, Shakeel et al 2018)
In developmental stages, the maximum and minimum dispersion of Ct values were identified for 7.43 cycles of Glucose-6-phosphate 1-dehydrogenase (G6PDH) and 2.72 cycles of β-TUB (Fig. 2A)

Summary

Introduction

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. In many studies of gene functional analysis, housekeeping genes including actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), elongation factor 1 alpha (EF1α), and β-tubulin (TUB) have been utilized heavily as the internal control in many different species without proper validation. To avoid misinterpretation of gene expression data, it is essential to check the expression stability of reference genes before normalizing target gene expression (Lu et al 2015, Shakeel et al 2015)

Methods

Results

Conclusion