Abstract
Reference genes are the key to study gene expression patterns using quantitative real-time PCR (qRT-PCR). No studies on the reference genes of Athetis dissimilis, an important agricultural pest, have been reported. In order to determine the reference genes for qRT-PCR normalization in A. dissimilis under different conditions, 10 candidate genes [18S ribosomal protein (18S), 28S ribosomal protein (28S), arginine kinase (AK), elongation factor 1 alpha (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L32 (RPL32), ribosomal protein L40 (RPL40), alpha-tubulin (α-TUB), beta-actin (β-ACT), and beta-tubulin (β-TUB)] of A. dissimilis were selected to evaluate their stability as reference genes under different biotic and abiotic conditions by using five tools, geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. Furthermore, CSP1 and superoxide dismutase (SOD) were used as target genes to validate the candidate reference genes. The results showed that different reference genes were needed under different experimental conditions, among which, EF-1α, RPL40, and 18S are most suitable reference genes for studying genes related development stages of A. dissimilis, RPL40 and α-TUB for larval tissues, α-TUB and 28S for adult tissues, EF-1α and β-ACT for insecticidal treatments, β-ACT and 28S for temperature treatments, EF-1α and β-ACT for starvation treatments, RPL40 and 18S for dietary treatments, and 18S, 28S, and α-TUB for all the samples. These results provide suitable reference genes for studying gene expression in A. dissimilis under different experimental conditions, and also lay the foundation for further research into the function of related genes in A. dissimilis.
Highlights
In molecular biological research, gene expression analyses provide the information concerning gene regulatory mechanisms and functions associated with different biological processes (Zhao et al, 2018)
The β-ACT, ribosomal protein L32 (RPL32), ribosomal protein L40 (RPL40), EF1-α, α-TUB, β-TUB, 18S ribosomal protein (18S), 28S, and arginine kinase (AK) genes in A. dissimilis were cloned as described below using the primers listed in Supplementary Table S1, and the sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
All the melting curves showed single dissociation peaks (Supplementary Figure S3), which confirmed that the primers designed for the selected reference genes were highly specific and could be used for further quantitative realtime PCR (qRT-PCR) analyses
Summary
Gene expression analyses provide the information concerning gene regulatory mechanisms and functions associated with different biological processes (Zhao et al, 2018). It is important to evaluate the validity of the candidate reference gene before qRT-PCR tests (Radonic et al, 2004; Huggett et al, 2005; Fleige and Pfaffl, 2006; Guenin et al, 2009; Xie et al, 2021). An ideal reference gene should have steady expression levels under all the experimental conditions (Brym et al, 2013; Janska et al, 2013). The expression profiles of the widely used reference genes are not always constant under different experimental conditions (Selvey et al, 2001; Glare et al, 2002; Radonic et al, 2004). Numerous studies have demonstrated that even the same reference gene exhibited different expression levels under different experimental conditions, such as organismal developmental stages (Sun et al, 2009; Nakamura et al, 2016), tissues (Huis et al, 2010), cells (Nelissen et al, 2010), and temperatures (Mahanty et al, 2017). It is crucial to identify suitable reference genes and evaluate their expression stability in certain target species under specific experimental conditions before the qRT-PCR data normalization of target gene expression levels using the reference genes (Guo et al, 2016; Wan et al, 2017; Renard et al, 2018)
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