Reference Gene Validation for Quantitative PCR Under Various Biotic and Abiotic Stress Conditions in Toxoptera citricida (Hemiptera, Aphidiae)

Abstract

The regulation of mRNA expression level is critical for gene expression studies. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is commonly used to investigate mRNA expression level of genes under various experimental conditions. An important factor that determines the optimal quantification of qRT-PCR data is the choice of the reference gene for normalization. To advance gene expression studies in Toxoptera citricida (Kirkaldy), an important citrus pest and a main vector of the Citrus tristeza virus, we used five tools (GeNorm, NormFinder, BestKeeper, ΔCt methods, and RefFinder) to evaluate seven candidate reference genes (elongation factor-1 alpha [EF1α], beta tubulin [β-TUB], 18S ribosomal RNA [18S], RNA polymerase II large subunit (RNAP II), beta actin (β-ACT), alpha tubulin, and glyceraldhyde-3-phosphate dehydrogenase) under different biotic (developmental stages and wing dimorphism) and abiotic stress (thermal, starvation, and UV irradiation) conditions. The results showed that EF1α and 18S were the most stable genes under various biotic states, β-ACT and β-TUB during thermal stress, EF1α and RNAP II under starvation stress, and RNAP II, β-ACT, and EF1α under UV irradiation stress conditions. This study provides useful resources for the transcriptional profiling of genes in T. citricida and closely related aphid species.

Keywords: agricultural pests; brown citrus aphid; normalization; quantitative reverse transcription PCR; reference gene.