Abstract
Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, convenient, and widely used methods for detecting gene expression in different biological samples [1,2,3]
The accuracy of the RT-qPCR primers was confirmed by the presence of a single peak in the melting curve analyses (Figure S2) and a single amplicon on 1% agarose gel
Our results indicate that EF1-α, RPS13, and RPL27 had the greatest stability across various developmental stages, while RPS13 and TATA binding protein (TBP) had the greatest stability across various adult tissues of T. absoluta
Summary
Real-time quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, convenient, and widely used methods for detecting gene expression in different biological samples [1,2,3]. It is the most reliable method for detecting and quantifying transcriptional abundance [4]. Many studies have demonstrated that the stability of different reference genes can change in response to different experimental conditions [9,10]. Using different normalization reference genes can lead to significant errors due to differences in sampling methods, total RNA extraction, efficiency of complementary DNA (cDNA)
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