Abstract
BackgroundBefore studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.ResultsThe experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene.ConclusionsThis is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.
Highlights
Before studying gene expression of different organisms, it is important to determine the best reference gene
Our research provided the best reference genes for Quantitative real time PCR (RT-qPCR) analysis of T. ciliata under different conditions, laying a foundation for studying molecular mechanisms in T. ciliata through gene expression analysis
Amplification efficiency, and expression profiles of candidate reference genes Reverse-transcribed complementary DNA (cDNA) from each sample was used as a template with primers for standard PCR amplification
Summary
Before studying gene expression of different organisms, it is important to determine the best reference gene. The most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR) With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. In previous research, it has been found that T. ciliata is very susceptible to the moth pest Hypsipyla robusta Moore [9] that eats mainly the young stems and causes the hollow branches to fail to grow and die in some cases. This pest is a regional issue in China, and a worldwide problem. Nor are there any literature references about the housekeeping genes in T. ciliata, which can be used for the standardization of gene expression
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