Abstract
Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages. Numerous studies have shown that light is a necessary environmental condition to control the growth and metabolism of C. sinensis. However, the reference genes of quantitative real time polymerase chain reaction (qRT-PCR) for systematic analysis of light-induced transcription mechanisms are still not available in C. sinensis. In this research, we identified actin family genes that are always used as reference genes with high frequency and without distinction for various expression experiments in C. sinensis. Six pairs of distinctive primers (corresponding to CsACT1, CsACT2, CsACT(3-4), CsACT(5-6), CsACT(7-8), and CsACT(9-10) genes) were designed to evaluate their expression stability in response to light quality (LQ), light intensity (LI), and photoperiod (PD). Simultaneously, six other family members (CsUBC1, CsCLATHRIN1, CsGAPDH, CsTBP, CsTIP41, and CseIF-4α) of C. sinensis commonly used as reference genes were also investigated. The stability rankings of gene expression were calculated by the statistical algorithms of geNorm, BestKeeper, NormFinder, and RefFinder softwares. CsACT(5-6), CsTIP41, and CsACT(3-4) were the most stable genes for light quality (LQ), light intensity (LI), and photoperiod (PD) treatments, respectively. This study provides a basis for the selection of reference genes for future research on the transcription mechanism of light response in C. sinensis. The study on the expression stability of individual members of housekeeping gene family will help to guide the accurate design of detection primers and clarify transcription mechanism in expression experiments.
Highlights
Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages
This study provides a basis for the selection of reference genes for future research on the transcription mechanism of light response in C. sinensis
Thirteen homologous actin genes were obtained from the C. sinensis genome using Arabidopsis thalianaAtACT1 gene (AT2G37620.1) as the retrieval sequence (Additional file 1: Table S1)
Summary
Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages. Gene expression experiments are always performed to explore the transcriptional regulation mechanism of plants widely based on the technique of quantitative real time polymerase chain reaction (qRT-PCR). The screening and application of reference genes are necessary for the normalization of gene expression under specific conditions. Quantitative real time polymerase chain reaction (qRT-PCR) is a universal technique to analyze gene expression because of its high sensitivity, good repeatability, and fast speed [1, 2]. Some genes necessary for maintaining basic cell functions have been found in plants, called housekeeping genes, whose expression levels are less affected by environmental conditions and developmental stages [5, 6]. It is necessary to select the reference genes for specific conditions to ensure the accuracy in qRT-PCR experiments
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