Evaluation and selection of suitable qRT-PCR reference genes for light responses in tea plant (Camellia sinensis)

Abstract

Background: Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages. Tea has been proved to be bene cial to human health because it is rich in tea polyphenols and other active ingredients. Numerous studies have shown that light is a necessary environmental condition to control the growth and metabolism of C. sinensis. 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 speci c conditions. However, the reference genes for systematic analysis of light-induced transcription mechanisms are still not available in C. sinensis.Results: In this research, we identi ed 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.Conclusions: 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. Moreover, the analysis of actin family members of C. sinensis will help to understand the individual transcription mechanism of housekeeping family. BackgroundQuantitative 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]. In the process of using qRT-PCR to analyze gene expression, the purity and concentration of the sample cannot be absolutely guaranteed that may cause experimental errors [2,3]. In order to make the results more accurate, it is necessary to use the reference gene as a reference to standardize the expression level of the target gene and correct the discrepancy of transcription e ciency and cDNA usage [2,4]. Stable expression is the basis for selection of reference genes because of the selection of unstable reference genes will lead to test deviation. 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]. The housekeeping genes o...

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