Abstract
The selection of reliable reference genes (RGs) for normalization under given experimental conditions is necessary to develop an accurate qRT-PCR assay. To the best of our knowledge, only a small number of RGs have been rigorously identified and used in tea plants (Camellia sinensis (L.) O. Kuntze) under abiotic stresses, but no critical RG identification has been performed for tea plants under any biotic stresses till now. In the present study, we measured the mRNA transcriptional levels of ten candidate RGs under five experimental conditions; these genes have been identified as stable RGs in tea plants. By using the ΔCt method, geNorm, NormFinder and BestKeeper, CLATHRIN1 and UBC1, TUA1 and SAND1, or SAND1 and UBC1 were identified as the best combination for normalizing diurnal gene expression in leaves, stems and roots individually; CLATHRIN1 and GAPDH1 were identified as the best combination for jasmonic acid treatment; ACTIN1 and UBC1 were identified as the best combination for Toxoptera aurantii-infested leaves; UBC1 and GAPDH1 were identified as the best combination for Empoasca onukii-infested leaves; and SAND1 and TBP1 were identified as the best combination for Ectropis obliqua regurgitant-treated leaves. Furthermore, our results suggest that if the processing time of the treatment was long, the best RGs for normalization should be recommended according to the stability of the proposed RGs in different time intervals when intragroup differences were compared, which would strongly increase the accuracy and sensitivity of target gene expression in tea plants under biotic stresses. However, when the differences of intergroup were compared, the RGs for normalization should keep consistent across different time points. The results of this study provide a technical guidance for further study of the molecular mechanisms of tea plants under different biotic stresses.
Highlights
The selection of reliable reference genes (RGs) for normalization under given experimental conditions is necessary to develop an accurate quantitative real-time polymerase chain reaction (qRT-PCR) assay
A few housekeeping genes have been rigorously identified and used as RGs in tea plants under abiotic stresses, such as cold, barrenness, drought, photoperiod and exogenous application of plant hormones[25,26,28,32,33,34], leaf developmental stages and even different organs[26,35]. These results demonstrate that identifying appropriate RGs for target gene expression analysis under different experimental conditions is an essential prerequisite for developing a qPCR assay of tea plants
To the best of our knowledge, the present study is the first to define the proper RGs for qRT-PCR analysis in tea plants under infestations of different herbivorous pests and their related biotic stresses
Summary
The selection of reliable reference genes (RGs) for normalization under given experimental conditions is necessary to develop an accurate qRT-PCR assay. Normalization scalar in studies of relative quantification of plant target genes, some of which (EF-1α, GAPDH, ACTIN) have been identified as reliable RGs in certain plants under given experimental conditions[7,8,9,10]. To avoid missing or overemphasizing potential biological changes of target gene expression, it is essential to identify optimum stable RGs for the proposed research object, for different tissues of the same species, for the same tissue of the same species under different biotic or abiotic stresses and their processing time
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