Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plants under differential biotic stresses

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.With the increasing popularity of gene expression analysis in biological research, quantitative real-time polymerase chain reaction (qRT-PCR) has become a critical and powerful tool for rapid and reliable quantification of mRNA transcriptional expression levels of target genes due to its high-throughput screening, sensitivity, simplicity, specificity and accuracy 1,2 . Relative quantification of target gene expression under certain stresses has been widely studied since the beginning of this century 3 . An accurate assay of gene expression through qRT-PCR relies on every step of sample preparation and processing, e.g., the integrity of purified RNA, the efficiency of reverse transcription, and the overall transcriptional activity of the tissues or cells analysed 4 ; each step needs to be accurately normalized by stably expressed reference genes (RGs) 5,6 . Therefore, the selection of reliable RGs for normalization under given experimental conditions is a requirement for developing an accurate qPCR assay.Housekeeping genes, such as the glyceraldehyde 3-phosphate (GAPDH), the actin gene (ACTIN), translation elongation factor EF-1 alpha (EF-1α), 18 s r...

Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

“…Since its publication in 2018, this article by Shakeel and colleagues on insects has been cited 22 times by articles in PubMed. Fourteen of these citing articles are those evaluating candidate reference genes [ 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 ]. We hope that our critical review will similarly stimulate future research on experimentally validating reference genes for gene expression studies in Aspergillus (and in fungi in general) using RT-qPCR.…”

Evaluation and Selection of Suitable qRT-PCR Reference Genes for Light Responses in Tea Plant (Camellia Sinensis)

“…In order to detect whether there is any difference in the expression stability of actin family members when C. sinensis is exposed to various light conditions, six pairs of distinctive primers (corresponding to CsACT1, CsACT2, CsACT(3-4), CsACT(5-6), CsACT (7)(8), and CsACT(9-10) genes from C. sinensis genome) were investigated in this study. In addition, six common reference genes of C. sinensis from other families, including CsUBC1, CsGAPDH, CsTBP, CseIF-4α, clathrin adaptor complex subunit (CsCLATHRIN1), and tap42-interacting protein of 41 kDa (CsTIP41) were evaluated together [28,30]. The expression stability of these candidate genes was calculated and evaluated to obtain the most suitable reference genes in response to light quality (LQ) (red light and blue light), light intensity (LI) (shading and darkness), and photoperiod (PD) (0-24 h) based on four different statistical algorithms (geNorm [32], NormFinder [33], BestKeeper [34], and RefFinder [35]).…”

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

“…In the expression experiments of C. sinensis, housekeeping genes, especially ACT gene, were the most frequently used reference genes [16,17,20,26]. Suitable reference genes in C. sinensis have been screened and evaluated under biotic and abiotic stress, biotic and abiotic stresses, tissue development, and hormone treatment [27][28][29][30][31]. In general, the stable expression of reference genes under certain experimental conditions may not be stable under other conditions.…”