Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant  (Camellia sinensis (L.) O. Kuntze)

Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

doi:10.3390/ijms151222155

Cited by 157 publications

(107 citation statements)

References 56 publications

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“…Normalizing results with one or more appropriate internal RGs is a simple and popular method for controlling error in qRT-PCR assays. To date, 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 (auxin, ABA, GA, IAA, MeJA and SA) 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.…”

Section: Discussionmentioning

confidence: 99%

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

Xü

Dong

et al. 2020

Sci Rep

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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...

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Section: Discussionmentioning

confidence: 99%

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

Xü

Dong

et al. 2020

Sci Rep

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“…Gene expression was analyzed by quantitative (q) RT-PCR using gene-specific primers (Table S1), and the qRT-PCR analysis was performed with an Applied Biosystems 7500 Sequence Detection System using SYBR ® Premix Ex Taq™ II (Takara Bio Inc., Otsu, Japan). According to Hao et al (2014) recently described, the polypyrimidine tract-binding protein (PTB1) gene was used as an internal control.…”

Section: Gene Expression Analysismentioning

confidence: 99%

Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season

et al. 2015

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Sugar plays an essential role in plant cold acclimation (CA), but the interaction between CA and sugar remains unclear in tea plants. In this study, during the whole winter season, we investigated the variations of sugar contents and the expression of a large number of sugar-related genes in tea leaves. Results indicated that cold tolerance of tea plant was improved with the development of CA during early winter season. At this stage, starch was dramatically degraded, whereas the content of total sugars and several specific sugars including sucrose, glucose and fructose were constantly elevated. Beyond the CA stage, the content of starch was maintained at a low level during winter hardiness (WH) period and then was elevated during de-acclimation (DC) period. Conversely, the content of sugar reached a peak at WH stage followed by a decrease during DC stage. Moreover, gene expression results showed that, during CA period, sugar metabolism-related genes exhibited different expression pattern, in which beta-amylase gene (CsBAM), invertase gene (CsINV5) and raffinose synthase gene (CsRS2) engaged in starch, sucrose and raffinose metabolism respectively were solidly up-regulated; the expressions of sugar transporters were stimulated in general except the down-regulations of CsSWEET2, 3, 16, CsERD6.7 and CsINT2; interestingly, the sugar-signaling related CsHXK3 and CsHXK2 had opposite expression patterns at the early stage of CA. These provided comprehensive insight into the effects of CA on carbohydrates indicating that sugar accumulation contributes to tea plant cold tolerance during winter season, and a simply model of sugar regulation in response to cold stimuli is proposed.

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“…Quantitative real-time reverse transcription PCR (qRT-PCR) is generally regarded as a convenient and efficient tool to analyse gene expression, but the RNA quantification, the reverse transcription reaction efficiency and other uncontrolled factors may limit the accuracy and stability of the final results (Huang et al 2014; Hao et al 2014). Thus, it is necessary to apply reliable reference genes to normalize the data.…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

Zhang

Hou

et al. 2017

AMB Expr

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Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek–Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.

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Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

Cited by 157 publications

References 56 publications

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

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

Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

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