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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).

ABSTRACT: Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ?CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

SUBMITTER: Hao X

PROVIDER: S-EPMC4284700 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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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).

Hao Xinyuan X Horvath David P DP Chao Wun S WS Yang Yajun Y Wang Xinchao X Xiao Bin B

International journal of molecular sciences 20141202 12

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stabilit ...[more]

PMID: 25474086

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Project description:BackgroundDrought is one of the major factors reducing the yield of many crops worldwide, including the tea crop (Camellia sinensis (L.) Kuntze). Calcium participates in most of cellular signaling processes, and its important role in stress detection and triggering a response has been shown in many crops. The aim of this study was to evaluate possible effects of calcium on the tea plant response to drought.MethodsExperiments were conducted using 3-year-old potted tea plants of the best local cultivar Kolkhida. Application of ammonium nitrate (control treatment) or calcium nitrate (Ca treatment) to the soil was performed before drought induction. Next, a 7-day drought was induced in both groups of plants. The following physiological parameters were measured: relative electrical conductivity, pH of cell sap, and concentrations of cations, sugars, and amino acids. In addition, relative expression levels of 40 stress-related and crop quality-related genes were analyzed.ResultsUnder drought stress, leaf electrolyte leakage differed significantly, indicating greater damage to cell membranes in control plants than in Ca-treated plants. Calcium application resulted in greater pH of cell sap; higher accumulation of tyrosine, methionine, and valine; and a greater Mg2+ content as compared to control plants. Drought stress downregulated most of the quality-related genes in both groups of tea plants. By contrast, significant upregulation of some genes was observed, namely CRK45, NAC26, TPS11, LOX1, LOX6, Hydrolase22, DREB26, SWEET2, GS, ADC, DHN2, GOLS1, GOLS3, and RHL41. Among them, three genes (LOX1, RHL41, and GOLS1) showed 2-3 times greater expression in Ca-treated plants than in control plants. Based on these results, it can be speculated that calcium affects galactinol biosynthesis and participates in the regulation of stomatal aperture not only through activation of abscisic-acid signaling but also through jasmonic-acid pathway activation. These findings clarify calcium-mediated mechanisms of drought defense in tree crops. Thus, calcium improves the drought response in the tea tree.

Dataset Information

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).

Publications

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).

Similar Datasets

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