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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:BACKGROUND:Tea plant (Camellia sinensis) is one of the most popular non-alcoholic beverages worldwide. In tea, lateral roots (LRs) are the main organ responsible for the absorption of moisture and mineral nutrients from the soil. Lateral roots formation and development are regulated by the nitrogen and auxin signaling pathways. In order to understand the role of auxin and nitrogen signaling in LRs formation and development, transcriptome analysis was employed to investigate the differentially expressed genes involved in lateral roots of tea plants treated with indole-3-butyric acid (IBA), N-1-naphthylphthalamic acid (NPA), low and high concentrations of nitrogen. RESULTS:A total of 296 common differentially expressed genes were identified and annotated to four signaling pathways, including nitrogen metabolism, plant hormone signal transduction, glutathione metabolism and transcription factors. RNA-sequencing results revealed that majority of differentially expressed genes play important roles in nitrogen metabolism and hormonal signal transduction. Low nitrogen condition induced the biosynthesis of auxin and accumulation of transcripts, thereby, regulating lateral roots formation. Furthermore, metabolism of cytokinin and ethylene biosynthesis were also involved in lateral roots development. Transcription factors like MYB genes also contributed to lateral roots formation of tea plants through secondary cell wall biosynthesis. Reversed phase ultra performance liquid chromatography (RP-UPLC) results showed that the auxin concentration increased with the decreased nitrogen level in lateral roots. Thus, tea plant lateral roots formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation. CONCLUSION:This study provided insights into the mechanisms associated with nitrogen and auxin signaling pathways in LRs formation and provides information on the efficient utilization of nitrogen in tea plant at the genetic level.

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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