Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses

PLoS One. 2017 Apr 28;12(4):e0175863. doi: 10.1371/journal.pone.0175863. eCollection 2017.

Abstract

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.

MeSH terms

  • Camellia sinensis / drug effects
  • Camellia sinensis / genetics*
  • Camellia sinensis / physiology*
  • Gene Expression Profiling / standards*
  • Genes, Plant / genetics*
  • Metals / pharmacology*
  • Reference Standards
  • Stress, Physiological / drug effects*
  • Stress, Physiological / genetics*

Substances

  • Metals

Grants and funding

This work was supported financially by the National Natural Science Foundation of China (31570689, 31470690), http://www.nsfc.gov.cn/; the China Earmarked Fund for Modern Agro-industry Technology Research System (CARS-23), http://www.moa.gov.cn/; the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), http://www.ec.js.edu.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.