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

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.

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

“…Different organs Nitrogen stress Fe stress Sun et al 29 ; Liu et al 20 ; Wang et al 24 2 CsCLATHRIN1…”

“…Wu et al 26 ; Wang et al 24 ; Zhou et al 27 Table 1. Ten housekeeping genes frequently used for qRT-PCR of tea plant.…”

Isolation of Partial Housekeeping Genes on Tuntun Angin (Elaeocarpus floribundu)

“…However, for the purposes of gene expression studies, more than one reference gene are needed as internal control (Rebouças et al, 2013;Wang et al, 2017;Bao et al, 2016). Moreover, the most widely used housekeeping genes as internal control are 18S rRNA, 26S rRNA, EF1α, GAPDH, actin, and beta tubulin (βTUB) in plants like tea (Camellia sinensis), jute (Corchorus capsularis), and olive (Olea europaea) (Kozera & Rapacz, 2013;Ray & Johnson, 2014;Niu et al, 2015;Wang et al, 2017). Therefore, the objective of this research was to isolate the partial housekeeping genes in E. floribundus such as genes encoding 18S rRNA, 26S rRNA, elongation factor 1 alpha (EF1α), and beta tubulin (βTUB).…”

Validation of reference genes for gene expression analysis using quantitative polymerase chain reaction in pea lines (Pisum sativum) with different lodging susceptibility

“…We have chosen five reference genes ( Phosphoprotein phosphatase 2A [ PP2A ], histone H3 [ H3 ], β‐tubulin [ TUB ], transcription factor IIA [ TF2A ] and actin [ ACT ]). All of them were previously used as reference genes in research concerning gene expression in plants (Guerriero et al, ; Wang et al, ; Imai et al, ). The chosen genes showed the most stable expression in pea leaf and root tissues, under different conditions (Saha & Vandemark, ; Die et al, ).…”