Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

Zhimin Yang,Baoyun Hu,Bingru Huang,Yu Chen,Zhiqun Tan,Zhulong Chan

doi:10.1371/journal.pone.0119569

Abstract

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

Highlights

Determination of gene expression patterns and quantitative levels is a commonly-used approach for discovering genes controlling plant traits in various plant species responding to different environmental factors [1,2,3,4]
The Cq values of nine reference genes were obtained by quantitative real-time polymerase chain reaction (qRT-PCR) analysis in 144 samples and variations of each gene were shown in the box-chart (Fig. 2)
The results showed that glyceraldehyde3-phosphate dehydrogenase (GAPDH) and TUB with same M values were the two best reference genes for pooled samples including leaves and roots of all stress treatments or PEG-treated leaves (PL), SAND and TUB in salt-treated roots (SR), PEPKR1 and TUB in salt-treated leaves (SL), TUB and TIP41-like family protein (TIP41) in PEG-treated roots (PR), PEPKR1 and TIP41 in cold-treated roots and leaves (CR and CL), clathrin adapter complex subunit family protein (CACS) and TUB in heat-treated roots (HR), and ACT and TIP41 in heattreated leaves (HL)

Summary

Introduction

Determination of gene expression patterns and quantitative levels is a commonly-used approach for discovering genes controlling plant traits in various plant species responding to different environmental factors [1,2,3,4]. Reference Genes for q-PCR Analysis of Gene Expression. The accuracy of qRT-PCR analysis is strongly influenced by the stability of reference genes, quantity and purity of the mRNA templates, enzymatic efficiency in cDNA synthesis, and PCR amplification [5,6]. Among those factors, the first consideration of qRT-PCR for data normalization should be the selection of reference genes as the internal control of stable expression under different experimental conditions [5]. The use of proper reference genes in qRT-PCR assay is critically important for the accuracy of qRT-PCR results

Objectives

Methods

Results

Conclusion