Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

Xiaoping Niu,Jiantang Xu,Jianguang Su,Aifen Tao,Gaoyang Zhang,Jianmin Qi,Pingping Fang

doi:10.3389/fpls.2015.00848

Abstract

To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.

Highlights

With the increase of global environmental awareness, more and more people are actively purchasing goods made from ecologically friendly materials
The results showed that EF1α could sever as a stable reference gene for normalization but ubiquitinconjugating enzyme like protein (UBC) unstable under NaCl stress condition (Figure 4A)
ACT was proved unsuitable for normalization in different flax tissues (Huis et al, 2010), during abiotic and biotic stress in S. tuberosum (Nicot et al, 2005), and in viral-infected N. benthamiana (Liu et al, 2012). These findings indicate that large numbers of experimental data on gene expression should be acquired to investigate the transcript stability of commonly used reference genes under different experimental conditions

Summary

Introduction

With the increase of global environmental awareness, more and more people are actively purchasing goods made from ecologically friendly materials. Many of these materials are intrinsically unrecyclable, including many of the predominantly used polymers. Many natural fibers such as jute and kenaf possess properties that are comparable to more traditional composites, including stiffness, flexibility, impact resistance, and elasticity. Fiber components are biodegradable, renewable, and result in low energy consumption. These characteristics lend natural fibers to being logical substitutes for non-renewable synthetic fibers (Oksman et al, 2003; Corrales et al, 2007)

Methods

Results

Discussion

Conclusion