Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses.

Man Zhou,Junming Zhao,Yu Meng

doi:10.3390/genes11050502

Abstract

Switchgrass (Panicum Virgatum L.) has been recognized as the new energy plant, which makes it ideal for the development of phytoremediation on heavy metal contamination in soils with great potential. This study aimed to screen the best internal reference genes for the real-time quantitative PCR (RT-qPCR) in leaves and roots of switchgrass for investigating its response to various heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and arsenic (As). The stability of fourteen candidate reference genes was evaluated by BestKeeper, GeNorm, NormFinder, and RefFinder software. Our results identified U2AF as the best reference gene in Cd, Hg, Cr, and As treated leaves as well as in Hg, Pb, As, and Cr stressed root tissues. In Pb treated leaf tissues, 18S rRNA was demonstrated to be the best reference gene. CYP5 was determined to be the optimal reference gene in Cd treated root tissues. The least stable reference gene was identified to be CYP2 in all tested samples except for root tissues stressed by Pb. To further validate the initial screening results, we used the different sets of combinatory internal reference genes to analyze the expression of two metal transport associated genes (PvZIP4 and PvPDB8) in young leaves and roots of switchgrass. Our results demonstrated that the relative expression of the target genes consistently changed during the treatment when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, or 18S rRNA/ACT12 were combined as the internal reference genes. However, the time-dependent change pattern of the target genes was significantly altered when CYP2 was used as the internal reference gene. Therefore, the selection of the internal reference genes appropriate for specific experimental conditions is critical to ensure the accuracy and reliability of RT-qPCR. Our findings established a solid foundation to further study the gene regulatory network of switchgrass in response to heavy metal stress.

Highlights

Real-time quantitative PCR (RT-qPCR) has become the leading technique applied in gene expression analysis due to its advantageous characteristics, such as high-throughput, high sensitivity, and specificity, along with great repeatability
Our study reports the first validation of housekeeping genes in switchgrass allowing the identification of the most suitable reference gene(s) for normalization of real-time quantitative PCR (RT-qPCR) in different plant tissues and different time-courses subjected to heavy metal treatments such as by Cd, Pb, Hg, Cr, and As
Our results demonstrated that the target genes exhibited a general expression pattern in response to heavy metal stress when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, and 18S rRNA/ACT12 were used as the internal reference genes, while irregular patterns were shown with CYP2 selected to be the reference gene for RT-qPCR analysis

Summary

Introduction

Real-time quantitative PCR (RT-qPCR) has become the leading technique applied in gene expression analysis due to its advantageous characteristics, such as high-throughput, high sensitivity, and specificity, along with great repeatability. Reference genes with stable expression levels are used as the standard markers to calibrate and ensure the accuracy and validity of results from RT-qPCR [1,2]. Genes 2020, 11, 502 identify the appropriate and stable reference genes associated with various situations in the analysis of gene expression profiles. The remediation of heavy metal polluted soils, especially phytoremediation, has gained increasing attention from both academia and industries due to its lower cost and fewer side effects than conventional chemical and physical techniques [6]. Though the hyperaccumulator has become the hotspot for ecological restoration in recent years, utilization of energy plants for remedying heavy metal contaminated soil can achieve a win–win situation for the production of biomass raw materials as well as the management of the polluted soil [7]

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